MCB4203 Exam 1 SG

Know how "old" bacterial infections can reoccur. Be able to describe mutations and their importance.

"Apparently new diseases" emerge or "old disease"s can reemerge: • bacteria can acquire new traits via mutations and HGT • our own activities create new opportunities for pathogens: a) Antibiotic use in hospitals: AMR Clostridium difficile, Klebsiella pneumoniae, MRSA, Streptococcus pneumoniae b) Tuberculosis came back in 1990s (dismantled infrastructure to contain the spread, presence of susceptible individual groups in prisons, emergence of AIDS) • Diseases were around for a long time but causative bacteria just recognized (e.g Helicobacter pylori)

Know the factors leading to smallpox's eradication

(1) It is a human-specific disease with no known animal reservoir for the pathogen, such that person-to-person transmission can be prevented, with no risk of reintroduction through animal exposure. (2) There are excellent sensitive and specific diagnostic and surveillance tools available for rapid identification and epidemiologic tracking. (3) Survival from infection or vaccination confers lifelong protective immunity. (4) There is a high international health care and financial burden associated with the disease, such that commitment to its elimination and eradication will be highly beneficial on a global scale.

Know what IRB and IACUC are and what these units are responsible for.

*IRB*- maintains guidelines for human volunteer research research is approved and overseen by Institutional Review Board (IRB), which approves, monitors, and reviews all biomedical and behavioral research involving humans IRB answers to FDA (Food and Drug Administration) and DHHS (The United States Department of Health & Human Services) *IACUC* - ethical considerations for animal models Use of vertebrate models (zebrafish, mice, etc) must be approved and regulated by IACUC (Institutional Animal Care and Use Committee

Know the importance of Langerhans, keratinocytes, and SALT in the skin

*Langheran cells*- serve as phagocytic cells keratinocytes (skin cells) are highly specialized epithelial cells skin -associated lymphoid tissue (SALT)->skin immune system cutaneous immunity Dermal dendritic cells • Lymphocytes: T cells, B cells, natural killer (NK) cells • Mast cells

Know the methods that are used to identify pathogens in tissues.

. Molecular: PCR-based tests 2. Immunohistochemistry 3. Enzyme-linked immunosorbent assay (ELISA) 4. Electron microscopy 5. Special stains, microbiology (culture-based methods)

Know the molecular Koch postulates and their difficulties

1. Gene (or its product) should only be found in bacterial strains that cause disease and not in bacteria that are avirulent 2. Gene should be "isolated" by molecular cloning and sequencing 3. Mutation/disruption of the gene should reduce virulence in vivo: loss of function (Or introduction of gene into avirulent strain should confer virulence) 4. The pathogenicity or phenotype is restored when the gene is reintroduced into the mutant bacterium and that the gene is expressed by the bacterium when it is in an animal or human volunteer at some point during the infectious process Caveats: 1. Not easy to demonstrate for pathogens that have an "arsenal" of virulence factors (e.g. Staphylococcus aureus) 2. Genetic manipulation of some pathogens difficult or impossible 3. Cannot be easily applied to polymicrobial/microbiota shift diseases

Know how to target mucosal immunity

1. Injection-Free Vaccines • intranasal vaccination to stimulate MALT • vaccine patch -> stimulation of SALT) 2. Edible vaccines • plant-based oral vaccines • low cost of production • ability to confer both mucosal (IgA) and systemic (IgG) immunity • issues with variable dosing low level expression of antigen • issues with introducing vaccine with food - > possible tolerance? 3. Vaccine Vector Technology (next slides) 4. Toxins to Stimulate Mucosal Immunity (next slides)

Know the four questions scientists sought to answer (slide 11)

1. Is spontaneous generation of microbial life possible? 2. What causes fermentation? 3. What causes disease? 4. How can we prevent infection and disease?

Understand the four postulates described by Koch

1. Microorganism should be found in all cases of the disease in question, and its distribution in the body should be in accordance with the lesions observed 2. The microorganism should be grow in pure culture in vitro for several generations 3. When such a pure culture is inoculated into susceptible animal species, the typical disease must result 4. The microorganism must again be isolated from the lesions of such experimentally produced disease

COMPLEMENT - FUNCTIONS

1. promotion of opsonization (engulfment) of invading bacteria by coating them with complement components (C3b) 2. enhancement of chemotaxis of phagocytes to attract them to the site of infection by releasing chemokines (C5a); 3. enhancement of vascular permeability - > transmigration of phagocytes from blood vessels to the site of infection by releasing vasodilators (C3a, C4a, and C5a) that cause degranulation (release of histamine and heparin) of basophils and mast cells 4. promotion of agglutination of invading bacteria ->increased phagocytosis and clearance from the system 5. direct killing of Gram-negative bacteria (holes in membranes, binding of C5b to LPS and formation of the membrane attack complex; MAC) Opsonization of a bacterium by activated complement component C3b and antibodies -> enhancement of the uptake of the bacterium by phagocytes opsonization = process of marking phagocyte targets

Know what are contigs.

1. simultaneous determination of millions of base pairs of DNA sequences in single reaction run 2. bioinformatic -> rapid assembly of sequences into large regions of overlapping sequences (contigs) 3. contigs mapped into a complete genome

Know how the peptides permeabilize the bacterial membrane.

1. α-defensin expressed as an inactive propeptide • processed (activated) by proteases (MMP7 or trypsin) 2. REG3α expressed as an inactive propeptide processed by proteolytic cleavage • binds to bacterial peptidoglycan • forms a hexameric pore in bacterial membranes. 3. Cathelicidins bind to bacterial membranes • form α-helical structures (presence of lipids) • insert into the bacterial membrane to form pore

Remember the function and binding partners of TLR4 and TRL5 and overall function what TLRs do and that some are intra- and others extra-cellular. (No need to remember all TLRs names and functions, but know there are more than one, and what they bind and why (NFkappaB stimulation -> inflammation).

10 Toll-like receptors (TLR) in human modulate cellular responses to pathogens: TLR4 - recognizes LPS • TLR1/TLR2 or TLR2/TLR6 TRL5- recognizes bacterial flagellin TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are exposed on the surface of the phagocytic cell while TLR3, TLR7, TLR8, and TLR9 reside within endosomes • TLR contains a leucine-rich repeat (LRR) domain which binds the PAMP ligand and induces dimerization. • The intracellular domain the Toll/IL-1R (TIR) domain conveys the signal detected by the LRR domain binding to a PAMP ligand to the interior of the phagocytic cell -> signal transduction cascade • activation of signaling pathways leads to expression of inflammatory genes or induction of phagocytic functions (maturation and secretion of cytokines, stimulation of the oxidative burst)

Know the terms: antigen, immunogenicity, adjuvant, APC, antibody, superntigen, epitope.

A substance (protein, lipid, carbohydrate) that can provoke the immune response, such as the production of an antibody • Immunogenicity - ability to induce a humoral and/or cell-mediated immune responses; dictated by: • Foreignness: "self" vs "non-self" • Size: < 10 kDa weakly immunogenic, amino acids (AAs) non-immunogenic • Hapten: molecule that is only immunogenic when joined to a suitable carrier molecule • Chemical/Structural complexity: i.e. immunogenicity of AA homopolymers < heteropolymers • Host genetics: differential response to the same antigen by two members of the same species (different alleles) • Dosage/Route/Timing of Ag administration: optimization of these variables -> altered immune response • Adjuvant - facilitates uptake into Antigen Presenting Cells (APCs) • Antibody (Ab): protein produced as a result of interaction with specific Ag

CLOSING

AMR is most certainly a Global Health issue—it takes the combined effort of all communities to gain control over the spread of deadly bacteria. • One Health, however, is just as important. For Global Health regulations to be mandated, local communities must be in control and aware of how to reduce the spread of AMR "Anything that improves global equality—increasing the wealth and dignity of people around the world—where AMR is emerging due to shortage of resources will benefit everyone with respect to AMR.

AMR TERMINOLOGY

AMR: Anti Microbial Resistance • ARB: Antibiotic Resistant Bacteria • ARG: Antibiotic Resistant Genes

Know that the route of the delivery influences the first microbiota of a baby.

After birth: • microbiota profile of vaginally delivered infants ~ mother's vagina • microbiota profiles of infants delivered via C section ~mother's skin microbiota

Number 2

Although many advances in culturing techniques have been made, there are still pathogens that are difficult or impossible to culture in vitro (i.e. Treponema pallidum) • More media formulations, specialized incubators • Genome sequencing -> metabolic prediction -> custom media formulation • Eukaryotic cell lines for intracellular bacteria Not all diseases are caused by a single-species of bacteria (i.e. polymicrobial disease, microbiota shift diseases) • Use of culture-independent/molecular techniques (polymerase chain reaction, immunohistochemistry) to identify pathogens in diseased tissue

COMPETITION ASSAYS

Animal infected with a mixture of wild-type and mutant bacteria • "input ratio" = (CFUmutant/CFUwild-type) • Each strain must be "trackable" i.e. antibiotic resistance gene • CFU plating of organs on selective media to determine output ratio (CFUmutant/CFUwild-type) Competitive Index (CI) = output ratio/input ratio • CI = 1.0; no difference in virulence • CI > 1.0; mutant outcompetes wild-type; aka mutant more virulent • CI < 1.0; wild-type outcompetes mutant; aka mutant less virulent • trans effects may complement mutant phenotype • If mutant is slow-growing, it will also likely be "less fit" in animal model

Know what type of animals can be used as models of human disease

Animal models of human disease should have • a route of infection • severity of disease • morbidity and mortality levels ...that are similar to the human course of the disease

Know that there is a crosstalk between adaptive and immune system - importance of DCs, complement, antibodies

Antibody can activate complement or adhere to mast cell or MAC T cell can activate MAC DCs can present T cells

Know the difference between MHCI and MHCII. Be able to explain Ag presentation on MHC.

Antibody: A substance (protein, lipid, carbohydrate) that can provoke the immune response, e.g. has ability to induce a humoral or cell-mediated immune responses • antigen peptide epitopes are displayed on the surface of APC (antigen presenting cells) on protein complex - the major histocompatibility complex (MHC). • Two main classes of MHC molecules, MHC I and MHC II • MHC I are produced by all nucleated cells in the body • MHC II produced by professional phagocytic APCs, such as DCs, macrophages, B cells, and certain activated epithelial cells • In the case of peptide antigens, the type of MHC used to display the epitope determines the type of immune response

Know two routes (one for each MHC) of Antigen display on MHC - one via class I (proteasome, ER) and one via class II MHC (endocytosis ER).

Antigen Presenting Cell (APC), e.g. dendritic cell or macrophage, takes up antigen (or infectious pathogen) 2. Activation of APC via Pattern Recognition Receptors (PRRs), induction of co-stimulatory molecules, release of cytokines. 3. APC degrades antigen into peptides (if needed) and presents them on MHC molecules to T cells 4. T cells require cytokines and co-stimulatory molecules for activation and proliferation

Know that they recognize antigens, and are stimulated by CD40-CD40 ligand (Th cell) interaction. Know what are memory B cells and their function.

Antigen recognition (direct binding of Ab to Ag) • internalization, processing, MHCII presentation ("APC") • T-cell interaction (TCR-MHC II) • Stimulation of B cells through interactions of CD40 on the B cells with CD40 ligand expressed by Th cells and through cytokines produced by Th cells • plasma cell differentiation and Ab production • Memory B cells ensure rapid response to second infection

IMPORTANCE OF ONE HEALTH TO AMR

Antimicrobial resistance (AMR) is a growing problem throughout the world: • Overuse of antibiotics • Millions of cases and deaths • Increased costs and time for medical treatment • Global Health requires One Health because local communities need to be corrected first; One Health requires Global Health because it needs an overarching plan of communication and cooperation.

Be able to explain what the pleomorphic theory and miasma theory were proposing.

Antoine Béchamp (1816 - 1908): microzymas ("tiny enzymes", "little bodies" are fundamental "building blocks" of life, can assume different "forms") • healthy body: microzymas repair and nourish cells • In a diseased body (i.e. acidic pH): microzymas morph to destructive bacteria. • Specific disease states dictate the type of "germ" that form • These "destructive" forms attack the host body • -> "germs" are the result of host disease, not the cause Predominant from Middle Ages to 19th century • infectious diseases were caused and transmitted by foul vaporous emanations that were associated with decaying matter and bad sanitary conditions. • "malaria" (1740) literally means "bad air" in Italian. • Max von Pettenkofer (1818-1901): improved sanitation → less smells → reduced disease • Wrong scientific reasons, but miasma theory still promoted public health reform and improved hygiene

Know who made their own microscopes and examined single-celled organisms

Antonie van Leewenhoek examined water and visualized fungi, algae, and single-celled protozoa "animalcules"

Know what autophagy is and its function in the clearance of intracellular bacteria.

Autophagy enables the breakdown and recycling of dysfunctional cellular components • sequestering unwanted components into a double-membrane autophagosome, which fuses with a lysosome (now: autophagolysosome) • Intracellular pathogens can be targeted for autophagy • Bacteria that escape into the cytosol of the cell become ubiquitinated • Modified bacteria then recruit adaptor proteins which bind ubiquitinated proteins and LC3-PE on phagophores • Lysis of bacteria

Bacteria!

Bacteria are formidable opponents: first form of life on Earth 3.5-4 billions years ago • Master recyclers: created first oxygen, making life on Earth possible • Developed a whole spectrum of metabolic capabilities to survive in various conditions (metabolize by oxidizing sulfides, reducing sulfate, oxidizing ammonia, reducing nitrate etc.) • Plasticity of genomes • Found anywhere on earth

AMR APPLIES TO BACTERIA SINCE "BACTERIA, NOT HUMANS OR ANIMALS, BECOME ANTIBIOTIC-RESISTANT", YET....

Bacteria are not the only problem: • Viruses • Fungi • Parasites 'antibiotic-resistant patients' and 'antibiotic-resistant infections' terms can be used • For this paper and discussion, AMR and ARG will be used, as similar ideas apply to other organisms.

Bacteria's lives are intertwined with their hosts

Bacterial endosymbionts formed mitochondria and chloroplasts: function in respiration and regulation of metabolism (mitochondria) as well as acquiring photosynthesis by plants (chloroplasts) • Human and animals serve as rich niches for bacteria i.e. bacterial heaven (temperature is stably maintained, lifestyle is to collect food and water). Human/animal as a HOST • Microbiome (microbial community) as our organ: microbes colonized intestinal tracts, skin, mouth , vagina, and other parts of our body • Helicobacter pylori: example of how sharing ancestral relationship with pathogen can prevent ulcers and cancer. The longer human co-evolved with pathogen, the less likely that bacterium caused diseases

BIOPHOTONIC IMAGING ("IVIS")

Bacterial strains to be studied are engineered to express luciferase (lux operon from Vibrio sp.) • Supersensitive digital camera can detect and quantify light produced by the bacterial infection • Technology can be adapted for detecting fluorescence • Many time points can be taken, without having to sacrifice animals for CFU plating

Know the important physical barriers: skin, mucous membranes, epithelial cells, endothelial cells, etc.

Barrier defenses • physical and chemical barriers that prevent pathogens from entering and infecting the body. • consists of protective mechanisms of the skin, mucosae, and chemical secretions against pathogens • The barrier system can fail when the skin breaks or upon pathogen invasion into the mucosal epithelium mucous membranes: epithelium for internal structures that secretes mucus and provides some barrier immune system function. The skin: interface with the environment and is the first line of defense skin + mucosae = barrier system

Know what Belmont report was

Belmont report (1979) - basis of current ethical guidelines and federal regulations concerning human subject research

Be aware of the location of cells of innate and adaptive immunity in different organs - e.g. know that spleen and lymphatics/nodes are major locations of T cells, know where macrophages and DCs reside and what cells are present in the blood.

Bone marrow and thymus: where immune system cells develop into functional cells • Immune tissues (concentrated immune cells) include the blood, spleen, and lymphatic tissues • Blood: various immune cells, clotting factors (plasma), antibodies, and other proteins • Lymphatic system: network of vessels and nodes that circulate lymph (plasmaderived liquid) • Lymph nodes: immune cells survey lymph for presence of foreign agents (transported from tissues by other immune cells) • Spleen: "blood filter"; antibody production, removes antibody-coated cells, "monocyte reservoir"

Th17 cells

CD4+ Th cells and CD8+ cytotoxic T cells that produce IL-17 • stimulate proinflammatory reactions that lead to recruitment of PMNs and neutrophils in tissues • produce IL-22 -> antimicrobial peptide production by epithelial cells • protect against extracellular bacteria and fungi.

Know Carl Linnaeus and his work

Carl Linnaeus (1707-1778) developed taxonomic system for naming plants and animals and grouping similar organisms together • Leeuwenhoek's microorganisms were grouped into six categories: • Fungi • Protozoa • Algae • Bacteria • Archaea • Small animals

Know the function of microbiota in the colon (anabolic and catabolic)

Catabolism • fermenting the dietary polysaccharides (Bacteroides): mucopolysaccharides, mucins, complex, undigested carbohydrates (cellulose, xylan, starch) Anabolism: • synthesize nutrients and vitamins • Production of CO2, H2, and short-chain fatty acids (SCFAs; acetate, propionate, and butyrate), nutrients absorbed by intestinal cells • The products of fermentation (acetate, CO2, and H2) - carbon and energy sources of methanogenic archaea (methane -> breath and flatus) • Sulfate-reducing bacteria use H2 to produce Sulfides (odor of feces) • obese individuals have less Bacteroidetes (5% vs 25%) and more Firmicutes (85% vs 75% Firmicutes) • Effect on emotional behavior (anxiety, depression, and pain)-> therapeutic potential of probiotics?

Know the importance of One Health (and the difference between One Health and Global health - graduate students)

Category A: • can be easily disseminated or transmitted from person to person; • result in high mortality rates and have the potential for major public health impact; • might cause public panic and social disruption; and • require special action for public health preparedness. Agents/Diseases: Anthrax (Bacillus anthracis) -> anthrax attacks in 2001 Botulism (Clostridium botulinum neurotoxin) Plague (Yersinia pestis) Smallpox (variola major) Tularemia (Francisella tularensis) Viral hemorrhagic fevers, including Filoviruses (Ebola, Marburg) Arenaviruses (Lassa, Machupo) Category B: are moderately easy to disseminate; • result in moderate morbidity rates and low mortality rates; and • require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance Agents/Diseases: Brucellosis (Brucella species) Epsilon toxin of Clostridium perfringens Food safety threats (Salmonella species, Escherichia coli O157:H7, Shigella) Glanders (Burkholderia mallei) Melioidosis (Burkholderia pseudomallei) Psittacosis (Chlamydia psittaci) Q fever (Coxiella burnetii) Ricin toxin from Ricinus communis (castor beans) Staphylococcal enterotoxin B Typhus fever (Rickettsia prowazekii) Viral encephalitis Water safety threats (Vibrio cholerae, Cryptosporidium parvum)

Know the three types of Yersinia pestis infections.

Causes plagues Bubonic (up to 80% death rate) septicaemic (~100% death rate) pneumonic (~95% death rate) plague

Know what types of methods can be used to study the infections in animal models, be aware of any pluses/weaknesses of the methods, what they exactly measure, and how they can be used

Choice of bacterial species, animal model, and route of infection • Infectious dose, time points, and other parameters must be optimized Common methods • Colony-forming units (CFUs) to determine bacterial loads • Survival Curve Analysis • LD50 and ID50 • Biophotonic Imaging • Competition Assays

Know how the trade and changing environments brought Yersinia pestis into Europe.

Climate change in Asia -> rodents with infected fleas moved to populated areas Aftermath: • Malnutrition • Poverty • Wars • Price control on grain and exported goods • Inflation • Population reduction • Deforestation leading to likely climate change • Religious persecution

Waterborne infections

Common culprits: - Legionella pneumophila - E. coli - Campylobacter Legionella pneumophila • discovered after an outbreak in 1976 (Philadelphia convention of the American Legion) • causes pneumonia (lung infection), Legionnaires' disease, Pontiac fever

Know what cells these two MHCs present antigens to

Cross-presentation: ability of certain antigen-presenting cells (APCs) to take up, process and present extracellular antigens with MHC class I molecules to CD8 T cells (cytotoxic T cells). First detected as genetic locus encoding glycoprotein molecules ("transplantation Ags") responsible for tissue graft rejection • MHC molecules bind peptide Ags and present them to T cells • Antibodies (Abs) interact directly with Ag; T-cell receptor only recognizes peptide Ags presented by self-MHC molecules on antigen-presenting cells (APC; MΦ, dendritic cell, B cell). i.e. T-cell specific for both Ag AND MHC allele • Large # alleles/genes for each MHC type

Know various methods that are used in tissue culture models: pluses, minuses, limitations, know what they measure

Cultured mammalian cells •More easily-controlled system (less complex) •Less expensive, not subject to IUCAC or IRB (unless harvesting/collecting human cells) •Primary culture vs. Immortalized cells •Many different limitations to consider

Know the pluses and weaknesses of tissue culture models

Cultured mammalian cells • More easily-controlled system (less complex) • Less expensive, not subject to IUCAC or IRB (unless harvesting/collecting human cells) • Primary culture vs. Immortalized cells • Many different limitations to consider

Know the principle of PCR

DNA isolated from infected tissue (mixture of host and bacterial DNA) Detection of product 1. Agarose-gel electrophoresis 2. Quantitative real-time PCR Must include appropriate positive and negative template controls: Positive control: Bacterial DNA isolated from pure culture Negative control: DNA from healthy (uninfected) tissue - highly sensitive for detecting minute quantities of the pathogen - specific for a particular pathogen - Multiplexing is possible (saves time and conserves sample) - Issues: positive controls are needed to rule out false negatives - real-time PCR-based pathogen detection vs endpoint, one-step RT-PCR

Know who is the father of germ theory

Diseases are caused by living micro-organisms • *Louis Pasteur* (1822-1895): processes of fermentation, food spoilage ("pasteurization"), and silkworm disease were caused by the growth of micro-organisms • "father of germ theory of disease" • particles in the air, rather than the air itself (spontaneous generation), leads to microbial growth • vaccination (smallpox, anthrax)

Know the role of 'microbiota shift' in inflammation and disease.

Disruption or elimination of normal microbiota leads to pathologies • antibiotic treatment -> vaginitis, yeast infection, diarrhea • "Microbial shift disease" vs "Opportunistic infection" A) Infections -->activation of inflammatory microbiota and pathogen-specific T cells B) inflammation in the GI tract--> shifts in the microbiota C) Shifts in the microbiota -->chronic inflammation and interference with healing D) immunological scarring, chronic inflammation and dysbiosis--> inflammation in the adipose tissue

Know the principle of antibody-based methods and how they can be used for pathogen identification and other purposes

ELISA, immunohistochemistry or immunofluorescence microscopy

Know the history of vaccination and innate immunity in Europe - E. Jenner example and Elie Metchnikoff example.

Edward Jenner (1796): milkmaids previously infected with cowpox were unlikely to succumb to smallpox • Used pustular material from milkmaid cowpox lesion to inoculate young boy -> smallpox pustular material -> no disease Élie Metchnikoff identified cells in larvae of starfish - white blood cells gather at the site of inflammation (ca. 1882), founder of cellular immunology • theory that white blood cells engulf and destroy bacteria was met with skepticism (Louis Pasteur and others) • Paul Ehrlich: side-chain theory explains the specificity of the antigen-antibody reaction (humoral immunity) • Paul Ehrlich and Élie Metchnikoff: joint Nobel Prize in 1908 • most of our current understanding of innate immunity comes from the last 100 years

Know the difference between Elimination and Eradication

Elimination - the reduction of new disease cases to zero in a specified geographical area • Eradication - worldwide elimination

Know Escherichia coli O157:H7 and its importance. What is a cell wall structure? How did it transmit? Is it dangerous?

Escherichia coli O157:H7, Shiga-toxin producing (STEC). Foodborne bacterium, causes kidney failure and death. In 1993: outbreak resulting in 4 deaths, followed by outbreak related to unpasteurized juice e.g. 2019: Fresh Express Sunflower Crisp Chopped Salad Kits - E. coli O157:H7 Romaine Lettuce - E. coli O157:H7 People infected with the outbreak strain of E. coli O157:H7, by state of residence, as of January 13, 2020 (n=167) At A Glance •Reported Cases: 167 •States: 27 •Hospitalizations: 85 •Deaths: 0 •Recall: Yes Structure: O antigen: cell wall antigen, LPS antigen H antigen: protein antigen in flagella K antigen: polysaccharide antigen in capsule

Know the ethical considerations of animal experiments.

Ethical Considerations: • Use of vertebrate models (zebrafish, mice, etc) must be approved and regulated by IACUC (Institutional Animal Care and Use Committee • Truly compelling rationale - no alternative model can be used or is available • Bacterial genetics and physiology must be thoroughly characterized in vitro • Hypothesis-driven research • Experimental design (minimum # of animals for statistical significance) • Care of animals before, during, after experiment • Anesthesia, minimization of pain and discomfort • Euthanasia once animals are moribund • Annual review of all approved protocols

Know the difference between prokaryotes and eukaryotes

Eukaryotes (multicellular organisms): fungi, protozoa Prokaryotes (single-celled): bacteria and archaea

Know the definition of an exposome and that it is linked to our understanding of the microbiome

Exposome: "the measure of all the exposures of an individual in a lifetime and how those exposures relate to health."

Know that intracellular PAMPs can also activate NLRs. Know what NRLs are. Know what inflammasome is and its function (IL-1b production, which induces more inflammation, pyroptosis). Know NOD1 binds PG.

Extracellular PAMPs • PAMP activation of TLRs and binding of the proinflammatory IL -1β and IL -18 to cognate receptors stimulate NF - κ B signaling -> expression of pro -IL -1β and pro -IL -18 Intracellular PAMPs: • Infection with intracellular pathogens induces assembly of the NLRs, adaptor protein (ASC), and caspase -1 into inflammasome complexes • caspase -1 activation/cleavage and secretion of IL -1β and IL -18 • induction of pyroptosis • NOD1 binds the bacterial peptidoglycan derived D -Glu -meso -diaminopimelic acid (DAP) • NOD2 binds muramyl dipeptide (MDP) to form a nodosome complex -> NF - κ B activation

Be able to explain what the fecal transplant is

Fecal microbial transplantation (FMT), stool transplantation, bacteriotherapy: • procedure in which fecal matter, or stool, is collected from a healthy donor and placed into the gastrointestinal tract of a patient • therapy to successfully treat severe, refractory or recurrent Clostridium difficile infection (CDI)

Know the Three Founders of Bacteriology and their importance

Ferdinand Cohn (1828-1898): father of modern bacteriology Robert Koch (1843-1910): German physician - studied causative agents of disease (anthrax, cholera, TB) - Isolated and grown Anthrax bacillus can be transmitted to other cattle - invented methods of selectively growing organisms in pure culture - pick a colony - Koch's postulates must be satisfied before we agree that specific bacteria cause particular disease - Nobel Prize - 1905 Louis Pasteur (1822-1895): - Worked on issues faced by French wine makers (fermentation to acetic acid instead of alcohol by bacteria-contaminated yeast cultures) - His research challenged 'spontaneous generation' claims - Germ theory, vaccination (chicken cholera)

Know the differences between foodborne, waterborne, and post-surgery infections.

Foodborne: within food Waterborne: in water Post-surgery (MRSA)

Know who setup the "meat-jar" experiment to disprove spontaneous generation

Francesco Redi designed experiment to test the spontaneous creation of maggots by placing meat in sealed/unsealed jars ....maggots do not spontaneously arise from decaying meat -> animals can not arise spontaneously

GENERALIST VS SPECIALIST

Generalist vs specialist terms • AMR in animals impacts our health if animals and human microbiomes share the same ARB species • host niche adaptation limits transmission between humans and food animals: 'commensal opportunistic pathogens' (e.g. extraintestinal pathogenic Escherichia coli (ExPEC) or Staphylococcus aureus) and 'frank pathogens'/true pathogens that cause foodborne zoonotic infections (Salmonella sp., Campylobacter jejuni) - generalist will do better! • Cross-species AMR transmission is difficult to demonstrate, only a few examples exist • the risk of animal-based AMR transmission to humans appears linked to particular generalist clones -> shuttles of AMR • Animal->human spread. Example: ExPEC clone acquired plasmid in poultry in 1940s->entered human food chain carrying ARGs; or example of MRSA (livestock -> humans transition happened 40 yrs ago!) • DT104 Salmonella Typhimurium: most DT104 transmission events occurred within each host population, small proportion of livestock-to-human transmissions.

Science and its new "weapons" to study the pathogens

Genomics: DNA sequencing technologies and bioinformatics: we can now rapidly study bacterial genomes • Genome annotation: Open reading frame (ORF) studies to assign gene a function • Other 'omics approaches: Transcriptomics (RNA study), Proteomics (protein study), Metabolomics (metabolite studies), Lipidomics (lipid study) • Pathogen evolution analysis: mathematical modeling to gain understanding of pathogen physiology, ecology or disease transmission • Modeling Host-pathogen interactions in animals: mouse model, Knock-out mutations, Danio rerio (fish), Caenorhabditis elegans, fruit fly Insects and worms lack adaptive immune system - cannot be used to study inflammation or humoral (antibody) responses • Correlation studies - statistical analysis of microbial and human/animal populations -> correlation between traits of the organism and outcomes of disease

Know the models that are used to study the microbiome and their limitations

Germ-free mice: the gold standard for demonstrating causal roles for the microbiota in shaping host physiology and disease Another approach: use secondary abiotic mice, such as antibiotic-treated normal mice Gnotobiosis: all the forms of life present within an organism can be accounted for.

WHAT IS GLOBAL HEALTH?

Global Health focuses on international/global impacts of reducing disease while One health focuses on geographically close ecosystems • One Health Communities together make up the Global Health initiative.

Know the difference between Gram-negative, Gram-positive, Mycobacteria, and Mycoplasma.

Gram-negative bacteria have plasma membrane and outer membrane (separated by periplasm). These bacteria contain LPS (lipopolysaccharides). Gram-positive bacteria have only plasma membrane, contain lipoteichoic acid (LTA) Mycobacteria (acid-fast bacteria) resemble Gram-positive bacteria but contain also glycolipids, such as mycolic acids Mycoplasma has no cell wall

THE S PREAD AND MAI NTENANCE OF A RG S DEPEN DS ON THEI R I N TEG RATI ON I N TO H IERA RCH ICALLY ORG A N IZ ED SYSTEMS , S UC H A S I N TEG RONS , A N D ON I N TERAC TI ON NETWORKS B ETWEEN ECOLOG I C ALLY CON N EC TED BAC TERIAL POPULATI ON S

Health institution-Person-Gene Exchange- Bacterial Clone-Plasmid-Integron-Gene Much like the nesting doll idea, AMR consists of many problems that all relate to ARG (the layers of selection relate to one another) • ARG can be considered the basis of AMR, as without those genes, bacteria possess few methods to overcome antimicrobials • Surveillance can lead to early identification and possible prevention, as described with Salmonella Typhimurium DT104, as well as many other incidents. • These outbreaks can be traced to the emergence of their genes.

Be able to list conditions for ethical research using human volunteers

Human volunteer research is only ethical under these conditions: • disease is not life-threatening • disease is easily treatable or can be prevented with a vaccine • disease treatment (usually antibiotics) does no harm to participants (i.e. ulcers, atherosclerosis)

Know the requirements for the animal model of disease

Ideal model requirements: • Disease symptoms and distribution in body mimic human infection • acquires the disease by the same route as in humans

Know different types of antibodies and the overall functions of each Ig (particularly "special" jobs). Know the difference between IgG1 and IgG2 - IgG2 does not opsonize well and does not stimulate complement. IgM and IgG - primary and secondary responses to infection roles. Role of IgE in degranulation of mast cells. Monomers/dimers of IgA - roles. Maternal immunity - two IgGs that play a role.

IgG: Bacteria and viruses; Secondary immune response IgM: Primary immune response; on surface of all uncommitted B cells; highest binding capacity; bacteria and viruses; IgE: bound to mast cells, basophils, eosinophils; allergic reactions Maternal immunity: IgG and IgA

Be able to tell the functions of antibodies as well as their architecture (chains and regions). Know different types of antibodies and the overall functions of each Ig (particularly "special" jobs). Know the difference between IgG1 and IgG2 - IgG2 does not opsonize well and does not stimulate complement. IgM and IgG - primary and secondary responses to infection roles. Role of IgE in degranulation of mast cells. Monomers/dimers of IgA - roles. Maternal immunity - two IgGs that play a role.

Immunoglobins that recognize and bind (noncovalently) foreign Antigen • 20% of plasma proteins • Different classes, common structural features • Variable region: recognizes/binds Ag • Constant region: various biological roles (offer host specificity and interacts with host cells) Protective functions: 1. Enhanced phagocytosis (opsonization) 2. Toxin neutralization 3. Complement-mediated lysis 4. Antibody-dependent cell cytotoxicity (ADCC) - promotes interaction of infected cells with NK cells

Know the importance of immunosuppressed/immunocompromised individuals

Immunosuppressed patients become infected with "old" bacteria, never thought to cause disease before Therapies leading to immunosuppression (Cancer therapy, transplants) New diseases (HIV) • Accidents during procedures (preformation of tissue during surgery, ventilator tubes, e.g. for CF patients, catheters) • Longer lifespan Rise in opportunistic pathogens: pathogens that do not normally cause disease but are infectious if normal defense mechanisms of the host are ineffective. Often gains access to sterile regions. Important in the developed countries *True pathogen*: causes disease in healthy individuals, often associated with specific diseases Common culprits: - Burhkholderia cepacia - Acinetobacter baumannii - Pseudomonas aeruginosa -Klebsiella pneumoniae

Know what CD1 pathway is - importance, what it binds, what cells are involved.

Important for the recognition of antigens in e.g. Mycobacterium tuberculosis • CD1 binds lipid or glycolipid antigens • Uptake of foreign glycolipid or lipid antigens occurs through multiple pathways • in ER, microsomal triglyceride transfer protein (MTP) facilitates the loading of the self-lipids onto CD1 • The antigen-CD1 complex traffics to the cell surface and is recognized by the CD1-specific T cell receptor

Know that IL4 and IL-10 Is anti-inflammatory while TNFa, IL-1beta, IL-6 are proinflammatory cytokines.

Inflammation, "to set on fire," caused by the release of proinflammatory cytokines: C3a and C5a generated by the complement cascade; tumor necrosis factor alpha (TNF-α), IL-1 and IL-6 and subsequent production of prostaglandins and leukotrienes. • inflammation can cause tissue damage: redness, swelling, heat, and pain • increased blood flow due to vasodilation, increased vascular permeability -> blood fluids to leak out of the vessels, edema • anti-inflammatory cytokines, e.g. IL-1 receptor antagonist, L-4, IL-10 down-regulate action of proinflammatory cytokines IL-1 and TNF-α interact with hypothalamus and adrenal gland -> fever and somnolence, anorexia. • TNF-α stimulates muscle cells to increase their metabolic rate for optimal host defenses. Increased metabolism of muscle cells -> chills • combination of anorexia and muscle cell breakdown -> weight loss and muscle wasting • tissue destruction: lysosomal enzymes released into the surrounding area, self-killing of PMNs, NK cells, and macrophages, release of lysosomal granules from PMNs. Pus: dead PMNs and tissue cells -> abscess

Be able to explain the weaknesses and pluses of using invertebrate models that were discussed during the lecture. Caenorhabditis elegans model

Info: Several models of infection (i.e. gut colonization model) • Many human pathogens have been tested • Accumulation of pathogen in worm gut, distension of the lumen • "Slow-killing": Slow killing by live pathogen accumulation in the gut • "Fast-killing": Rapid death due to toxin production by pathogen Positives: Fully-sequenced genome, many genetic tools • Rapid generation time (3 days) • Relatively easy and "cheap" to propagate in the lab • Targeted by many virulence factors/pathogenic organisms • Suitable for rapid highthroughput screening in vivo • "Permissive" legislation: Not regulated by IACUC Negatives: Innate immune system only (NO Adaptive Immunity) • Wouldn't normally encounter certain pathogens in its natural environment • Cannot grow at 37°C • It's a worm (different physical barriers - i.e. cuticle)

Know which cells are of the adaptive immune system (see slide 8) and most important sites of their location (spleen, lymphatics/lymph nodes, blood, thymus)

Innate immune system: Auxillary cells: - Magakaruocyte-- platelets; Mast cells Granulocytes - Basophil DCs - cells of innate immune system also play important role in adaptive immunity Phagocytes (macrophage and DC's) Adaptive Immune system: Antibody-producing cells: B cell- Plasma cell or Memory B cll T cell- effector T cell or Cytotoxic T cell or Memory T cell (Lymphocytes)

CATEGORIES OF IMMUNE SYSTEM FUNCTION

Innate: always on, first responders, rapid, non-specific, short Humoral: activation of B cells, production of antibody Cell-mediated: activation of T cells, production of cytokines

Know John Needham's and Lazzaro Spallanzani's experiments

John Needham experiments reinforced the idea of spontaneous regeneration (~1745) Lazzaro Spallanzani (~1765) refuted this claim: - Needham failed to heat vials sufficiently to kill all microbes or had not sealed vials tightly enough - Microorganisms exist in air and can contaminate experiments - Spontaneous generation of microorganisms does not occur Critics: vials did not allow enough air for organisms to survive and that prolonged heating destroyed "life force

Know the difference between the keystone pathogen and dominant pathogen

Keystone pathogen: microbial pathogen that is of low abundance can drive inflammatory disease by remodeling a normally benign microbiota into dysbiotic microbiota. Dominant pathogen (e.g. Salmonella Typhimurium) directly induces inflammation and microbial dysbiosis by overgrowing and outcompeting the normal microbiota.

Issues with #1

Koch had issues with the identification of M. tuberculosis -difficulties with staining Some pathogens colonize many people ('carrier-state'), but only cause disease in some (i.e. Helicobacter pylori, Staphylococcus aureus) • H. pylori: not all cases of colonization with H. pylori are associated with symptoms of disease Microbes as a risk factor for disease, rather than a cause (i.e. Chlamydophila pneumoniae & atherosclerosis)

number 3 issues

Koch's experiment: • spores of B. anthracis but not of B. subtilis caused anthrax in animals • M. tuberculosis (guinea pig model) • Issues with V. cholerae animal model- von Pettenkofer's self-experiment failure • Most difficult to satisfy this postulate for many pathogens • A "human model" of infection is usually not allowed • A "good" animal model may not be available

Know what LD50 is and what it measures

LD50 - 50% lethal dose (amount of the microbe that can lead to killing of exactly ½ of exposed animals)

Know what T cell immunity is and major cells in this immunity. How these cells are stimulated?

LECTURE

Th1/Th2 division - know the cells and functions, differences between them. Th1 produce IL2 and IFNgamma, Th2 produce IL4 and IL10...

LECTURE

Be able to calculate dilutions in gentamicin protection assay, understand what CFU is and how to interpret it, know what numbers of CFUs are within the countable range, be able to understand the difference between adhesion and invasion frequency

LECTURE! Used to calculate adhesion and invasion frequencies MOI = multiplicity of infection; Ratio of bacterial (or viral) cells to mammalian cells Adhesion frequencyCell-associated CFU: Total CFU Invasion frequencyInvasion CFU: Cell-associated CFU Graph: plot the log(MOI/cell) versus the log(invaded bacteria/cell) • minimal MOI (minMOI) required to obtain the maximum number of internalized bacteria per mammalian cell (BImax) • Strains with lower minMOIs are more invasive

Know the basic structure of LPS

LPS (lipopolysaccharides): "endotoxin" present on Gramnegative bacteria: Lipid A secures LPS in the OM responsible for the toxicity of Gram-negative bacteria -> Septic shock Parts: 1. Lipid A (endotoxin) 2. Core polysaccharide 3. O-antigen

CELLULAR RECOGNITION OF LPS THROUGH TOLL-LIKE RECEPTOR SIGNALING

LPS is released from outer membranes after bacterial lysis • LPS binds to LPS-binding protein (LBP) and is delivered to CD14 on the surface of macrophages • Transfer of LPS to the transmembrane signaling receptor TLR4 and accessory protein MD2. • TLR4/LBP/CD14/MD2 complex triggers a cellular signal transduction pathway • activation of the transcription factor NF-κB • NF-κB translocates to the nucleus • Induction of inflammatory cytokine gene expression

Know the difference between vaccine types (slide 16)

Live-attenuated vaccines: • use a weakened (or attenuated) form of the microbe that causes a disease, strong and lasting immune response • typically 1-2 doses required for lifelong protection • issues for immunocompromised • they need to be stored at low temperature • Measles, mumps, rubella (MMR combined vaccine), Rotavirus, Smallpox, Chickenpox, Yellow fever Inactivated vaccines • use the killed version of the germ that causes a disease • don't provide immunity (protection) that's as strong as live vaccines, boosters often needed • Hepatitis A, flu shot, polio shot, rabies Subunit, recombinant, polysaccharide, and conjugate vaccines • Use specific part of the microbe such as protein, sugar, capsid • Strong immune response that is targeted • Can be used for immunocpromised • Often requires boosters and needs to be designed • Haemophilus influenzae, hepatitis B, HPV, Whooping cough, pneumococcal diseases, meningococcal, shingles Toxoid vaccines • Use toxin made by bacterial pathogen, immunity to the toxins and not the bacteria • Often require boosters • Diphtheria, Tetanus

Know the mucosal antibacterial proteins

Lysozyme: cleaves bacterial peptidoglycan between NAG-NAM linkages: • 1° active against Gram + bacteria • Can also act on Gram -bacteria if outer membrane is disrupted (detergents, bile salts) • Lactoferrin: Binds iron; makes it unavailable ("sequesters") to bacteria •Lactoperoxidase: heme-containing peroxidase, present e.g., in milk - OSCN- attacks exposed thiol groups of enzymes/proteins found in bacterial membranes, disrupts their functionality Phospholipase: degrades cytoplasmic membrane to lyse bacterial cells • Antimicrobial peptides (AMPs): e.g. in Paneth cells, highly cationic regions enable them to insert and/or depolarize bacterial cell membranes • Form channels/holes, collapse proton motive force (PMF) • Examples: defensins, cathelicidins, histatins • Are not functional in all body sites (i.e. inhibited by salt and serum); may be more important/active inside phagocytes (immune cells)?

MACROPHAGE POLARIZATION

Macrophages either stimulate inflammation, or decrease inflammation via specific signaling molecules (cytokines) • M1 macrophages: metabolize the amino acid arginine into a reactive nitrogen species (nitric oxide) and stimulate inflammation, • M2 macrophages metabolize arginine into ornithine for cycling through the urea cycle to dampen the inflammatory response. M2 promote repair of damaged tissue.

ARGS AND AMR IN GLOBAL HEALTH continued

Many other factors facilitate the rise of AMR: • Climate patterns, climate change (-> Effect on cyanobacteria; Surprisingly, integrons found in cyanobacteria, indicating they share ARG acquisition platform with ARBs • Large-scale changes in microbial communities • Human-made creation of selection pressure • While surveillance networks are useful for detecting new resistance, it does nothing to prevent the development of AMR. • Essentially, humanity is perpetually playing "catch-up" with bacteria and other microorganisms • AMR-driven consequences for human health are more relevant in countries where health services are poor

Know what plaque assay measures and for what types of bacteria this assay would be useful

Measures cell-cell spread of intracellular bacteria or viruses • Cleared zone of killed/lysed TC cells = Plaque • Agar/Gentamicin overlay applied to TC monolayer to prevent diffusion of bacteria through the medium

Know what memory T cells are (importance of TCR)

Memory T-cells: Persist for long periods of time in the body; allow a rapid response to a second encounter with a pathogen TCR makes Memory T cells

Understand what are microbiota shift diseases and polymicrobial infection, know the examples of these diseases

Microbiota Shift Disease = caused by shifts in the composition of bacterial populations of the body (the microbiota) • Examples: • Koch postulates need to be adjusted: pure culture replaced by shift from one population to another (dysbiosis) • Third postulate is a challenge (inducing specific population shifts in animals is difficult) • Use of selective antibiotics as evidence

Know what villi/microvilli are and their functions.

Microvilli on the surface of epithelial cells: increase the cell's surface area and thus facilitate the absorption of ingested food and water molecules.

Know the characteristics of monocytes/macrophages, be able to distinguish the functions of M1/M2 macrophages (focus on functions of M1, M2 types, know that M1 is stimulated by LPS/TNF/IFNgamma, while M2 by IL10 or TGFb, know that M1 macrophage produces TNFa, iNOS and IL-1b, while M2 produces more IL-10)

Monocytes & Macrophages (MΦ) aka leukocytes (WBCs) • Monocytes circulate in blood, mature into macrophages • Found in almost all tissues • Kupffer cells (Liver), Alveolar MΦ (lung) • Phagocytic, engulf and kill pathogens; do not typically self-destruct • Process and present antigens - activate B/T cells • Regulate innate immunity by producing cytokines and chemokines • Cytokine: potent low MW cell-signaling molecules produced transiently and locally by numerous cell types; involved in a variety of immune responses • Chemokine: low MW proteins that stimulate leukocyte movement

FINAL THOUGHTS ON MEASURING INFECTIVITY AND VIRULENCE

Most models (in vitro and in vivo) have their pro's and con's; investigators need to be aware of potential limitations and interpret data accordingly • No one model fits all pathogens; choice of model dictated by microbe under study • Ever-evolving breakthroughs in model systems (i.e. 3-D TC, organ culture models, donated tissue) • Communication between "basic" scientists and clinicians -> documenting and understanding disease progression/pathology in humans

Mucosal surface defenses

Mucosal cells - rapidly dividing, constantly shed • Mucus: mixture of glycoproteins/mucins produced by Goblet cells (cell in epithelial cell lining of small and large intestines or lungs) • Traps bacteria • Constant production, shed and expelled • Binds antibacterial proteins • Cilia (Respiratory, fallopian tubes)

Know that some pathogens (Salmonella, Rickettsia, Mycobacterium) escape phagolysosomal pathway. Know autophagy ism mediated by ubiquitination of bacteria and recruitment of autophagy machinery followed by lysis of bacteria. Know what autophagosome is and its appearance.

Mycobacterium tuberculosis and Salmonella enterica escape phagolysosomal pathway by modifying the phagosome into a specialized vacuole that does not fuse with lysosomes (e.g. Salmonella-containing vacuole, SCV) Rickettsia conorii escape the phagosome and reside within the host cytosol.

Know the granulocytes characteristics - focus on functions. Know what NETs are and which cells make them. Know that basophils, eosinophils produce granules with toxic enzymes etc.

Neutrophils (aka polymorphonuclear leukocytes, PMNs): most common; short half-life, phagocytic, destroy pathogens within intracellular vesicles • Found in blood • Engulf and kill pathogens • Usually undergo apoptosis once pathogen is destroyed • Produce cytokines • Produce NETs - web-like structure composed of chromatin, histones, and antimicrobials (neutrophil extracellular traps) • Eosinophils, Basophils: less abundant • Granules containing enzymes and toxic proteins that are released upon cell activation • Important for defense against parasites

Know difference between oxidative and non-oxidative killing. Know the organelles involved.

Non-oxidative killing: • Lysosomal vesicles contain various macromolecule-degrading enzymes that destroy bacterial components • Enzymes lead to the destruction of the surface and membrane components of bacteria, bacterial DNA and RNA • Defensins, antimicrobial peptides -> pore creation in bacterial membrane Oxidative killing: • formation of toxic reactive oxygen and reactive nitrogen species • Lysosomes contain myeloperoxidase to produces reactive forms of oxygen toxic to bacteria • The generation of toxic forms of oxygen by phagocytes is called the oxidative (respiratory) burst • Myeloperoxidase is activated when it is in contact with an NADPH oxidase in the phagosomal membrane and upon low pH • cytokines stimulate increased production of these lysosomal enzymes

Mast cells: role and activation functions.

Non-phagocytic • Congregate around blood vessels • In presence of foreign material, release granules containing histamine • Vasodilation of blood vessels "leaky" • Aids in the movement of circulating PMNs and monocytes to site of infection

Know examples of obligate and facultative intracellular bacteria and be aware of how it affects postulate #2.

Obligate: rickettsia sp, chlamydia sp, anaplasma sp, ehlichia sp., coxiella burnetti, mcyobacterium leprae Facultative: francisella tularensis, listeria monocytogenes, mcyobacterium sp, salmonella sp, legionella pneumophila

Know what are PAMPs and give some examples (LPS, flagellin, lipoteichoic acid, PG, DNA/RNA)

PAMPSs: Pathogen associated molecular patters • lipopolysaccharide (LPS), peptidoglycan or flagellin -> Gram-negative bacteria, • Peptidoglycan, lipoteichoic acid (LTA) -> Gram-positive bacteria • DNA (CpG-rich DNA) and RNA

THE MICROBIOTA SHIFT DISEASE PROBLEM IN PERIODONTAL DISEASES

Periodontal disease: biofilm-associated inflammatory disease of the tissues surrounding the teeth (periodontium) • can lead to gingivitis, progressive loss of the bone around the teeth, dental decay, tooth loss • microbes -> dental plaques -> calcification -> inflammation (role of host immunologic response) -> periodontitis • 700 different bacterial species. • microbial population shift that favors some Gram-negative anaerobes is responsible for the initiation of the microbial dysbiosis

DCs: know the appearance, functions, role in the adaptive immune response. Be aware that they can become stimulated to mature dendritic cells by PAMPs and special cytokines, when they express more MHCII, proinflammatory cytokines and have reduces phagocytic capacity. Know which cells they interact with.

Phagocytic, can degrade pathogens • "stand guard" in various tissues that are in contact with the external environment • types (i.e. Langerhans cells in dermis) • primary role is activation of B/T cells (adaptive immune response) • "antigen-presenting cell" (APC) • production of cytokines - Dendritic cells are activated upon recognition of bacterial PAMPs • Migration through the lymphatic system to lymph nodes • mature DCs present antigens to cells of the adaptive defense system: T helper cells (Th cells), cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs), and B cells.

Know the differences between the physical barrier, innate, and adaptive systems. Be able to list the cells of each immune system arm.

Physical barrier: external agents used for the reduction and elimination of bacteria - cleaner - sanitizer - disinfectant -virucide - sterilant Mechanical barriers and Host Immunity: Innate immunity (within hours of infection): epithelial barriers, phagocytes, dendritic cells, complement, NK cells Adaptive immunity (within days of the infection): B lymphocytes, Plasma cells, antibodies; APCs, t Lymphocytes, effector T cells

PLASMID PROBLEM

Plasmids are circular formations of bacterial DNA, not a chromosomal DNA, replicates independently • Plasmids can encode "extra" DNA for the function of the organism, including virulence, and, in some cases, encode extra mechanisms for AMR. • Plasmids can be transferred through reproduction, transduction, conjugation, or simply transformation. • Mobile genetic elements, MGEs can be transferred between bacteria : plasmids encoding extended spectrum b-lactamases

Understand the presented model of Porphyromonas gingivalis and how it affects the periodontal diseases.

Porphyromonas gingivalis acts as a keystone pathogen • P. gingivalis secretes a cysteine protease that cleaves complement protein C5 into C5a -> inflammatory responses and impairment of leukocyte killing of bacteria • overgrowth and changes in the composition of the microbiota -> microbial dysbiosis -> inflammation • Drug antagonists targeting specific components of complement allowed the recovery

Point out the difference between probiotics and prebiotics, give an example of probiotics.

Probiotics (i.e. live-culture yogurt, freezedried pellets); Lactobacillus and/or Bifidobacterium sp.; cultures from commercial preparations do not colonize the colon well Prebiotics (i.e. fructo-oligosaccharides) foster the growth of "good" gut bacteria in the large intestine

Understand the difference between prospective and respective studies.

Prospective study: clinical trial involving inoculation of subjects with disease agent and/or treatment of infected humans "present -> future" Retrospective study: Study of disease outbreaks that have already occurred and are studied in retrospect; disease transmission i.e. TB transmission on school bus

Be able to list qualitative methods of microbial communities and know the principle of each one

Qualitative method (microbial census, DNA profiling) what species are present? 16s rRNA sequencing Metagenomic sequencing -> study of total DNA from samples, "NextGen" sequencing

Know the principle of transcriptomic and what it can be used to study.

RNA Isolation • RNA selection/depletion -> only mRNA present • cDNA synthesis • sequencing

ARGS AND AMR IN ONE HEALTH

Reduce Antimicrobial use: • Agriculture (80% of antibiotics in US used for animals)[1] • Hospitals (over-prescribed for non-bacterial conditions) • Society (not using drugs as prescribed) Develop alternative treatment methods: • Bacteriophages • Combined therapies • Host-directed therapies • Antibodies

Post-surgical

Relaxed hygienic practices in hospitals to cut costs - AMR - aftermath increased risks and higher financial costs of insurance and increased liability of hospital War-related infections Common culprits: Acinetobacter baumannii ('iraquibacter') - soil bacterium, identified in wounds, now present in hospital settings, pan-resistant Pseudomonas aeruginosa, burn victims

Know that the neutrophils are directed from bloodstream to tissues by cytokines, and also selectins that alter the binding and motility.

Resident macrophages and DCs localize in specific areas of the body • neutrophils (PMNs) and monocytes, produced in the bone marrow and migrate constantly through the bloodstream -> require signals for transmigration • Selectins expressed on the surface endothelial cells promote loose reversible binding -> rolling motility • IL8 -> tighter binding • The PMNs stop moving, flatten against the vessel wall, move across the endothelial wall • PMNs move chemotactically along a C5a gradient

Know who first coined the term cells

Robert Hooke has not seen single-celled organisms but used this term First description of microorganisms

Know that permeabilization during fluorescent microscopy can be used to study intracellular/extracellular bacteria

Sample permeabilization with detergents to stain intracellular bacteria or bacteria in intercellular compartments 1. Stain extracellular bacteria 2. Permeabilize 3. Stain intracellular bacteria 4. Permeabilize vacuolar membranes 5. Stain intravacuolar bacteria

Understand how different types of ELISA tests work

Sandwich ELISA is the most effective/specific - use of 2 antibodies • Immobilization of antigen or antibody on a plate, depending on the ELISA type • target antigen: bacterial cell components or bacterial toxins from the sample • Can be used to recognize antibody titer in patients as well • Sandwich ELISA is useful for applications that require a high accuracy. • High throughput ELISA tests available (e.g. VIDAS, Assurance EIA) Direct vs indirect vs sandwich

Know what to consider when choosing the animal model of disease

Several considerations when choosing an animal model of infection • Ease and expense in maintaining animals • Insights consistent with disease in humans • Genetic manipulation of animal model (transgenic mice- knock-out and knock-in) • Differences in physiology, anatomy, behavior (coprophagy in mice) • Gnotobiotic ("germ-free"), specific pathogen-free animals • Ethical considerations

Roles of cytotoxic T cells. Know the different names of this cell type. Know what these cells produce to kill cells.

Similar function to NK cells and PMNs • Interact with MHC I on APCs, then kill them • Release cytotoxic granules -> perforin, granzymes (kills host via apoptosis but not microbes which are phagocytized) or poreforming cytolysin named granulysin (primarily kills bacteria) • apoptosis is a programmed cell death activated by caspase-3/7 activation • Similar process used when recognizing and killing tumor cells • Important for killing intracellular pathogens

Know the difference between stratified and simple epithelia

Simple squamos: Lining of cavities (heart, lung, mouth), material can pass via diffusion or filtration, secretion of substances Simple cuboidal:Kidney tubules, gland ducts, secrets and absorbs Stratified squamos: Skin, cervix Protects against abrasion Simple columnar: Stomach, intestine, absorbs and secretes mucous and enzymes Ciliated columnar: Respiratory tract, fallopian tubes Connective tissues: Epithelial layers have no blood vessels -> receive nourishment via diffusion from the underlying connective tissue (e.g. basal lamina) SUMMARY: Most surfaces exposed directly to the environment (skin, mouth) are covered by stratified epithelia simple epithelia found in internal areas (intestinal tract, lungs), more vulnerable to bacterial invasion than stratified epithelia,

TRIGERRING THE INNATE IMMUNITY

Skin- and mucosal-associated lymphoid tissue (SALT and MALT) trigger the general innate immune system • secretory immunoglobulin A (sIgA) that binds to mucin and the microbe (MALT) • The innate immune system cells are always present -> first responders to pathogen invasion. • innate immune system becomes activated upon recognition of PAMPs - components of foreign invaders • recognition of structural components and other molecules specifically found in bacteria - quasi "bacterial barcodes" - pathogen-associated molecular patterns (PAMPs) • production and release of cytokines and chemokines • complement system organizes and regulate the activities of immune cells.

Know the different levels of disease intensity (Slide 10)

Sporadic - disease that occurs infrequently and irregularly. Endemic - constant presence and/or usual prevalence of a disease or infectious agent in a population within a geographic area. Hyperendemic - persistent, high levels of disease occurrence. Epidemic - increase, often sudden, in the number of cases of a disease above what is normally expected in that population in that area. Outbreak - same as epidemic but is often used for a more limited geographic area. Cluster - aggregation of cases grouped in place and time that are suspected to be greater than the number expected, even though the expected number may not be known. Pandemic - epidemic that has spread over several countries or continents, usually affecting a large number of people.

Be aware that the severity of the disease depends on the microbial factors as well as host factors

Status of the immune system • Congenital or genetic predispositions to infectious disease • Microbiome • Environmental factors (diet, stress, ecology) • Pathogen-host interaction is complex and can result in several outcomes: • pathogen clearance, • asymptomatic carriage • disease symptoms

Know specialized defenses of GI tract

Stomach lumen: acidic (pH ~ 2) • Although protective against many bacteria, DNA found from 128 species of bacteria occupying the stomach! • Food-borne pathogens (E. coli, Salmonella, Campylobacter) have acidtolerance response (ATR) (pH 4); ingestion with foods also provides them protection • H. pylori the most well-studied • Lives in the mucin layer (near normal pH) that covers and protects stomach lining • Can protect itself from high stomach acidity long enough to reach the mucin Bile salts (intestine, colon): steroids with detergent-like properties • Produced in liver, stored in gall bladder, released into GI tract via bile duct • Neutralize stomach acid, aid in fat digestion • Disrupt bacterial membranes • Rapid flow of contents (intestine) • Normal resident microbiota (NOT present in uterus, upper genital tract, urinary tract) • Mucosal defense systems (MALT, GALT) • Secretory IgA (apical side of epithelium) increases stickiness of mucin by attaching to mucin sugars at one end and trapping bacteria at antigen-binding ends)

Skin

Stratified squamous cells (keratinocytes) • Keratin protein: not easily degradable by bacteria • Desquamation/shedding (remove bound bacteria) • Inhospitable environment for many pathogens (↓pH, ↓temperature, arid) Hair follicles and glands • Simple epithelial cells; potential breach areas (boils, acne) • Protected by lysozyme, toxic lipids Resident microbiota (microflora): • 1° Gram-positive rods/cocci • Staphylococcus epidermidis, Propionibacterium acnes • Physical, metabolic competition • Production of toxins/growth inhibitors e.g. bacteriocins • -> colonization by pathogens usually transient permanent colonizers • Possibility for opportunistic pathogens

Know the major accomplishments of Robert Koch in the field of microbiology

Studied Bacillus anthracis in his home (1876) • Improved pure-culture isolation and staining methods; animal models of disease • Best known for his discovery of Mycobacterium tuberculosis (Nobel Prize 1905) • This work helped formulate his postulates, a systematic approach for establishing the causal relationship between a particular microorganism and a specific disease.

Know the difference between adherence, invasion, and evasion of host defenses

Survival outside the host • Adherence to host tissues • Penetration of host physical barriers, invasion • Evasion of host defenses, persistence strategies • Production of toxins, other virulence factors/strategies • Antimicrobial resistance • Transmission

Know Koch's Postulates and be able to describe/define them

The Germ Theory of disease: 1876: Koch provided proof that a bacterium causes anthrax and provided the experimental steps (Koch's postulates) used to prove that a specific microbe causes a specific disease. Koch's Postulates are a sequence of experimental steps to relate a specific microbe to a specific disease.

Phagocytosis: know what it is. Be able to explain oxidative burst in phagolysosomes.

The phagocyte forms pseudopods that engulf the bacterium • Phagocytosis (engulfment) requires dynamic rearrangements of actin (cytoskeleton component of the host) • bacterium is encased in an endocytic vesicle called phagosome • ATPases on phagosomal membrane pump protons into the phagosome interior -> increase in acidity • antibacterial proteins are stored in an inactive form in lysosomes • Fusion of a lysosome with a phagosome into phagolysosome • Release of the lysosomal enzymes and other molecules into the phagolysosome -> activation of enzymes by the low pH of the phagolysosome interior • Lysis of bacteria • Debris is released by exocytosis.

epithelial cells

The tight binding of epithelial cells to one other prevents bacterial pathogens from transiting an epithelial layer. basolateral surface: The membrane surface of an epithelial cell that faces toward the interior tissues of the body, attached to other cells or connective tissues apical surface: membrane surface that faces outward - apical is outward while basolateral is inward These surfaces have a different protein composition -> cell polarization Intestinal epithelial cells showing tight, junctional, and adherens junctions, and desmosomes. JAM, junctional adhesion molecules.

Be able to explain the principles of human volunteer research

Three principles must be maintained: with IRB consent 1. Respect for persons (Informed Consent) 2. Beneficence (Maximize Benefit and Minimize Harm) 3. Justice (Fair Treatment and Risk Distribution)

Know some of the "new weapons" for fighting/studying pathogens.

Treatment-based approach versus prevention: costs and risks Treatment: - Waiting for a patient to get sick - Prophylactic use in patients to prevent post-surgical infection - Dangers of AMR: pressure on bacteria to develop resistance against frontline antibiotics - Side-effects of antibiotics - Issues with diagnostics - Sepsis can kill a patient in a few hours Prevention: - more effective and less expensive - knowledge of epidemiology of disease (disease patterns, geographic distribution, determinants of health-related states) - Importance of surveillance programs (monitor appearance of new diseases, incidence of existing diseases, AMR occurrence) - Sometime diseases are not reported efficiently, require funding One Health: collaborative, multisectoral, and transdisciplinary approach. The goal is to achieve optimal health outcomes recognizing the interconnection between people, animals, plants, and their shared environment. CDC's One Health Office leads the agency's One Health efforts in the United States and abroad

Know the specialized defenses of the respiratory tract

Upper respiratory tract pathogens: Neisseria memingitidis, Streptococcus pyogenes Lower tract: Bordetella pertussis Streptococcus pheumoniae ciliated epithelial cell layer + goblet cells ( source of mucin) • specialized ciliated columnar cells • protrusions (cilia) continuously wave in the same direction to propel mucus blobs out • Airway epithelial cells secrete collectins (bind to bacterial LPS), defensins and other antimicrobial peptides • comatose patients lack normal cough reflex (velocity up to 150 cm/sec!) and reduced mucociliary clearance -> increased susceptibility to respiratory infections • ventilators, intubation: introduce air with water droplets (sometimes contaminated with pathogens) directly into the lung, bypassing mucociliary defenses lower respiratory tract: mostly lacks microbiota • Upper respiratory tract: a few bacteria present (Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and nontoxigenic Corynebacterium diphtheriae) -> upper and lower respiratory infections if the immune system is impaired • asthma, cystic fibrosis, chronic obstructive pulmonary disease: Pseudomonas aeruginosa, Burkholderia cepacia, and Mycoplasma pneumoniae. • Bordetella pertussis (lower respiratory tract) -> whooping cough

and how H. pylori can survive

Urease: generates ammonia from urea, localized raise in pH • Urease activity facilitates flagellar motility through the mucous layer by changing the properties of gastric mucins (gel -> viscoelastic solution) • Chemotaxis and cell shape also facilitate H. pylori translocation through mucus • cell shape mutants that have lost helical twist and/or curvature exhibit attenuated colonization • chemotaxis mutants have altered localization, including lower numbers of bacteria that are in close association with gastric epithelial cells

BASIC STRUCTURE OF AN ANTIBODY

V(D)J recombination and somatic hypermutation to increase diversity of antibodies • Similar overall structure for: IgG, IgM, IgD, IgE, and IgA/sIgA but some differences (see next slide Fab- antigen-binding Fc - constant region (Ab-isotype specific) Antigen-binding: within variable sites Complement binding site (binds C1q) Phagocyte receptor-binding site (Fc receptor)

Know antigen choice for making ideal vaccine

Vaccine has to enhance immunogenicity • Vaccine has to elicit protective immune response -> long-lasting cell-mediated immunity (T cells and B cells) Antigen choice • Is antigen conserved, prone to mutations? • epitope for Th1 or Th2? (Th1: intracellular pathogens, Th2: extracellular pathogens) • not all antigens elicit an immune response. • not all immunogen (antigen that elicits an immune response) elicit protective immune responses • not all immunogenic epitopes (part of the antigen recognized by the antibody; antigenic determinant) elicit protective immune responses. Epitope may be inaccessible to the antibody • Booster might be needed: protection dependent on the ratio of antibody to antigen: antibody titers have to be high, memory Th2/B cells have to be high. Cost issue, more infrastructure required to maintain the booster immunization program • Epitope might be difficult to manufacture if discontinuous: the strongest and most protective immunogenic epitope can be against a structural conformation or multiple segments of the antigen forming a topological surface

Know the history of vaccine development (slide 1)

Vaccine: a substance containing a virus or bacterium in a form that is not harmful, given to a person or animal to prevent them from getting the disease that the virus or bacterium causes Variolation - inoculation of individuals with material from smallpox pustule, 2-3% risk of infection • China - 15th century • India - 18th century • Europe: Edward Jenner study 1797 - vaccination • Complete eradication of smallpox 1980. Campaign by WHO1960s-1970s; Last case was in Somalia in 1977 Other infections: Louis Pasteur 1880 (fowl cholera), 1881 (anthrax), rabies (1885) - attenuated pathogens

Know the difference between virulence, pathogenesis, and disease

Virulence: the ability/capacity of a microorganism to cause disease • Initial stage of host-pathogen interactions • "Virulence factor": a bacterial product or strategy that contributes to the organism's ability to survive in the host and/or cause damage to the host • Qualitative or quantitative term • LD50 - 50% lethal dose (amount of the microbe that can lead to killing of exactly ½ of exposed animals) • Pathogenesis: the process by which microbes cause disease in a host • Follow virulence, determined by virulence factors • Qualitative term

Slide 38: know humoral/cellular paths, and roles of these cells, do not memorize each cytokine, only what I emphasize in other slides. Know the division of Th2 cells and Th1 cells. Know the difference between TH17 and Tregs (functions).

WATCH LECTURE

Know that APC and CD4/CD8 interact and how - know that several receptors are involved, including MHC epitopes and CD80/86 on APCs, and CD4/CD8, TCR, CD28 on T cells etc.. Importance of specific TCR heterodimers. Two signals are needed for the proliferation of T cells and production of cytokines.

WATCH LECTURE APC displays MHC I-epitope or MHC II-epitope complex on surface to specific immature precursor T cells with TCR (The T-cell receptor) capable of recognizing particular MHC-epitope combination • the complex formed between the epitope-MHC II complex on the APC and the CD3-TCR complex on the CD4+ Th • CD4 recognizes MHC II complexes and CD8 recognizes MHC I complexes on APCs • Two stimulating signals needed to induce proliferation and production of cytokines • signal 1 occurs via specific interaction of the epitope-MHC II with the CD4-TCR and CD3 complexes • signal 2 occurs upon costimulation through the CD28 binding to CD80 or CD86 on the APC

Know what the SCFAs are, and how are they made?

We need bacteria to eat our food: intestinal microbiota help digest food: Short-chain fatty acids (SCFAs) • Microbiota synthesize vitamins (K, B12 and other B vitamins) • Microbiota protect us from pathogen colonization (real estate and dining) • Training of immune system (cross-reactive antibody production -> prevention of infection by related pathogens)

Know general information about the mucous membranes and their defenses for the body

Where do we find mucosal membranes? - Eyes - Respiratory tract (lungs, trachea) - GI tract - Urogenital tract • Only one epithelial layer • 37°C, neutral pH, humid • Many specialized chemical and physical barriers Basal lamina contains ECM (extracellular matrix) components such as collagens and laminin (->adhesions for pathogens) • Most mucosal membranes are polarized - apical (exposed to environment) vs. basolateral (basal lamina) surfaces: different properties/protein composition • Intestinal epithelial cells showing tight, junctional, and adherens junctions, and desmosomes. • JAM, junctional adhesion molecules- protein structures that connect the cells • Fibroblasts produce ECM - coat surface of implanted materials and can facilitate attachment by certain pathogens

Know the functions of complement. Know that complement includes three pathways, and know the differences between them (mannose, antibody and C3b opsonization). Know that C3b is opsonizing, C5a is a chemokine and vasodilator, and C3 is convertase leading to cleavage. Know what MACs are and their function.

blood proteins marking targets for phagocytosis and directing the inflammatory response • complement system proteins circulate in the blood, often as zymogens (i.e. have to be activated by cascade of proteolytic cleavage). • Nine of core components labeled with 'C', activated product labeled with 'a' or 'b' • Main function is rapid pathogen clearance Triggered by foreign and altered surfaces • upstream of most defense and homeostatic systems • mediator in pathophysiological processes Mannose-binding lectin pathway and alternative pathway are antibodyindependent - Classical pathway requires antibodies - works when adaptive immunity is active too - LPS or Lipoteichoic acid , LTA as complement-triggering molecule (-> alternative pathway) - Mannose-binding groups: mannose molecules found on surface of bacteria but not host - Antibodies can also trigger complement

Know that next-generation sequencing can be quantitative as well and be able to identify the workflow of Illumina sequencing

can be quantitative, can identify mutations • requires bioinformatics and statistical software to assemble reads, analyze data • Many different technologies available • Illumina, IonTorrent, PacBio • Each technology has specific sequencing chemistries/detection • trade-off between length of sequencing "read" and accuracy

Table information

chemokines are a type of cytokine, direct the migration of white blood cells to infected tissues. - complement -cytokines -chemokines IL-IB IL-6 IL-17 IFN-gamma TNF-a

Know why 16S rRNA genes are used for sequencing (variable vs. conserved regions)

choose a gene that is common to all bacteria of interest • 16S rRNA genes, component of the 30S subunit of prokaryotic ribosomes: • large enough - enable discrimination • small enough - good for sequencing • some regions are highly conserved, others contain sequence signatures • used in phylogenetic studies to identify bacteria in a community • Conserved regions - targeted my primers for PCR amplification • Variable regions -species-specific, identify WHAT species are in community after sequencing

PATHOGEN ADAPTATION TO THEIR HOSTS IS OFTEN CONCURRENT WITH DE-ADAPTATION TO AN ALTERNATIVE HOST

collapse in animal and plant genetic diversity • urban spread, habitat destruction and the anthropogenic selection of a limited range of varieties of economic interest → collapse in animal/plant genetic diversity • homogenization of hosts -> dissemination of ARGs among common microbial communities is more possible

Be able to explain the weaknesses and pluses of using invertebrate models that were discussed during the lecture. Drosophila melanogaster (fruit fly)

considered the most versatile invertebrate infection model; can be infected by pathogenic bacteria, viruses and and parasites (latter two cannot be modeled in C. elegans) • small size, short generation time • fully sequenced genome, pre-existing libraries of mutants • Extensively studied innate immune response (similar to mammals) via TLRs, IL-1 • High inoculums of bacteria sometimes needed (TLR mutant flies often used)

Know what sepsis is and how it can be triggered (importance in TLR4-LPS, cytokine storm etc.). Know the stages of sepsis. Importance of antimicrobial treatment and early detection.

cytokine storm - overwhelming inflammatory response • Toxic shock Triggering of proinflammatory cytokine release by Gram-negative LPS and its role in septic shock. TNF-α, IL-1, IL-6, and IL-8 are proinflammatory cytokines. • Septicemia: condition where bacteria infect and grow in the bloodstream - leading cause of septic shock • occurs in 500,000 to 750,000 patients per year in United States • Survivors may have long-term aftereffects: stroke, damage to lungs, kidneys, liver, or other organs. • systemic inflammatory response syndrome (SIRS): temperature >38°C or <36°C, elevated heart rate, elevated respiratory rate, abnormally high or low neutrophil count. • Sepsis: culture-documented infection • severe sepsis: organ dysfunction and very low blood pressure (hypotension). • septic shock: low blood pressure despite intravenous fluid administration, death rate 30-50% or 70-80% upon upon disseminated intravascular coagulation (DIC, due to activation of blood clotting)

SURVIVAL CURVE ANALYSIS

determines the median survival time of animals following infection with a wild-type bacterial pathogen and mutants that are being tested for virulence • Complemented controls can be used (Complementation - two different sources of genetic information (e.g., different gene loci encoding proteins of different function) together each provide something the other lacks.) • Animals euthanized when they reach moribund state • Advantage: statistically robust results by using small animal cohort

Know what difficulties were faced by researchers studying pylori as a causative agent of ulcers.

discovered by Dr. J.R. Warren • Dr. Barry Marshall - worked with Dr. Warren; early proponent of the hypothesis that gastric ulcers are caused by H. pylori • Lack of animal model at that time • Barry Marshall drank culture of bacteria isolated from lesions of patients • H. pylori as a causative agent of ulcers • Warren and Marshall won Nobel Prize in 2005

Physical Barriers

epithelial cells - cover all surfaces (internal, external) of the body exposed to the environment • Skin • Mucosal epithelia (respiratory, intestinal, urogenital tracts, mouth, eye) • In general, difficult to breach (tightly-packed cells) endothelial cells - line surfaces of interior of body, • blood and lymphatic vessels • relatively easy to breach (loosely packed -> immune cell movement) epi: on endo: in

SPECIAL DEFENSES OF THE UROGENITAL TRACT

female and male urogenital tracts: different environments and different associated bacteria; sexual exchange leads to the exchange of bacteria • epithelial layer of the urinary tract system (kidneys, ureters, bladder, and urethra) protected by secretion of mucin • bladder is typically sterile • The urethra sphincter prevents further ascent of the bacteria to the bladder and kidney. • Urine is antiseptic - helps remove bacteria • The prostate gland in men secretes defensins • Certain conditions (pH changes, obstructions) facilitate urethra colonization with pathogens (e.g., E. coli, Proteus mirabilis, Staphylococcus saprophyticus, and Klebsiella) -> urinary tract infections (UTIs) • Vagina: a cervical plug protects from invasion by bacteria, vagina lined by a stratified epithelium, which produces lysozyme, lactoferrin, small antimicrobial cationic peptides, and proteins • The resident microbiota (Lactobacillus) protects against pathogens in the female genital tract, such as HIV.

memory t cells

few of the T cells become quiescent memory T cells • persist for long periods • present in higher numbers than naïve T cells with the same TCR ' • easily stimulated to proliferate and produce cytokine responses upon encounter with specific epitopes on APCs (next infection, or after vaccination) • Fast and stronger response to a second encounter with a particular pathogen

Be able to explain the weaknesses and pluses of using invertebrate models that were discussed during the lecture. Galleria mellonella (wax moth larvae)

highly sensitivity to infection by pathogenic bacteria → melanization • Can use purified bacterial toxin • larval stage able to survive at 37°C • genome has not yet been sequenced • lacks well-established methods for generating mutants • susceptible to a number of bacterial pathogens

Know the factors that affect the microbiome.

host and environmental factors -> microbiota composition towards a stable community at equilibrium (resistance to pathogen overgrowth), or unstable community (predisposed to infection and inflammation)

Understand how to read a phylogenetic tree and know the terms associated with this approach

inferred evolutionary relationships among microbial communities based upon sequence similarities Lines: • phylogenetic distance between each of the samples • measure of the relationship node : a taxonomic unit e.g. a taxon branch : defines the relationship between the taxa in terms of descent and ancestry. branch length : number of changes that have occurred in that branch. root : is the common ancestor of all taxa. distance scale :represents the number of differences between sequences (e.g. 0.1 means 10 % differences between two sequences)

Know the Clostridium difficile infection model and possible therapeutic treatment

intestinal microbes do not normally infect:sloughing of intestinal mucosal cells, intestinal immune system • Bacteroides, E. coli, Enterococcus, Clostridium -> infections upon trauma • colonic bacteria act as reservoirs of antibiotic resistance and virulence genes (reservoir hypothesis), issues with food supply, resistance genes can be transferred to bacteria permanently or temporarily residing in the human colon • Perturbation of the normal healthy microbiota (dysbiosis - microbial imbalance, impaired microbiota) • inflammatory and autoimmune diseases: inflammatory bowel disease (IBD) and Crohn's disease • increased susceptibility to opportunistic pathogens such as Clostridium difficile

Know the applications of rRNA gene sequencing

isolate the total genomic DNA from the microbial population • Use polymerase chain reaction (PCR) to amplify the bacterial 16S rRNA genes -> amplicons • Sequencing • bioinformatics: compare sequences of the rRNA genes (output sequence reads) to publicly available DNA sequence database • identify the nearest bacterial relatives and taxon

ORGAN CULTURE MODELS

multiorgan platforms: connecting medium diffuses from one organ to the other in transwells, microtunnels, monolayer patterns, or large pores in wells. Schematic representation of epithelial tissues with their native 3D architecture, conventional 2D cell cultures, and novel 3D cell culture approaches.

Be aware of different models that can be used in tissue culture: polarized monolayers, organoid and other 3D cultures, cell culture, transwell, and other microscale platforms

multiorgan platforms: connecting medium diffuses from one organ to the other in transwells, microtunnels, monolayer patterns, or large pores in wells. Schematic representation of epithelial tissues with their native 3D architecture, conventional 2D cell cultures, and novel 3D cell culture approaches.

Know the "best defense" against pathogens (slide 4)

natural agents: antibiotics, alcohols, and natural acids such as vinegar or citrus juice artificial agents: organic chemicals, drugs, and detergents agents used externally on inanimate objects (e.g., disinfectants, sanitizers) - agents used on body surfaces (e.g., antiseptics, germicides) - agents safe to ingest or inject (e.g., antibiotics, other antimicrobials). - personal hygiene (handwashing, alcohol-based antiseptic gels)

IMMUNOHISTOCHEMISTRY (IHC) AND IMMUNOFLUORESCENCE

permits rapid agent identification. - specific antibodies localize to the antigens of the pathogen - Compatible with formalin-fixed tissues (retrospective studies are possible, lack of exposure to infectious agents) - sensitive and specific test - Direct visual evidence of the presence of a pathogen in tissues - Detection of organisms difficult to culture - information for clinical diagnosis as well as for the study of pathogenesis Salmonella (red) intracellular SCV-like microcolonies of Salmonella E-cadherin (green), wheat germ agglutinin (WGA, white) Dapi (blue) Lamp1 (red) -> SCV Salmonella (green) E-cadherin (white) Dapi (blue

Treg pathway (Th3 pathway)

produce additional regulatory compounds that control the differentiation of Th0 cells maintain tolerance to self-antigens modulate autoimmune responses -> suppress other immune cells after pathogen is cleared direct the outcome of an immune response Th1 vs Th2

INTESTINAL EPITHELIUM

provides a physical barrier that separates commensal bacteria in intestinal lumen from lamina propria and other intestinal layers • composed of 4 cell lineages obtained from pluripotent stem cell progenitor: • absorptive enterocytes (most intestinal epithelial cells, IECs), • mucus-producing goblet cells • hormone-producing enteroendocrine cells • Paneth cells (antimicrobial peptides or lectins secretion). • Lamina propria (LP): The layer of mucosal tissue directly under the mucosal epithelial cell surface of the gastrointestinal tract, in which mucosal effector immune cells reside (Stromal cells, B cells (especially IgA-producing plasma cells), T cells, macrophages and dendritic cells, DCs ) • subsets of T cells (intraepithelial lymphocytes, IELs) and some DCs are positioned to sample the luminal contents • Beneath the lamina propria (LP) there are two layers of smooth muscle to propel nutrients along the digestive tract.

Know the role of sIgA.

sIgA: mucosal immunity Dimer: secretory IgA (sIgA) attaches to microbes or toxins to trap them in the mucus layer

Know the role of sIGA and where it is produces, how it works.

secretory immunoglobulin A (sIgA) that binds to mucin and the microbe (MALT) ...more on Triggering the Innate Immunity card

Know what type of microbiota live in the colon, skin, nasopharynx/oropharyngeal, genitalia - categories and highlighted examples, and how some of them are linked to a particular disease

small intestine: • microbiota poorly characterized • In mice adherent bacteria are mostly Clostridium l ower intestine (colon): • higher concentrations of adhering and bacteria

Be able to list the processes that happen in the small and large intestines.

small: Faster transit time, less microbes colon: Slower transit time, more microbes

Th1 cells:

stimulated to proliferate by IL-12 and IL-2 released by APCs • release IL-2, IFN-γ, and TGF-β • Recognition of MHC II-epitope by CD4+ Th1 cells stimulates the production and release of IL-2 and IFN-γ -> activation of macrophages. • cellular immunity most effective against intracellular pathogens • only specific binding of an MHC-epitope complex to its cognate TCR will result in Th cell activation.

Th2 cells

stimulated to proliferate by IL-4 • release IL-4, IL-5, IL-9, IL-10, and IL-13to modulate other immune cells • MHC II-epitope stimulation of CD4+ Th2 cells stimulates naïve B cells to proliferate into B cells -> antibodyproducing plasma cells • Function in humoral immunity and production of memory B cells • IL-4 stimulates B cells to produce IgE -> mast cell stimulation (histamine, serotonin, and leukotrienes)

Know the definition of adjuvants and what they do

strategy to improve the immunogenicity of vaccines • components added to the vaccine formulation that serve as both antigen delivery systems and immune potentiators to induce stronger immune responses than the antigen alone. • can be used to help direct the immune system toward a desired Th1, Th2, and/or Th17 response • subunit vaccines often require the addition of adjuvants to improve delivery and immunogenicity or specific effects on particular immunity • E.g. peptide-based vaccines need adjuvants as alone they do not elicit much of an immune responseand epitope can be ignored (induced tolerance, anergy, a consequence of antigen-induced cell death of activated, antigen-specific T cells) • Adjuvants increase the half-life of activated T cells

Know what "fifth postulate" adds to the Koch postulates.

sufficient microbial data should allow scientists to treat, cure, or prevent thespecific disease • design of effective therapeutic or preventative measures for eliminating the pathogen • May provide more convincing evidence if other postulates are not "conclusive" • Antibiotic therapy that eliminates the bacteria and at the same time cures the disease • Vaccination to prevent disease

Know general information about herd immunity and complications that can occur with it

unvaccinated person in a population that is mostly vaccinated will be protected from disease - diseases needs a sufficient number of susceptible people in the population for transmission • Minimum 70% for polio, diphtheria • 95% for highly infectious diseases (influenza, mumps, and measles) • Issues with international travel to places where infections are endemic

Know examples of how the microbiome in the colon can affect the brain

vagus nerve activation • SCFAs have immunomodulatory properties, cross BBB, regulate microglia • Tryptophan metabolism -> required for serotonin

Plasmid Problem continued

vertical gene transfer, the transfer of genetic material is from parents to offspring Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between organisms other than by the ("vertical") transmission

Know B cells can also be activated independently of T cells (e.g. polysaccharide molecule) and the difference between Th-dependent stimulation

via direct binding of structures on b cell surfaces - Antigens typically nonpeptide,such as polysaccharide (capsule), DNA, RNA, lipids, glycolipids - leads to proliferation, maturation, and production of antibodies (mostly IgG2 and IgM - low opsonization) - Cytokine binding often required as second signal to stimulate B cells to proliferate/differentiate - antibodies produced this way have typically lower affinity than those produced via T-celldependent activation of B cells - Important for control of bacteria that have capsule and cannot be phagocytosed

Know why AMR is such a threat to the world and be able to describe it

~1970s: danger of antibiotic resistance: • Anti Microbial Resistance (AMR): ability of bacteria to resist the effects of antibiotics • infections with such resistant bacteria are difficult to treat • simply using antibiotics creates resistance use of antibiotics in people and in food animals as a contributing factor to resistance • Inappropriate and unnecessary use of antibiotics is a large contributing factor and it should be limited

Understand why microbiota in the colon varies from microbiota in the small intestine.

• # microbes colon >> intestine • Colon microbes 1° anaerobic • Colon microbes as reservoir for opportunistic infections • Shift from "good" to "bad" microbiota may be responsible for diseases (diabetes, obesity, asthma)

number 4 issues

• Assuming postulates 1-3 have been met, this postulate is usually straightforward • Demonstrates that the disease lesions of the intentionally-infected host contain the microbe (similar to what occurs in the "natural" disease)

Know what humoral immunity is and list the cells there. Know what B cells are and what their job is, know plasma cell function. Know that the immature B cell expresses IgM.

• B cell 'job': production of antibodies (Abs) • over10^10 pool of different B-cells, each expressing a single immunoglobin receptor (aka B-cell receptor, BCR) • Immature, naïve B-cells express IgM • Terminal differentiation into plasma cells • Also contain receptors for Fc portion of Abs, complement factors • Ab production stimulated by T-cell activation OR directly independently of T cells, by antigen (i.e. capsule, LPS; no memory B cells produced)

Know the adjuvant examples (slide 21)

• Freund's Adjuvant. 1940s, inactivated Mycobacterium tuberculosis emulsified in mineral oil or paraffin. Stimulates cell-mediated immunity. Stimulates production of the cytokine tumor necrosis factor (TNF), -> pain and tissue damage. Freund's incomplete adjuvant has no mycobacterial components • Aluminum Salts. Might promote antigen uptake by DCs or stimulate Th2 response (humoral activity e.g. IgG1, IgE, IL-4). Vaccines with alum cannot be sterilized by filtration, freezing or lyophilization. • Triterpenoid-based Adjuvants. Plant-derived triterpene glycosides (saponins). Elicit strong, long-lasting Th1, Th2, and cell-mediated immunity, including CTL responses. High toxicity and undesirable hemolytic side effects, instable in aqueous solutions. • Virosomes. Influenza virus-derived antigen carrier and adjuvant. Virosomes are vesicles (nanoparticles) composed of a lipid membrane with membrane-bound viral proteins, mostly hemagglutinin and neuraminidase. Lack the viral genetic material. Help with the delivery of encapsulated antigens to APCs. Present antigens via both MHC-I and MHC-II -> humoral antibody and cell-mediated immunity. • Liposomes and Microspheres. Liposomes incorporate the antigens and adjuvant components in synthetic lipid membranes. Microspheres (microcapsules) made of microparticles of biodegradable polymers such as poly(lactide-co-glycolide) to carry protein or polysaccharide antigen. Controlled release possible to increase exposure time with the immune system. Memory response might be elicited with 1-2 injections.

Know the difference between LD50 and ID50.

• LD50: bacterial dose required to elicit moribund state in 50% of animals • ID50: bacterial dose required to colonize/initiate disease in 50% of animals • 50% used because it is more accurate (area of graph where maximal change occurs) • These methods can lack sensitivity in studying bacterial virulence factors • Large #s of animals needed • Can be used to compare different strains (or wildtype/mutant) of the same species, but NOT different diseases (i.e. cholera vs. bacterial dysentery)

NK functions: role and what they produce (IFNgamma), know that they kill other cells.

• Large, granular lymphocytes, look similar to T-cells; nonphagocytic • Make up 10-15% of leukocytes in blood • Provide protection against viruses, intracellular pathogens • Can lyse tumor cells (malignant transformation) • Can kill cells infected with virus (altered antigen presentation) • 1° source of interferon gamma (IFN-γ) -> potent antiviral and immuno-regulating cytokine • Intersects with adaptive immune response

What is One Health?

• One Health consists of preventing disease and increasing overall health in the following: • Environment • Animals • Humans • One Health is a community-focused initiative, as both preventative and treatment options rely on these above-mentioned factors

ARGS AND AMR IN GLOBAL HEALTH

• The world is connected in numerous ways, making ARGs and AMR much easier to spread. • Surveillance is one of the most-used methods as there is no enforced standardization of AMR control between nations. • "The emergence of similar resistance traits in different locations

Be able to tell which body sites are typically considered sterile (slide 6)

• blood • cerebrospinal fluid (CSF), pleural fluid, peritoneal fluid, pericardial fluid • Bone, bone marrow • joint fluid, synovial fluid • internal body sites (lymph node, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovary, vascular tissue) • Central nervous system • Fetus...