Bacterial pathogenesis and immune evasion

Question 1

Define the terms: virulence, pathogenicity, immunopathology

Answer 1

virulence

pathogenicity

immunopathology

Question 2

List and describe the Mechanisms of immunopathogenicity

Answer 2

Question 3

Virulence Factors are important for evasion, tissue damage and, colonisation and adherence

List Virulence Factors of bacteria

Answer 3

Adherence Factors:
colonize mucosal sites by using pili (fimbriae) to adhere to cells.

Invasion Factors:
Surface components and secreted effector proteins

Capsules:
polysaccharides - protect from opsonization and phagocytosis.

Endotoxins:
lipopolysaccharide on Gram-negatives; lipoteichoic acids G+ves cause fever, changes in blood pressure, inflammation, lethal shock

Exotoxins:
protein toxins and enzymes produced and/or secreted e.g. cytotoxins, neurotoxins, and enterotoxins.

Siderophores:
iron-binding factors to compete with the host for iron hemoglobin, transferrin, and lactoferrin

Question 4

Stages of Infection of bacteria

Answer 4

Acquisition
Colonisation – adherence
Penetration
Multiplication and Spread
Immune avoidance
Damage
Transmission
Resolution

Question 5

The first lines of defense do not depend
on antigen recognition

Describe the first line of defence

Answer 5

the skin and exposed epithelial surfaces
have non-specific or innate protective systems, which limit the entry of potentially invasive organisms. Intact skin is impenetrable to most bacteria. Additionally, fatty acids produced by the skin are toxic to many organisms. Indeed, the pathogenicity of some strains correlates
with their ability to survive on the skin.

Epithelial surfaces are cleansed, for example, by ciliary action in the trachea or by flushing of the urinary tract.

Many bacteria are destroyed by pH changes in the stomach and vagina, both of which provide an acidic environment. In the vagina, the epithelium secretes glycogen, which is metabolized by particular species of commensal
bacteria, producing lactic acid.

Question 6

Describe how commensals can limit pathogen invasion?

Answer 6

Commensal bacteria occupy an ecological niche
that would otherwise be occupied by pathigenic bacteria.

The normal flora protect against pathogens by competing more efficiently for nutrients, by producing antibacterial proteins termed colicins and by stimulating immune responses which act to limit pathogen entry.

The host attempts to minimize contact between the bacteria and the epithelial cells of the gut lumen by production of mucins, and by effector molecules including antimicrobial peptides and secretory IgA.

Question 7

The second line of defense is mediated by
recognition of bacterial components

LPS is the dominant activator of innate immunity in Gram-negative bacterial infection

Describe this

Answer 7

Injection of pure LPS into mice or even humans is sufficient to mimic most of the features of acute Gram-negative infection, including massive production of proinflammatory cytokines, such as IL-1, IL-6, and tumor necrosis factor (TNF), leading to severe shock.

Recognition of LPS is a complex process involving molecules that bind LPS and pass it on to cell membrane-associated receptors on leukocytes, and endothelial and other cells,
which initiate this proinflammatory cascade.

Binding of LPS to TLR4 is a critical event in immune activation.

The LBP and CD14, which bind LPS, are also involved in recognition of lipid-containing bacterial components from mycoplasmas, mycobacteria, and spirochetes.

Question 8

Q. List some examples of soluble molecules, cell surface receptors, and intracellular molecules that recognize PAMPs.

Answer 8

A. Collectins and ficolins, the Toll-like receptors, the mannose receptor, and the NOD-like receptor proteins all recognize PAMPs.

Question 9

Q. How does release of proinflammatory cytokines cause shock?

Answer 9

A. These proinflammatory cytokines (such as TNF and IL-1 from macrophages) act directly on endothelium to increase vascular adhesiveness,facilitate the passage of more phagocytes into inflamed tissue and indirectly activate other plasma enzyme systems to release vasoactive peptides and amines leading to a drop in blood
pressure

Question 10

Other bacterial components are also potent
immune activators

Describe this

Answer 10

Gram-positive bacteria do not possess LPS yet still induce intense inflammatory responses and severe infection via the actions of other chemical structures such as peptidoglycans
and lipotechoic acids of their cell wall, which can
be recognized by TLR2, often in cooperation with TLR1 or TLR6.

Most capsular polysaccharides are not potent activators of inflammation but they shield the bacterium from host immune defenses.

Other bacterial molecules that trigger innate immunity include lipoproteins (via TLR 2/6), flagellin (via TLR5), and DNA (due to its distinct CpG motifs) via TLR9.

Most pattern recognition receptors are expressed on the plasma membrane of cells, making contact with microbes during the process of binding and/or phagocytosis.
However, others are designed to detect intracellular pathogens and their products inside phagosomes (such as TLR9) or in the cytosol.

Question 11

Q. Which proteins can recognize pathogens in the cytosol, and which pathogen components?

Answer 11

A. NOD-1 and NOD-2 proteins are members of the larger family of NOD-like receptors (NLRs) and recognize peptidoglycans of both Gram-positive and Gram-negative bacteria

Question 12

How does the host determine a real pathogenic threat?

Answer 12

Epithelial cells of the gut and lung can have few TLRs on their luminal surface, but can be triggered by pathogens that:
* actively invade the cell (such as Listeria spp.);
* inject their components (such as Helicobacter pylori); or
* actively reach the basolateral surface (e.g. Salmonella.
This helps to explain why constant exposure to nonpathogenic microbes in the intestine and airways does not induce a chronic state of inflammation – the host waits until they move beyond the lumen, signifying the presence of a
real pathogenic threat.

Question 13

Lymphocyte-independent effector
systems

Describe how the Complement is activated via the alternative pathway to kill bacteria

Answer 13

Complement activation can result in the killing of some bacteria, particularly those with an outer lipid bilayer susceptible to the lytic complex (C5b–9).

Perhaps more importantly, complement activation releases C5a, which attracts and activates neutrophils and causes degranulation
of mast cells. The consequent release of histamine and leukotriene (LTB4) contributes
to further increases in vascular permeability.

Opsonization of the bacteria, by attachment of cleaved derivatives of C3, is also critically important in subsequent interactions with phagocytes.

Question 14

Q. To which strains of bacteria are individuals with C9 deficiency more susceptible?

Answer 14

A. Neisseria

Question 15

How does the release of proinflammatory cytokines help in the immune response

Answer 15

The rapid release of cytokines such as TNF and IL-1 from macrophages increases the adhesive properties of the vascular endothelium and facilitates the passage of more phagocytes into inflamed tissue.

the release of chemokines such as CCL2, CCL3, and CXCL8 directs the recruitment of different
leukocyte populations.

IL-1, TNF, and IL-6 also initiate the acute phase response, increasing the production of complement components and proteins involved in scavenging material released by tissue damage and, in the case of CRP, an opsonin for improving phagocytosis of bacteria. When NK cells are stimulated by the phagocyte-derived cytokines IL-12 and IL-18 they rapidly release large quantities of interferon-g (IFNg).

Question 16

Lymphocyte-independent effector systems

Pathogen recognition generates signals that
regulate the lymphocyte-mediated response

Describe these signals

Answer 16

Dendritic cells:
Contact with bacteria in the periphery induces immature DCs to migrate to the draining lymph nodes and augments their antigenpresenting
ability by increasing their:
* display of MHC molecule–peptide complexes;
* expression of co-stimulatory molecules (such as CD40, CD80, and CD86)
* secretion of T cell differentiating cytokines.

Binding of bacterial components to pattern recognition receptors such as TLRs induces a local environment rich in cytokines such as IFNg, IL-12, and IL-18, which promote T cell differentiation down the TH1 rather than TH2
pathway.

Question 17

Antibody dependent anti-bacterial defenses

Answer 17

Antibody deals with bacterial toxins:
*neutralizes diphtheria toxin by blocking the
attachment of the binding portion of the molecule to its target cells;
*it may block locally acting toxins or extracellular matrix-degrading enzymes, which act as spreading factors.
*can also interfere with motility by binding to flagellae.

secretory IgA stop bacteria binding to epithelial cells – e.g. to the M proteins of group A streptococci gives type-specific immunity to streptococcal sore throats.

IgM antibodies bind to common bacterial structures such as phosphorylcholine, protecting against some bacteria (particularly streptococci) via their complement fixing activity.

Specific, high-affinity IgG antibodies are important, particularly for anti-toxin responses where the antibody must compete against the affinity of the toxin receptor on host cells.

The most efficient complement-fixing antibodies in humans are IgM, then IgG3 and to a lesser extent IgG1, whereas IgG1 and IgG3 are the subclasses with the highest affinity for Fc receptors

Question 18

Pathogenic bacteria may avoid the effects of antibody

Describe how

Answer 18

Neisseria gonorrhoeae is an example of a pathogenic bacterium that uses several immune evasion strategies and humans can be repeatedly infected with N. gonorrhoeae
with no evidence of protective immunity.

1) it fails to evoke a large antibody response, and the antibody that does form tends to block the function of damaging antibodies.
2) the organism secretes an IgA protease to destroy antibody.
3) blebs of membrane are released, and these
appear to adsorb and so deplete local antibody levels.
4) the organism uses three strategies to alter its antigenic composition:
(i) the LPS may be sialylated, so that it more closely resembles mammalian oligosaccharides and promotes rapid removal of complement;
(ii) the organism can undergo phase variation, so that it expresses an alternative set of surface molecules
(iii) the gene encoding pilin, the subunits of the pilus, undergoes homologous recombination to generate variants.
N. gonorrhoeae also impairs T cell activation by engaging a co-inhibitory receptor CEACAM-1
on the lymphocyte surface by one of its OPA proteins.

Question 19

Pathogenic bacteria can avoid the detrimental
effects of complement

Answer 19

*Some bacterial capsules are very poor activators of the alternative pathway.

*For other bacteria, long side chains (O antigens) on their LPS may fix C3b at a distance from the otherwise vulnerable lipid bilayer.

*smooth-surfaced Gram-negative organisms (Escherichia coli, Salmonella, Pseudomonas) may fix but then rapidly shed the C5b–C9 membrane lytic complex.

*Other organisms exploit the physiological mechanisms that block destruction of host cells by complement. When C3b has attached to a surface it can interact with factor B leading to further C3b amplification or it can become inactivated by factors H and I.

*Capsules rich in sialic acid (as host cell membranes are) seem to promote the interaction
with factors H and I. Neisseria meningitidis, E. coli K1, and group B streptococci all resist complement attachment in this way. The M protein of group A streptococci acts as an acceptor for factor H, thus potentiating C3bB dissociation. These bacteria also have a gene for a C5a protease.

Question 20

Bacterial components attract phagocytes
by chemotaxis

Describe this process

Answer 20

resident macrophages are constitutively present in tissues where exposure to pathogens
first occurs (e.g. alveolar macrophages,lung and Kupffer cells, liver). These macrophages have some killing activity, but need to be supplemented by recruitment of neutrophils and/or monocytes. Phagocytes are attracted by:
* bacterial components such as f-Met-Leu-Phe (which is chemotactic for leukocytes);
* complement products such as C5a; and
* locally released chemokines and cytokines derived from resident macrophages and epithelial cells

The cellular composition of this inflammatory response varies according to the pathogen and the time since infection. For instance:
* acute infection with encapsulated bacteria such as Streptococcus pyogenes give rise to tissue lesions rich in neutrophils
* at the other extreme, chronic infections with M. tuberculosis result in granulomas rich in macrophages, macrophage-derived multinucleated giant cells, and T cells;
* other organisms, such as Listeria and Salmonella spp., result in lesions of more mixed composition.

Question 21

The choice of receptors is critical

Answer 21

• direct binding is mediated by pattern recognition molecules including Toll-like receptors and scavenger receptors (such as SRA, MARCO), mannose receptor, and dectin-1;
• opsonization is mediated through complement receptors such as CR1, CR3, and CR4, which recognize complement fragments deposited on the organism via the alternative or classic complement pathways.

Complement can also be fixed by MBL present in serum, which can itself bind to C1q receptors and CR1.
Additionally, Fc receptors on the phagocyte (FcgRI, FcgRII, and FcgRIII) bind antibody that
has coated bacteria.

Question 22

Phagocytic cells have many killing methods

Answer 22

The phagosome helps to deliver antimicrobial molecules to the organism at high concentrations and reduces collateral damage to the host.

Maturation of the phagosome into a killing zone occurs by acquisition of microbicidal mediators
following fusion with other intracellular vesicles such as lysosomes. The killing pathways of phagocytic cells can be oxygen dependent, with the generation of reactive oxygen intermediates, or oxygen independent. In neutrophils, the oxidative burst may also act indirectly, by promoting the flux of K+ ions into the phagosome and activating microbicidal proteases.

A second oxygen-dependent pathway involves the creation of nitric oxide from the guanidino nitrogen of L-arginine. This in turn leads to further toxic substances such as the peroxynitrites, which result from interactions
of NO with the products of the oxygen reduction pathway.

Question 23

Defensins have antibiotic-like properties

Describe these actions

Answer 23

The defensins are found in phagocytes such as neutrophils, where they comprise 30–50% of the granule proteins.

They act by integrating into microbial lipid membranes and disrupting membrane function and structure, resulting in lysis of the pathogen.

Defensins also have important immunostimulatory properties including:
* promoting chemotaxis and phagocytosis;
* regulating cytokine production; and
* acting as adjuvants for adaptive immunity by promoting multiple facets of dendritic cell function including antigen uptake, processing and presentation as well as their migration and maturation.

Question 24

Immunopathological reactions induced by bacteria

All immune responses designed to kill invading pathogens have the potential for causing collateral damage to the host.

Describe how Excessive cytokine release can lead to endotoxin shock

Answer 24

If cytokine release is sudden and massive, several acute tissue-damaging syndromes can result and are potentially fatal.

One of the most severe examples of this is endotoxin (septicemic) shock, when there is massive production of cytokines, usually caused by bacterial products released during septicemic episodes. Endotoxin (LPS) from Gram-negative bacteria is usually responsible, though Gram-positive septicemia can cause a similar syndrome. There can be lifethreatening
fever, circulatory collapse, diffuse intravascular
coagulation, and hemorrhagic necrosis, leading eventually to multiple organ failure

Paradoxically, individuals who recover fromthe initial lifethreatening phase often overcompensate and switch from a hyper- to a hyporesponsive phase, in which excessive production of endogenous immune regulators such as IL-10 and TGFb results in immune
paralysis, making them susceptible to secondary infection.

Question 25

The toxicity of superantigens results from
massive cytokine release

Describe their action

Answer 25

superantigens bind directly to the variable regions of beta chains of antigen receptors
on subsets of T cells, and cross-link them to the MHC molecules of APCs, usually outside the normal antigen binding groove.

the toxicity of the massive release of cytokines (including IL-2, TNFa, and TNFb, together with
IL-1b from activated macrophages) due to the simultaneous stimulation of up to 20% of the entire T cell pool.

The staphylococcal toxins responsible for the toxic shock syndrome (toxic shock syndrome toxin-1 [TSST-1], etc.) operate in this way, though not all shock syndromes caused by staphylococci are the result of T cell activation.

Recent evidence suggests that streptococcal M protein, a known virulence factor of S. pyogenes, forms a complex with fibrinogen, which then binds to b-integrins on neutrophils, causing the release of inflammatory mediators, which also
result in massive vascular leakage and shock.