Bacterial Virulence Factors

Koch’s postulates

• set of criteria used to establish a connection between a particular microbe and particular disease

• established in 1880s by Robert Koch

challenges — Postulates assume that:

• disease symptoms depend entirely on bacterium, yet host factors play huge role — asymptomatic carriage in some patients

• a pathogen can be isolated rom infected animal and cultured in pure form — M. leprae, treponemo pallidum, chalmydophila pneumonia cannot be cultured.

• a good animal model (or human volunteer) exists for re-inoculation: not always so

• disease are caused by a single pathogen — but what about polymicrobial diseases i.e microbial shift diseases

examples of modern methods and additonal guidelines for providing evidence of cause and effect

• detection of pathogen in diseased but not healthy tissue via PCR/Immunohistochemistry/ELISA — chlamydia pneumoniae

• elimination of suspected pathogen from infected host should cure the disease — Helicobacter pylori

• vaccination can be used to prevent infection by suspected pathogen and thus prevent disease — HPV vaccination effective against genital warts

pathogenicity

the ability of a microorganism to cause disease

infection

the successful colonization of the body by a microorganism capable of causing damage to the host i.e a pathogen

virulence

the degree of pathogenicity of a microorganism, or its relative ability to overcome host defences

virulence factor

bacterial product or strategy that contributes to the ability of the bacterium to survive in the host and/or cause infection

types of virulence factors

• iron acquisition

• attachment to host tissues

• motility and chemotaxis

• invasion of host cells

• toxin production

• quorum sensing

• biofilm formation

• secretion and delivery of bacterial virulence factors

• anti-virulence antimicrobials

best model for studying human disease

• human volunteer

  • feasible when disease is non-life threatening, treatable or vaccine preventable

  • serious ethical issue are involved and there are now strict regulation

• Usually animal models as ‘gold standard’

  • animal host must be susceptible to the pathogen

  • infection of the animal should ideally mirror all of the disease symptoms that occur in human infections (not always possible)

  • must have some way to measure virulence in model

  • numerous ethical issues are involved and all protocols must be approved by an ethics committee

advantages of a tissue culture

• cultured mammalian cells are easily controlled system to investigate host-bacterium interactions — animals complex and variable

• cells grown in defined medium under reproducible conditions → improved reproducibility of results

• readily visualised by microscopy/sorted by flow cytometry

• cheaper and easier to maintain

limitations of tissue culture models

• many genes expressed in vivo will not be expressed in culture

• cultured cells lsoe normal distribution of surface antigens — pathogens may invade cultured cells derived from a tissue they do not normally infect

• cells in vivo are polarised — membranes on different sides of cells contain different sets of preteins; tissue culture monolayers not differentiated

biochemical approached to identify virulence factors

• bacterial protein toxins can be purified from filtered bacterial culture supernatants

• filtrated processed by chromatography and fractions can be tested for toxicity

• ion exchange columns — seperation based on charge

• gel filtetration columns — seperation based on molecular size

• amino acid sequence of a purified toxin can be determined using mass spec methods

molecular genetic approaches to identify virulence factors

• identify virulence genes via cloning and expressing them in avirulent bacterial strains

• very successful in some cases i.e cloning LLO haemolysin producitom from Listeria into avirulent lab strain of Bacillus

limitations of moleclar genetic approaches to identifying virulence factors

• works best if only one of a few contiguos genes give desired phenotype — standard cloning isolates only fragments of genome

• requires gene of interest to be expressed in E.coli or other avirulent bacterium being used

mutagenesis screening

• observe a phenotype in an organism

• mutagenize the organism

• isolate mutants that no longer have that phenotype

• work out what change(s) have occurred in the mutant’s DNA

• use traditional mutagens or transposon mutagenesis (popular now)

traditional mutagens

• chemicals i.e ethyl methan sulphonate (EMS) or ethidium bromide or UV irradiation

• produce random mutations in any gene → may cause too many DNA changes for easy analysis

transposon mutagenesis

• transposon insertion mutants are generated by nearly random insertion of a Tn into a pahtogens genome

• can generate a collection or library of mutants where each mutant contains a single Tn insertion and many of these insertions disrupt a gene

• resulting mutants are screened for loss of virulence

• Tn’s carry a selectable marker making it easier to find the mutated gene

limitations of transposon mutagenesis

• Tns can carry transcriptional terminators

• insertion mutants can only be obtained for genes that are not essential for growth in vitro → otherwise the mutant would not form a colony

• bacterial genes sometimes have a redundant function i.e if two homologues of a gene → knocking out one copy will not result in a mutant phenotype

iron acquisition as a bacterial virulence factor

• nearly all bacteria require iron for growth

• Iron deprivation is bacteriostatic — won’t grow without it

• extreme iron deficiency is bactericidal — kills bacteria

• Fe is central trace element in respiration and a co-factor in many enzymes

• Borrelia burgdorferi uses manganese instead of iron — lacks all iron dependent enzymes → virulence is ‘iron abstinence’

transferrin

• made in liver

• serum protein responsible for iron transport in blood and tissue

lactoferrin

• protein secreted at mucosal surfaces, found in milk, salive, tears

haemoglobin

• contains heme

• 70% of total body iron is in red blood cells

ferritin

• major protein used for intracellular iron storage

three main iron acquisition mechanisms

• siderophores

• direct contact

• 
  

siderophores

• low molecular weight compounds that chelate iron with very high affinity

• best studied of bacterial iron uptakke mechanisms

• wide spread among bacterial species

• two main chemical types — catechols and hydroxamates

is siderophore production a bacterial virulence factor

• not always

• loss of ability to produce siderophores ia sometimes associated with loss of virulence (i.e shown with mutagenesis fo E.coli, Pseudomonas etc)

• But Vibrio cholerae siderophore-negative mutatants are still virulent\

• many non-pathogens produce siderophores

why do bacteria produce more than one siderophore

• probably do different jobs in different environments or at different times during infection

• i.e aerobactin and enterobactin

• switching between apparently redundant iron uptake mechanisms is thought to be beneficial to bacterial survival → trading off efficiency versus cost of siderophore production

aerobactin (siderophore)

• induced rapidly, early stages of infection

• Fe affinity low (less efficient)

• re-usable

enterobactin (siderophore)

• induced slowly, when infection is established

• higher Fe affinity

• Non-reusable; degraded

Hemolysins

• pathogen lyses erythrocytes via hemolysin

• digests hemoglobin and assimilates the heme which contains iron

• i.e strep pneumo associtaed with pneumolysisn → binds cholesterol in host cell membranes and disrupts them by forming pores

• “systemic” e.coli produce alpha-hemolysin; faecal, non-invasive E.coli do not

exploitation of siderophores to deliver drugs into bacteria

• antibiotics can be covalently linked to siderophores

• following iron-scavenging → siderophore antibiotic hyrid is recognised by bacterial iron siderophore uptake pathways

• shown to be successfuul for cephosporins and other antibiotics

direct contact

bacteria bind directly to host transferrin or lactoferrin

• mechanism of iron uptake requires a number of membrane proteins

• bacteria have specific receptors for transferrin or lactoferrin of the hosts they infect — haemophilus influenzae binds to human transferrin

• at least 20 bacterial pathogens have this mechanism i.e Neisseria and Haemophilus well studied

colonisation

• establishment of stable bacterial population in host

• first critical step in establishment of infectious disease (for most but not all pathogens)

• epithelial tissues are primary sites of colonisation

potential outcomes of colonisation

• pathogen (mainly) confined to epithelial surfaces

• pathogen crosses epithelial surfaces → invades deepr tissues, some may cause systemic infection

examples of pathogens confined to epithelial surfaces

• Vibrio cholerae in intestinal tract → cholera

• Bordetella pertussis in repiratory tract → whooping cough

examples of pathogens that cross epithelial surfaces

• Salmonell enterica serover Typhi in intestinal tract → typhoid fever (systemic infection)

• Legionella pneumophila in respiratory tract → legionellosis (systemic infection)

Mucous

• secreted by goblet cells and subepithelial glands

• inhibit bacterial colonisation

• Major component — mucins (family of filamentous proteins)

• mucins bind to and trap bacterial pathogens → bacteria then expelled from body by physical mechanisms (i.e flushing)

glycosyl chains of mucins

• protect peptide from bacterial degradation

• are receptors for bacterial surface ligands

why must bacterial pathogens attach to host tissue

• overcome mechanical removal of bacterium from host by innate defences i.e flushing action of blinking, tearing, urination flushing action, etc

• allow colonisation and progression to disease

• often one of first critical steps in infection process

requirements of successful colonisation

• avoid being trapped in mucous layer

• penetrate mucous layer → reach epithelial cell surface via mobility or mucinases

• adhesion to epithelial cell receptors via specialised structures = adhesins

bacterial adhesins

• specialised surface structures that bind to specific host receptors

• “lock and key” interactions occur between adhesins and receptors

epithelial cell receptors

• glycoproteins

• glycolipids

• microbial adhesin binds to sugar moiety of receptor

Bacterial adhesion classes and their ligands

Fimbrial (pilus) adhesins

• Chaperone-Usher pili: P pili and Type 1 pili

• Type IV pili

• Curli pili

Afimbrial adhesins (non-fimbrial)

• Integrin and fibronectic-binding proteins

• autotransporters

• multi-valent adhesion molecules

afimbrial adhesins of gram negatives

• often outer member proteins (OMPs)

• OMPs of gram negs contribute to tighter binding following anchorage of the bacterium by pilus adhesion

• i.e neisseria meningitidis

• anchorage → tight adherence → invasion and transcytosis

pili (hair) / Fimbriae (fringe)

• hair like protein organelles

• 5-7 nm diameter

• 0.5-several micrometer long

• peritrichous or polar

• varied morphology

• a few to several hundred per cell

composition and structure of pili

• pilus shaft/rod made of protein subunits (pilins) assembled into helical array

• adhesive subunit(s) may be at pilus tip or along shaft

  • specialised tip structure (i.e. PapG adhesin at tip of Pap pili)

  • tip protein with no specialised structure

  • adhesive subunits along length of shaft

specialised tip structure of Pap pili

• Pap F, E, K proteins form a thin fibril at the end of the pilus

• PapG adhesin at tip of Pap pili, one monomer per pilus

Pap

• pyelonephritis associated pili

• associated with uropathogenic E.coli (UPEC)

• cause UTIs (bladder, kidney infection)

fibrillae morphology

• resemble pili but structure differs from pili

• finer fibrous organelles 2-3nm diameter

• long single multidomain protein attached at cell surface

  • an adhesive domain

  • multiple tandemly repeated domains making the ‘stalk’

• found widely in Gram +ve and -ve but less well-studied than pili

  • well-known fibrillae of streptococcus — M protein

receptor specificity of Pap pili of UPEC (UTIs)

• adhesin if PapG protein at pilus tip

• receptor — globotriasylceramide (a glycosphingolipid)

• gal-a1, 4-gal of receptor binds pilus tip

• tissue specific as uroepithelial cells (bladder & kidney) have this receptor

receptor specificity of type 1 pili of commensals and UPEC

• adhesin is FimH protein at pilus tip

• receptor — mannose-containing surface glycoproteins

• mannose exist on most mammalian cell types

• thus adhesion is tissue non-specific: type 1 pili were often considered unrelated to E.coli pathogenesis

• more recently found that UPEC strains attach to bladder epithelium via Type 1 pili — considered an important virulence factor for UPEC

chaperone-usher pili

• CU pili

• secreted into the periplasm by the sec transporter complex in the plasma membrane

• use chaperone proteins in the periplasm to reach OM → self-assemble into pilus

• most abundant type of pilus → often important virulence factors

• Pap pili (pap proteins), Type I pili (Fim proteins) of E. coli

type IV pili

• are assembled by large multisubunit complexes cross the whole bacterial cell envelope in a process requiring ATPase

• mediate ‘twitching motility’ → requires adherence to solid surface

• widespread in gram +ve and gram -ve pathogens

• type IV pili of N.gonorrhoeae, Ps. aeruginosa

Curli

• type of adhesive amyloid (insoluble polymeric protein stacked to form fibrils)

• 5-40% of species in biofilms possess adhesive amyloids

• Csg proteins secreted into the periplasm by the Sec tranporter complex in the plasma membrane

• CsgG forms a non-selective protein channel that allows diffusion of Csg proteins across OM

modern pilus classification

• based on genetics of pilus assembly systems

• based on sequence homology of genes encoding machinery required for pilus biogenesis

• so far proven more useful and reliable than earlier systems

• inclues CU pili, Type IV pili, Curli and F conjugative pili

Motility and flagella as bacterial virulence factors

• the capacity to move towards a host surface via chemotaxis could be beneficial to a pathogen

• number of cases where motility has been shown to be associated with virulene is increasing

• i.e helicobacter pylori, vibrio cholerae, salmonella and campylobacter spp.

integrins

superfamily of cell adhesion receptors

• transmembrane glycoproteins

• always have two components: alpha and beta chains (heterodimers)

• extracellular domain binds ligands

extracellular matrix proteins

• act as ligands

• include fibronectic, vitronectic, collagen, Iaminin

interaction of integrins with extracellular matrix proteins

triggers cytoskeleton changes via actin, controlling eukaryotic cell division, apoptosis, migration of cells, endocytosis to engulf food particle

Autotransporters

• family of diverse outer membran proteins (OMPs) in many gram -ve

• ‘self tranport’ through OM

• all share a conserved structure

  • Signal sequence SP directs protein to Sec system to cross the plasma membrane and enter periplasm

  • conserved C-terminal beta-barrel domain (anchor) inserts into OM to form pore

  • variable passenger domain with different functions in different spp. i.e adhesion — passes through pore; forms stalk and head structures

  • linker domain joins passenger and beta-barrel domains

multi-valent adhesion molecules (MAMs)

• participate in high affinity binding during early stages od infection by wide range of gram negative bacteria i.e e.coli, vibrio parahaemolyticus, shigella

• MAMs contain a N-terminal hydrophic region connected to 6 or 7 binding domains

bacterial flagella

• majority of motile prokaryotes are motile this way → allows to navigate in their microenvironment

• thread like appendages extending from the plasma membrane and cell wall

• slender rigid structures (20nm across and up to 20nm long) → not straight (helical) with constant and species characteristic wavelength

• filament rotation acts like propeller of a boat

flagellar arrangment

• monotrichous

• amphitrichous

• lophotricous

• peritrichous

monotrichous

single “hair”, polar flagellum

amphitrichous

“on both sides”; single flagella at opposite ends of cell

lophotrichous

“tuft”; flagella cluster at one or both ends

peritrichous

“around”; flagella spread over cell-surface

flagellar structure

• similar amongst bacteria and archaea

• filament

• hook

• basal body

filament

• hollow, rigid cylinder composed of ~20000 flagellin protein molecules

• synthesised by transfer of flagellin subunits through the hollow filament (spontaneous aggregation occurs)

• over 40 genes (fla, fli and flg) control synthesis and motility

• flagellum synthesis involves 20-30 genes

hook

composed of protein and acts as a flexible coupling between the filament and basal body

basal body

• attaches flagellum to the cell

• functions as the flagellum motor

• consists of rings attached to a central rod

basal body of gram negative bacteria

• four rings are connected to the central rod

  • L (lipopolysaccharide) ring

  • P (peptidoglycan) ring

  • MS (plasma membrane) ring

  • C ring attaches to the MS ring on the cytoplasmic side (a newer discovery, not shown in all figs)

• P and L rings act like bearings

• MS and C rings act as a motor — rotate within the membrane; turn the attached rod and filament

polar flagella

• cells swim forward or backward by reversing flagellar rotation (CCW rotaiton pushes, CW rotation pulls the cell)

peritrichous flagella

• counterclockwise rotation causes running

• clockwise rotation causes tumbling (dirupts a run)

• cell moves off in a new direction

flagella-mediated motility

• flagellar motor can rotate very rapidly

• i.e vibrio alginolyticus averages 1100 rps

• in a homogenous environment

  • the direction of bacterial motility is random

  • straight runs are interspersed with tumblems that result in a random directional changes when running resumes

taxis responses

• directed movement to attractants and from repellents

• chemotaxis, aerotaxis, phototaxis, thermotaxis, osmotaxis, magnetotaxis

helicobactr pylori motility as bacterial virulence factors

• causes inflammation of the stomach lining

• linked to gastric ulcers and stomach cancer

• call have tuft of 4-7 flagella - lophotrichous

• flagellum-negative fla-mutants are unable to colonise gnotobiotic (germ-free) piglets’ stomachs

vibrio cholerae motility as bacterial virulence factors

• infects small intestine → causing profuse watery diarrhea, vomiting, rapid dehydration, electrolyte loss

• cells comma-shaped, have single polar flagellum

• flagellum-negative (Fla^-) mutants had reduced virulence in animal models

invasiveness

• ability of a microbe to enter a host, grow and reproduce within the host and spread throughout the body

• entry and survival within phagocytic cells

• ability to enter non-phagocytic host cells i.e epithelial cells

collagenase

• clostridium

• breaks down collagen that forms connective tissues

fibrinolysin (staphylokinase)

• staphylococcus

• digests fibrin clots that form a mesh cover over plateley plugs at wound sites

hyaluronidase

• streptococcus

• depolymerises hyaluronic acid — the interstitial cement of connective tissue

DNase

• clostridium, staphylococcus, streptococcus

• destroys DNA, lowers viscosity of exudates/pus which enhances bacterial motility

how does invasion benefit the bacterial pathogen

• provides safe environment for bacterial growth

• pathogen gains nutrients from host

• no competition for nutrients from other microbes

• pathogen can evade host immune systemm

• pathogen can avoid antibiotics

two mechanisms for invasion of non-phagocytic cells

• zipper mechanism

• trigger mechanism

zipper mechanism

• bacterial invasins bind to host cell receptors at a number of points along the cells

• actin polymerization is triggered and bacteria are surrounded by tight-fitting pseudopodia and engulfed into phagosome

trigger mechanism

• bacteria inject molecules into host cell via Type III secretion apparatus

• these activate changes to host cell’s actin cytoskeleton causing membrane ruffles or “splash” → leading to uptake of bacterial cell by host cell

toxin

a microbial product or component that injures another cell or organism

• first bacterial virulence factors to be identified and studied in detail

• there is a huge diversity in toxin propertioes and modes of action

• inactivated form as vaccines (toxoids), as treatments (botox), as tool in cell biology

how are toxins classified

• endotoxin

• exotoxin

endotoxin

heat-stable lipid A portion of the lipopolysaccharide (LPS) of gram-neg bacteria, within the outer membrane

exotoxin

heat-labile protein that is usually released (secreted) from the bacterial cell

• term is gradually falling out of favour, some protein toxins are not secreted but released by cell lysis.

• others are injected directly as effectors from the bacterial cell into the human cell by secretion systems and bypass the extracellular fluid

adenylate cyclase

• toxin of bordetella pertussis enters eukaryotic cells and produces cAMP

• the cell loses abiliy to control flow of ions and also immune response is supressed

lecithinase/phospholipase C

• alpha toxin of clostridium perfringens (gas gangrene) hydrolyses lecithin in mammalian cell membranes

• an expanding zone of dead tissue results

how are extoxins names

• named for species that produces them/disease they are associated with

• named according to their enzymatic activity

• named according to the host cell types they attack

how are exotoxins detected and quantitated

• toxins can be assayed on appropriate transformed cell lines

  • limitation — cell line may not be relevant to infection in vivo

  • concentration of purified toxin used is often much higher than that produced during infection

• toxins can be directly inoculated into animals

  • toxin may not necessarily cause a similar effect as in human effection

  • a relevant animal mode may be available

  • expensive method, ethical considerations

Type I toxins

• bind to a host cell surface but do not enter host cell - act extracellularly

• secreted proteins → massive, non-specific inflammatory response

superantigens

• bind directly to MHC class II molecules on the surface of macrophages

• also bind to T cell receptors that interact with MHC

• many more macrophage — T cell pairs form than normal

• high levels of cytokines are released by the T-cells, causing nausea, vomiting, fever

staphylococcus type I toxins

• toxic shock syndrome toxin (TSST-1) — causes toxic shock; associated with tampons

• staphylococcus enterotoxins A-I: food poisoning - nausea vomiting

streptococcus pyogenes

• Spe exotoxins — streptococcal toxic shock syndrome

• Spe exotoxins — necrotising fasciitis

type II toxins

• act on eukaryotic cell membranes and detroy their integrity

• pore forming cytotoxins (PFTs)

• phospholipase enzymes that damage cell membranes

• many are also hemolysins and can be asayed conveniently using red blood cells

• many are toxic to many cell types → ‘cytolysin’ often used