Larry A. Hanson [email protected] - Home | Food and ...

Bacterial PathogenesisLarry A. Hanson

[email protected]

Aquatic AMR Workshop 1: 10-11 April 2017, Mangalore, India

FMM/RAS/298: Strengthening capacities, policies and national action plans on

prudent and responsible use of antimicrobials in fisheries

Host-Parasite Relationships: Pathogenesis of Infections

In any host-pathogen encounter, there are two determinants of the outcome:

1. Virulence of the parasite

2. Resistance of the host

In some cases, the host-pathogen relationship is very complex:

-Commensal but opportunistic

will take advantage of weakened host and invade tissues setting up a potentially life-

threatening infection

Examples include motile Aeromonads- natural inhabitants of intestine but cause

septicemia when fish is immune suppressed

o Bacteria cause disease by 2 basic mechanisms:1-Direct damage of host cells

2-Indirectly by stimulating exaggerated host inflammatory/immune response

Virulence factors are molecular components expressed by

a pathogen that increases its ability to cause disease

Virulence factors can be divided into two categories:• 1. Those that cause damage to the host (toxins)

• 2. Those that do not directly damage the host but promote colonization

and survival of infecting bacteria

A. Bacterial toxins

1. Exotoxin: protein molecule liberated from intact living bacterium.

a. They are antigenic and can elicit protective antitoxic antibodies. Many of these

toxins can be converted to nontoxic immunizing agents termed toxoids.

b. Three roles of exotoxins in disease:

i. Ingestion of preformed toxin (botulism)

ii. Colonization of wound or surface followed by toxin production (cholera and

diphtheria toxins)

iii. Exotoxin produced by bacteria in tissues to aid growth and spread

(Clostridium perfringens alpha-toxin)

d. Types of exotoxins:

i. A-B toxins (intracellular acting)

1) Composed of two parts: A and B portions

2) The B portion mediates binding to a specific host cell receptor.

3) After binding to the host cell, the A portion is translocated into host cells and has biological activity against an intracellular target

or

4) Examples:

a) Diphtheria toxin: ADP-ribosylation of host EF-2; host cells are killed by blocking translation.

b) Cholera toxin: ADP-ribosylation of a cAMP regulatory protein, which causes loss of ion regulation, water loss, diarrhea.

c) Shiga toxin cleaves host rRNA, which blocks translation and kills the host cell.

d) Clostridium botulinum- large subunit targets neurons, small subunit cleave snare proteins inhibiting neurotransmitter release from neurons- causes paralysis BoNT- E in fish (most toxic substance known)

ii. Membrane disrupting (surface damaging)

1) Cause damage or disruption of plasma membranes, which leads to osmotic lysis and cell death. Many were originally termed “hemolysins” because they lyse RBCs.

2) Three types of membrane disrupting toxins:

a) Enzymes that hydrolyze phospholipids: phospholipase, sphingomyelinase

b) Toxins with detergent-like surfactant activity that disrupt by membrane solubilization

c) Pore forming toxins (the most common): proteins that insert in the host membrane and form a hydrophilic pore

Aeromonas produces up to 4 hemolysins- aerolysin A (AeroA) and Heat labile hemolysin AHH1- work synergistically, also some aeromonads produce the pore forming toxin RtxA

Staphylococcus aureus

alpha hemolysin,

looking down the

central pore

iii. Superantigens

1) Toxins that bind directly to MHC II on macrophages (without being processed) and form a crosslink with T cell receptors.

2) Crosslinking causes stimulation of up to 1 in 5 T cells in the body (normal antigens cause stimulation of 1 in 10,000).

3) Excessive IL-2 production results from the massive stimulation of T helper cells,

4) Stimulation of other cytokines by IL-2 lead to shock.

Example: staphylococcal toxic-shock syndrome

iv. Extracellular enzymes: break down host macromolecules.

play an important role in disease development by providing a nutrients or aiding in

dissemination. Can cause extensive tissue damage

Examples:

a) Coagulase – clots fibrin, thus protecting the bacteria.

b) Hyaluronidases and proteases – aid in the spread of bacteria by degrading extracellular

matrix.

c) Collagenase – aids in dissemination

d) DNase – reduces viscosity of debris from dead cells (may help escape DNA net by

neutrophil).

A. hydrophila - Express diverse extracellular enzymes can contribute to virulence including

collagenase, elastase, enolase, lipases (heat stable lipase, pla and Plc), metallo protease, and

serine protease, Rnase R.

2. Endotoxin- released when cells die: lipopolysaccharide (LPS) produced by gram-

negative bacteria. In gram-positive bacteria peptidoglycan and teichoic acids.

a. LPS is bound by LPS binding proteins in plasma, which then binds CD14. This complex binds

Toll-like receptor 4 (TLR4) on macrophages and monocytes. TLR2 binds teichoic acids.

TLR1 binds peptidoglycan.

b. Macrophages and monocytes release cytokines (IL-1, IL-6, IL-8, TNF alpha, Platelet

Activating Factor), which subsequently trigger prostaglandin and leukotriene release

c. The complement and coagulation cascades are activated.

e. endotoxic shock occurs when bacterial products reach high enough levels in the blood to

trigger complement activation, cytokine release, and coagulation cascade activation in many

parts of the body. Circulatory system collapse followed by multiple organ system failure

occurs.

B. Bacterial invasion of host tissues

1. Host damage is caused during invasion by either:

a. direct disruption of function

b. an exaggerated immune response that compromises tissue function.

2. The invasive bacteria are classified as:

a. Facultative Intracellular Parasites

i. FIPs are not confined to cells

ii. Some can multiply in professional phagocytic cells.

iii. When a balance is established between the bacterium and phagocyte, the

bacteria may survive in this intracellular state for months or years (example:

Mycobacterium).

b. Obligate Intracellular Parasites; can only propagate inside host cells.

Examples include chlamydia and rickettsia

c. Extracellular parasites, which cause tissue damage while they are outside

phagocytes and other cells and do not have the ability to survive long

periods in cells.

3. Steps in bacterial invasion:

a. Motility

i. Flagella are the best characterized; adapted for low viscosity

fluids.

ii. Other types of motility: corkscrew type (Spirochetes--best in

viscous solutions), gliding motility (Flavobacterium columnare

and cytophagas, myxobacteria--movement over solid surfaces).

iii. Chemotaxis is directional swimming using a gradient (especially

nutrients).

A. hydrophila produce lateral flagella for surface movement and polar

flagella for movement in suspension. Glycosylation of polar flagella involved

in biofilm formation, binding to cells and mucosal adherence

b. Adherence

i. Two common strategies: fimbriae and monomeric protein adhesins.

ii. Fimbriae (pili): receptors are usually carbohydrate residues of glycoproteins or

glycolipids. Attachment is more fragile. Highly specific binding, often mediated by

adhesins, can be blocked by antibodies, often specific for host tissue

type/location.

iii. Monomeric protein adhesins: mediated by cell surface proteins, tighter binding to

host cell, may recognize proteins on host cell surface, may follow looser fimbrial

attachment.

Aeromonas-bundle-forming pilus (encoded by bfp) is a critical internal colonizing

factor

c. Invasion of host cells (intracellular pathogens)

i. Some invasive bacteria have mechanisms for entering host cells that are not

naturally phagocytic.

ii. Two types of bacterial-mediated invasion:

a. Zippering: bacteria present ligands on their surface allowing them to bind to

host cells and initiate the entry process. It is similar to FcR- and CR3-

mediated phagocytosis, which is characterized by the formation of inclusion

shaped by the bacteria they ingest (Yersinia pestis Ail).

b. Triggering: bacteria inject effectors into host cells via T3SS to regulate

phagocytosis (Salmonella).

iii. Following attachment to host cells, pathogens cause changes in host cell

cytoskeleton (actin) that cause the pathogen to be internalized.

iv. Some pathogens can utilize actin fibers intracellularly to move through host cells

(transcytosis).

v. Invasins may also mediate uptake of bacteria into professional phagocytic cells in a

way that bypasses normal phagosome formation.

d. Manipulation of host cell functionsi. Bacterial pathogens are often very manipulative of host cell functions; both extracellular

and intracellular pathogens will cause host cells to perform functions favorable to the

pathogen.

a. For example, leukotoxin produced by Mannheimia haemolytica (extracellular pathogen)

induces cytokine secretion.

b. Listeria monocytogenes (intracellular pathogen) produces a protein that mobilizes actin to

propel bacteria through the cell and into neighboring cells.

ii. Some bacterial pathogens have a specialized type III secretion system (TTSS) that forms a needle-like structure that injects effector proteins directly into the host cell cytoplasm.

a. In some cases, these effector proteins serve as receptors in the host membrane for bacterial attachment.

b. In some cases, these effector proteins can mobilize cytoskeleton to cause phagocytosis.

c. In some cases, effector proteins can induce or prevent apoptosis.

Aeromonas express type II, III and VI secretion

systems III and VI can inject effector proteins into

host cells (II is for extracellular release of proteins).

4. Obtaining nutrientsa. Pathogenic bacteria have intricate methods to obtain all essential nutrients.

b. Obligate intracellular bacteria have complex nutrient requirements and parasitize the living cell for an extended period.

c. Host cytoplasm is a very nutrient rich environment.

i. Extracellular pathogens often lyse cells to obtain nutrients.

ii. Intracellular pathogens will either escape from phagosomes to enter the nutrient rich cytoplasm or modify the vacuole so they can get nutrients from the cytoplasm (example: E. ictaluri).

d. Iron

i. Host tissues are very low in iron because it is bound to transferrin, lactoferrin, ferritin, and heme.

ii. Bacterial strategies for obtaining iron (often induced by low iron conditions):

1) Siderophores--low MW compounds that chelate iron with very high affinity; secreted and taken up by bacterial surface receptors

2) Direct binding of host transferrin, lactoferrin, ferritin, or heme by bacterial surface receptors.

3) Exotoxins that lyse host cells (can be used to obtain other nutrients as well).

5. Evasion of host immune responsea. Serum resistance

i. Serum resistance is defined as the ability to prevent bacterial lysis by the C5b-C9 membrane attack complex (MAC).

ii. Capsule mediates resistance to complement by:

1) preventing C3b binding

2) promoting C3bH complex formation instead of C3bBb (mediated by sialic acid in capsule-this inhibits complement cascade).

iii. Lipopolysaccharide--binds C3b and C5b. However, O polysaccharide can mediate resistance to complement by:

1) having sialic acid attached to promote C3bH formation

2) having long O polysaccharide side chains that prevent MAC killing after C5b binds (possibly too far from bacterial outer membrane).

iv. S-layer or outer membrane proteins

Aeromonas encodes an S-layer also and capsule, TagA cleaves C1-esterase inhibitor imparting serum resistance

b. Resistance to opsonization/phagocytosis

i. Capsule:

1) prevents C3b-mediated opsonization (by the same

mechanism used to avoid complement-mediated

killing)

2) prevents antibody-mediated opsonization by

masking (hyaluronic acid, sialic acid).

Aeromonas- capsule have anti-phagocytic activity,

provide increased resistance to the complement

system, and increased adherence

b. Resistance to opsonization/phagocytosis

ii. LPS O polysaccharide can prevent opsonization if it has sialic acid

iii. S-layer

iv. Extracellular products: enzymes that inactivate C5a chemoattractant (S.

pyogenes), toxins that kill phagocytes (leukotoxins) (Mannheimia

haemolytica), inhibit migration, or reduce oxidative burst.

c. Strategies for surviving phagocytosis:

i. Escape from phagosome before fusion with lysosome (example: Listeria

monocytogenes, mediated by listeriolysin)

ii. Prevent phagosome-lysosome fusion-use type 3 secretion system to influence

trafficking

iii. Express factors that allow survival in harsh phagolysosome conditions (catalase,

superoxide dismutase, surface polysaccharides, cell wall)

d. Evading antibody

i. Ig proteases

ii. Antigenic switching or phase variation

iii. Masking (sialic acid, hyaluronic acid, coating with host

proteins such as fibronectin).

6. Virulence factors expression and release are coordinated by

intricate gene regulation and regulated protein function

a. Regulon-coordinated control of group of virulence factors that are

activated or deactivated in response to environmental signal.

b. Allows bacterial pathogens to adapt to varying host conditions.

c. Virulence gene expression can be triggered when a pathogen

senses environmental cues from the host environment

(examples: pH, iron concentration).

d. Virulence gene expression is sometimes triggered when a pathogen

detects sufficient bacterial numbers are present: “quorum sensing”

i. Bacteria with quorum sensing capability secrete a small molecule (for example,

homoserine lactone)

ii. When the quorum sensing molecule reaches a critical concentration, gene

expression is stimulated.

iii. Sometimes quorum sensing regulates virulence genes.

advancedhealing.com

Aeromonads have elaborate quorum

sensing system that regulated biofilm

formation and virulence genes

Biofilm

Definition: a structured community of bacteria enclosed in

a self-produced polymeric matrix and adherent to an inert

or living surface. Can provide resistance to damage

outside of host, can protect against immune processes

inside the host and can provide transient antibiotic

resistance

Resistance is due to:

a. Slower growth rates of bacteria within biofilms

b. Decreased diffusion of antibiotics through the

biofilm (protective matrix)

c. Accumulation of enzymes that contribute to

resistance

Scanning electron

micrograph of E. coli

O157:H7

biofilm bacteria

Persistence in the presence of antibiotics- regulated phenotypes

Persisters are non- or slow-growing reversible phenotypic variants of the wild

type, tolerant to bactericidal antibiotics.

i. tolerance is due to inhibition of essential cell functions during antibiotic stress,

resulting in inactivity of the antibiotic target.

ii. Persistence occurs in E. coli, Pseudomonas aeruginosa, Mycobacterium

tuberculosis, and Staphylococcus aureus.

iii. Persistence requires coordinated metabolic changes; entry and exit from the

persister state is regulated by signal molecules (such as guanosine

pentaphosphate or ppGpp).

Summary

o Bacterial pathogens are highly adapted and specialized.

o Infection constitutes an organized, orchestrated attack.

o Toxins manipulate the host to the bacteria’s advantage.

o Specialized mechanisms are required to invade the host, obtain nutrients, and avoid the immune response.

Thank you