Unit 1, Lecture 3

Antigen

An antigen is a molecular shape that reacts with antigen receptors on lymphocytes to initiate an adaptive immune response.

State what color acid-fast bacteria stain after acid-fast staining.

- Acid-Fast Bacteria: resist decolorization with an acid-alcohol mixture during the acid-fast stain procedure, retain the initial dye carbolfuchsin and appear RED when observed through the microscope. - Common acid-fast bacteria of medical importance include Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium-intracellulare complex.

Innate Immunity

- An antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to almost any microbe. This is the immunity one is born with and is the initial response by the body to eliminate microbes and prevent infection.

State what color Gram-negative bacteria stain after Gram staining.

- Gram-Negative Bacteria decolorize during the Gram stain procedure, pick up the counterstain safranin, and appear PINK when observed through the microscope. - Common Gram-negative bacteria of medical importance include Salmonella species, Shigella species, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus species, and Pseudomonas aeruginosa.

State what color Gram-positive bacteria stain after Gram staining.

- Gram-Positive Bacteria retain the initial dye crystal violet during the Gram stain procedure and appear PURPLE when observed through the microscope. - Common Gram-positive bacteria of medical importance include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Clostridium species.

Invasins

- Molecules that activate the host cell's cytoskeletal machinery enabling bacterial entry into the cell by phagocytosis. By entering the cytoplasm of the host cell, it has a ready supply of nutrients and is able to protect the bacteria from complement, antibodies, and certain other body defenses. Many bacteria use injectosomes, such as a type 3 secretion system, to inject effector molecules into the host cell's cytoplasm. - Invasins cause the host cell to polymerize and depolymerize actin filaments. This cytoskeletal rearrangement is a key part of the formation of pseudopodia in phagocytic cells and is what enables phagocytes to engulf bacteria and place them in a vacuole.

Briefly describe how opsonizing antibodies can promote phagocytosis and how antibodies made against cell wall adhesins can block colonization.

- Opsonization, or enhanced attachment, refers to molecules such as the antibody molecule IgG or the complement proteins C3b and C4b attaching antigens to phagocytes. This results in much more efficient phagocytosis. - The Fab portion of the IgG binds with the epitopes of that antigen. The Fc portion of the IgG can then bind to phagocytic cells such as neutrophils and macrophages thus binding the antigen to the phagocyte, which then engulfs the organism and places it in a phagosome. - One of the ways bacteria colonize host cells is to use cell wall proteins called adhesins to attach to receptors on the surface of that host cell. Antibodies are made against the adhesins on the bacterial cell wall. The Fab portion of the antibodies combine with adhesins before they can interact with the receptor sites on the host cell, thus blocking bacterial attachment.

State the 3 parts of a peptidoglycan monomer and state the function of peptidoglycan in bacteria.

- Peptidoglycan, also called murein, is a vast polymer consisting of interlocking chains of identical peptidoglycan monomers. A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM. - Peptidoglycan forms a protective barrier around the cell membrane, as well as giving the cell its shape and strength. It also counteracts osmotic pressure from the cytoplasm, keeping the cell from rupturing.

Adhesin

- Surface proteins in the bacterial cell wall bind to receptor molecules on the surface of a susceptible host cell enabling the bacterium to make intimate contact with the host cell, adhere, colonize, and resist flushing.

Briefly describe how bacteria synthesize peptidoglycan, indicating the roles of autolysins, bactoprenols, transglycosylases, and transpeptidases.**

- The peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol. As discussed below, the bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and work with the enzymes discussed below to insert the monomers into existing peptidoglycan enabling bacterial growth following binary fission. Once the new peptidoglycan monomers are inserted, glycosidic bonds then link these monomers into the growing chains of peptidoglycan. These long sugar chains are then joined to one another by means of peptide cross-links between the peptides coming off of the NAMs. By linking the rows and layers of sugars together in this manner, the peptide cross-links provide tremendous strength to the cell wall, enabling it to function similar to a molecular chain link fence around the bacterium. 1) autolysins: a) Break the glycosidic bonds between the peptidoglycan monomers at the point of growth along the existing peptidoglycan; b) Break the peptide cross-bridges that link the rows of sugars together 2) The peptidoglycan monomers are synthesized in the cytosol and bind to bactoprenol.The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and interacts with transglycosidases to insert the monomers into existing peptidoglycan. 3) Transglycosylase (transglycosidase) enzymes insert and link new peptidoglycan monomers into the breaks in the peptidoglycan. 4) Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong.

Bactoprenol

- hydrophobic lipid alcohol - binds the peptidoglycan precursor - facilitates transport of NAG and NAM through the cytoplasmic membrane

Cell-mediated Immunity

- involves the production of cytotoxic T-lymphocytes, activated macrophages, activated NK cells, and cytokines in response to an antigens. These defense cells help to remove infected cells and cancer cells displaying foreign epitopes.

Adaptive (acquired) Immunity

-Refers to antigen-specific defense mechanisms that take several days to become protective and are designed to react with and remove a specific antigen. This is the immunity one develops throughout life.

Identify the parts of a bacterium and their physiological purpose:

1) a cytoplasmic membrane surrounded by a peptidoglycan cell wall and maybe an outer membrane; 2) a fluid cytoplasm containing a nuclear region (nucleoid) and numerous ribosomes; 3) and often various external structures such as a glycocalyx, flagella, and pili.

Gram-Negative Cell Wall

1. A thin, inner wall composed of peptidoglycan The peptidoglycan portion of the Gram-negative cell wall is generally 2-3 nanometers (nm) thick and contains just 2-3 layers of peptidoglycan. Chemically, only 10 to 20% of the Gram-negative cell wall is peptidoglycan. 2. An outer membrane: The outer membrane of the Gram-negative cell wall appears as a lipid bilayer about 7 nm thick. It is composed of phospholipids, lipoproteins, lipopolysaccharides (LPS), and proteins. Phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A embedded in the membrane and a polysaccharide portion extending outward from the bacterial surface. The LPS portion of the outer membrane is also known as endotoxin. In addition, pore-forming proteins called porins span the outer membrane. The porins function as channels for the entry and exit of solutes through the outer membrane of the Gram-negative cell wall. 3. The outer membrane of the Gram-negative cell wall is studded with surface proteins that differ with the strain and species of the bacterium. 4. The periplasm is the gelatinous material between the outer membrane, the peptidoglycan, and the cytoplasmic membrane. This periplasmic space is about 15nm wide and contains a variety of hydrolytic enzymes for nutrient breakdown, periplasmic binding proteins for transport via the ATP-binding cassette (ABC) system, and chemoreceptors for chemotaxis.

Gram-Positive Cell Wall

1. Consists of many interconnected layers of peptidoglycan and lacks an outer membrane. 2. Activates both the body's innate immune defenses and its adaptive immune defenses. 3. PAMPs (pathogen associated molecular patterns) associated with the Gram-positive cell wall include peptidoglycan monomers, teichoic acids, lipoteichoic acids, and mannose-rich sugar chains. 4. Peptidoglycan prevents osmotic lysis in the hypotonic environment in which most bacteria live. 5. Teichoic acids and lipoteichoic acids are interwoven through the peptidoglycan layers. 6. Surface proteins embedded in the cell wall can function as adhesins, secretion systems, and enzymes.

Functions of the Gram-Negative Cell Wall Components:

1. The peptidoglycan in the Gram-negative cell wall prevents osmotic lysis . 2. The outer membrane of the Gram-negative cell wall confers several functions: a. Like the cytoplasmic membrane discussed previously in Unit 1, is semipermeable and acts as a coarse molecular sieve. Many small molecules may pass through due to pores running through the membrane. These pores are composed of proteins called porins. b. Because of its semipermeable nature, the outer membrane helps retain certain enzymes and prevents some toxic substances, such as penicillin G and lysozyme, from entering. c. The LPS from the outer membrane of the Gram-negative cell wall is thought to add strength to the outer membrane, in a manner similar to the glycopeptides and teichoic acids of the gram-positive cell wall. d. The outer membrane may also form vesicles that contain quorum signaling molecules, enzymes, toxins, virulence factors, and even antibiotic resistance genes. These vesicles can then fuse with the outer membrane of other Gram-negative bacteria enabling them to communicate, obtain virulence factors, pick up resistance genes, or deliver toxins to human cells. 3. The surface proteins in the bacterial peptidoglycan, depending on the strain and species, carry out a variety of activities. a. Some surface proteins function as enzymes. b. Other proteins serve as adhesins. Adhesins enable the bacterium to adhere intimately to host calls and other surfaces in order to colonize those cells and resist flushing. c. Many bacteria involved in infection have the ability to co-opt the functions of host cells for the bacterium's own benefit. This is done by way of bacterial secretions systems that enable the bacterium to directly inject bacterial effector molecules into the cytoplasm of the host cell in order to alter its cellular machinery or cellular communication to the benefit of the bacteria. They do this by producing secretion systems such as the type 3 secretion system that produces hollow, needle-like tubes called injectisomes. Certain bacteria, for example, inject invasins into the cytoplasm of the host cell that enable the bacterium to enter that cell. The role of these cell wall surface proteins will be discussed in greater detail later in Unit 3 under Bacterial Pathogenicity. 4. The periplasm contains enzymes for nutrient breakdown as well as periplasmic binding proteins to facilitate the transfer of nutrients across the cytoplasmic membrane.

Acid-Fast Cell Wall

3. The acid-fast cell wall consists of a thin, inner layer of peptidoglycan linked to a layer of arabinogalactin, which in turn is linked to an outer membrane containing mycolic acids and overlaid with a variety of polypeptides and glycolipids. 4. Porins are required to transport small hydrophilic molecules through the outer membrane of the acid-fast cell wall. 5. The acid-fast cell wall activates both the body's innate immune defenses and its adaptive immune defenses. - Benefits of: peptidoglycan, arabinogalactin, mycolic acid, and porins... 1. Peptidoglycan prevents osmotic lysis. 2. The arabinogalactan layer is linked to both the peptidoglycan and to the mycolic acid outer membrane and probably provides additional strength to the cell wall. 3. The mycolic acids and other glycolipids also impede the entry of chemicals causing the organisms to grow slowly and be more resistant to chemical agents and lysosomal components of phagocytes than most bacteria. There are far fewer porins in the acid-fast cell wall compared to the gram-negative cell wall and the pores are much longer. This is thought to contribute significantly to the lower permeability of acid-fast bacteria.

Epitope

Are the actual portions or fragments of an antigen that react with antibodies and with receptors on B-lymphocytes and T-lymphocytes.

Humoral Immunity

B cells produce antibodies after exposure to specific antigens; type of adaptive immunity

Briefly describe how LPS and other PAMPs of the Gram-negative cell wall can promote inflammation:

In order to protect against infection, one of the things the body must initially do is detect the presence of microorganisms. The body does this by recognizing molecules unique to microorganisms that are not associated with human cells. These unique molecules are called pathogen-associated molecular patterns or PAMPs. (Because all microbes, not just pathogenic microbes, possess PAMPs, pathogen-associated molecular patterns are sometime referred to as microbe-associated molecular patterns or MAMPs.) LPS, porins, and fragments of peptidoglycan are PAMPs associated with the cell wall of Gram-negative bacteria. These PAMPS bind to pattern-recognition receptors or PRRs on a variety of defense cells of the body and trigger such innate immune defenses as inflammation, fever, and phagocytosis. The LPS aso activates the alternative complement pathway and the lectin pathway, defense pathways that play a variety of roles in body defense.

Briefly describe how antibiotics such as penicillins, cephalosporins, and vancomycin affect bacteria and relate this to their cell wall synthesis.*

Many antibiotics work by inhibiting normal synthesis of peptidoglycan in bacteria causing them to burst as a result of osmotic lysis. As just mentioned, in order for bacteria to increase their size following binary fission, enzymes called autolysins break the peptide cross links in the peptidoglycan, transglycosylase enzymes then insert and link new peptidoglycan monomers into the breaks in the peptidoglycan, and transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong. Interference with this process results in a weak cell wall and lysis of the bacterium from osmotic pressure. Examples include the penicillins (penicillin G, methicillin, oxacillin, ampicillin, amoxicillin, ticarcillin, etc.), the cephalosporins (cephalothin, cefazolin, cefoxitin, cefotaxime, cefaclor, cefoperazone, cefixime, ceftriaxone, cefuroxime, etc.), the carbapenems (imipenem, metropenem), the monobactems (aztreonem), the carbacephems (loracarbef), and the glycopeptides (vancomycin, teichoplanin). For example, penicillins and cephalosporins bind to the transpeptidase enzymes (also called penicillin-binding proteins) responsible for resealing the cell wall as new peptidoglycan monomers are added during bacterial cell growth. This blocks the transpeptidase enzymes from cross-linking the sugar chains and results in a weak cell wall and subsequent osmotic lysis of the bacterium.

Briefly describe how PAMPs of the Gram-positive cell wall can promote inflammation.

The body activates innate immunity by recognizing molecules unique to microorganisms that are not associated with human cells called pathogen-associated molecular patterns or PAMPs. PAMPs bind to Pattern-recognition receptors (PRRs) on defense cells to trigger the production of inflammatory cytokines.

Filamentous Temperature Sensitive Proteins

These proteins form the cell division apparatus known as the divisome and are directly involved in bacterial cell division by binary fission. The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum.

The function of the peptide cross-links in peptidoglycan is to:

connect one row of peptidoglycan monomers to the next row and one layer of peptidoglycan to another.