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Home , Exotoxin, Pathogen

FMM/RAS/298: Strengthening capacities, policies and national action plans on prudent and responsible use of antimicrobials in fisheries Bacterial Pathogenesis Larry A. Hanson [email protected]

Aquatic AMR Workshop 1: 10-11 April 2017, Mangalore, India -Parasite Relationships: Pathogenesis of

In any host-pathogen encounter, there are two determinants of the outcome: 1. 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

Examples include motile Aeromonads- natural inhabitants of intestine but cause septicemia when is immune suppressed o cause 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 () • 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 . Many of these toxins can be converted to nontoxic immunizing agents termed . b. Three roles of exotoxins in disease: i. of preformed (botulism) ii. Colonization of or surface followed by toxin production ( and toxins) iii. Exotoxin produced by bacteria in tissues to aid growth and spread ( 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) : ADP-ribosylation of host EF-2; host cells are killed by blocking translation. b) : ADP-ribosylation of a cAMP regulatory protein, which causes loss of ion regulation, water loss, . c) cleaves host rRNA, which blocks translation and kills the host cell. d) - 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 “” because they lyse RBCs. 2) Three types of membrane disrupting toxins: a) 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 AHH1- work synergistically, also some aeromonads produce the pore forming toxin RtxA

Staphylococcus aureus alpha hemolysin, looking down the central pore iii. 1) Toxins that bind directly to MHC II on (without being processed) and form a crosslink with receptors. 2) Crosslinking causes stimulation of up to 1 in 5 T cells in the body (normal 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 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) and proteases – aid in the spread of bacteria by degrading extracellular matrix. c) – aids in dissemination d) DNase – reduces viscosity of debris from dead cells (may help escape DNA net by ).

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: (LPS) produced by gram- negative bacteria. In gram-positive bacteria 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, 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 to trigger complement activation, 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 , the bacteria may survive in this intracellular state for months or years (example: ). b. Obligate Intracellular Parasites; can only propagate inside host cells. Examples include chlamydia and c. Extracellular parasites, which cause tissue damage while they are outside 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. 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 (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 , which is characterized by the formation of inclusion shaped by the bacteria they ingest ( pestis Ail). b. Triggering: bacteria inject effectors into host cells via T3SS to regulate phagocytosis (). iii. Following attachment to host cells, pathogens cause changes in host cell cytoskeleton () 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 formation. d. Manipulation of host cell functions i. 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 . b. (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 nutrients a. 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 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 response a. 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 ). 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 -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, ) 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.

Aeromonads have elaborate quorum sensing system that regulated biofilm formation and virulence genes

advancedhealing.com 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 resistance Resistance is due to: a. Slower growth rates of bacteria within biofilms b. Decreased diffusion of 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, aeruginosa, Mycobacterium , and 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