Major advisor Parampal Sahota Senior Microbiologist

Vainy Garg M.Sc Microbiology L-2011-BS-255-M

Capsule Lipid A Adhesins Invasins Toxins Plasmids Bacteriophages Infecting dose Route of infection Communicability Bacterial appendages

CAPSULE produce inflammatory cytokines. e.g. Streptococcus pneumoniae, Neisseria

meningitidis, and Pseudomonas aeruginosa.

differences in capsular polysaccharide chemical structure determine the meningococcal serogroups.

CELL WALL : contain toxic components involved in

bacterial septic shock, collapse of the circulatory system and multiple organ system failure.

Acts via the initiation of an inflammatory response through the stimulation of monocytes and macrophages.

ADHESINS :

Adherence of the pathogen to host surfaces.

MECHANISMS OF ADHERENCE TO CELL OR TISSUE SURFACES

Possible interactions and forces involved are:

hydrophobic interactions

electrostatic attractions

atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies

Brownian movement

recruitment and trapping by biofilm polymers interacting with the bacterial glycocalyx (capsule)

Receptor-ligand interactions promotes adherence involves protein-protein and protein-carbohydrate interactions.

molecules serve as host receptors for microbes include membrane-spanning proteins, surface immunoglobulin, glycolipids, glycoproteins, and extracellular matrix proteins.

Enterotoxigenic E. coli

Type-I fimbriae Intestinal epithelium

Diarrhea

Uropathogenic E. coli

Type I fimbriae Urethral epithelium

Urethritis

Uropathogenic E. coli

P-pili (pap)Upper urinary

tract Pyelonephritis

Bordetella pertussis

Fimbriae (filamentous

hemagglutinin)

Respiratory epithelium

Whooping cough

V. choleraeN-methylphenyl- alanine pili

Intestinal epithelium

Cholera

Treponema pallidum

Peptide in outer membrane

Mucosal epithelium

Syphilis

INVASION : Pathogens gain deeper access into the host to perpetuate the infection

cycle.

Extracellular invasion : allows pathogens to proliferate tissues, disseminate to other sites in the body, express toxins, and initiate inflammatory responses.

E.g. : β hemolytic streptococcus and S. aureus- secrets hyaluronidase streptokinase and staphylokinase, lipase, and nuclease .

Intracellular invasion : microbe penetrates the cells of host tissue and survives within this environment.

E.g. : Chlamydia spp, Rickettsia spp and Mycobacterium leprae : obligate intracellular lifecycle that requires mammalian cell for growth.

Invasin Activity

Hyaluronidase Degrades hyaluronic of connective tissue

Collagenase Dissolves collagen framework of muscles

Neuraminidase Degrades neuraminic acid of intestinal mucosa

Coagulase Converts fibrinogen to fibrin which causes clotting

Kinases Converts plasminogen to plasmin which digests fibrin

LeukocidinDisrupts neutrophil membranes and causes discharge of

lysosomal granules

StreptolysinRepels phagocytes and disrupts phagocyte membrane and

causes discharge of lysosomal granules

HemolysinsPhospholipases or lecithinases that destroy red blood cells

(and other cells) by lysis

Lecithinases Destroy lecithin in cell membranes

Phospholipases  Destroy phospholipids in cell membrane

Anthrax EF One component (EF) is an adenylate cyclase which causes

increased levels of intracellular cyclic AMP 

Pertussis ACOne toxin component is an adenylate cyclase that acts

locally producing an increase in intracellular cyclic AMP

Extracellular bacterial proteins that are considered invasins

Toxigenicity :

Exotoxins and Endotoxins

Classification of exotoxins:a. Membrane damaging toxins:

destroy the host membranes by inducing pore formation or by destabilising cytoplasmic membranes.

b. Superantigens: bind non-specifically, activate large numbers of T cells leading to shock.

c. A-B component toxins : The B component binds to specific host cell receptors and A is enzymaticcally active portion of the toxin.

PLASMIDS: Gene coding for virulent characteristics can be plasmidborne.

E.g. surface antigens responsible for colonisation of intestinal mucosa by E. coli and enterotoxin production by E. coli and S. aureus.

BACTERIOPHAGES : In diphtheria bacilli, gene for toxic production is present in beta or other toxic corynephages.

COMMUNICABILITY : Ability of parasite to spread from one host to another.

Highly virulent parasite may not exhibit high degree of communicability due to rapid lethal effect on host. E.g. respiratory and intestinal diseases.

BACTERIAL APPENDAGES: Bacterial surface antigens such as Vi antigen of S typhi and K antigens of E coli : prevents bacteria from phagocytosis and lytic activity of compliments.

INFECTING DOSE : Dosage estimated as minimum infecting dose (MID) or minimum lethal dose (MLD)

Route of infection : Modes by which different bacteria able to initiate tissue damage.

Cholera vibrios infect orally and unable to cause infection subcutaneously.

• SECRETED OR SURFACE-EXPOSED BACTERIAL PROTEINS IN BACTERIAL-HOST INTERACTIONS.

• Proteins secreted by the Type I system cross directly from the cytoplasm to the cell surface, bypassing the general secretory pathway completely.

• Type II-secreted proteins use the general secretory pathway to reach the periplasm and traverse the outer membrane through distinct channel proteins.

• Type III system : effector molecules move to the external surface of the bacterium, facilitate the pathogen’s ability to survive and replicate.

colonization of the intestinal tract, and penetrate M cells of Peyer’s patches.

replicate in extracellular form within micro-abscesses.

form microcolonies and resistant to phagocytosis by macrophages and neutrophils (Fabrega et al 2011).

six biotypes differentiated by physiochemical and biochemical tests (1A, 1B, 2, 3, 4, and 5)

more than 50 serotypes differentiated by antigenic variation in cell wall lipopolysaccharide.

virulent biotypes 1B and 2–5 has highly conserved 70-kb virulence plasmid, termed pYV/pCD and certain chromosomal genes.

The biotype 1A lack pYV plasmid encodes virulence factors {Yersinia adhesin A (YadA) and Ysc-Yop type III secretion system (TTSS)} and chromosomally borne virulence genes {ail, myfA, ystA, ysa, high pathogenicity island- (HPI-)}

Biotype 1B carry high-pathogenicity island (HPI),facilitates the uptake and utilization of iron by bacterial cells, promote growth under iron-limiting conditions in host tissues.

Virulence-associated determinants of pYV-negative strains includes:

cell surface lipopolysaccharide SodA (a superoxide dismutase) : bacterial survival

in tissues. urease, enhances bacterial resistance to stomach

acid and in nitrogen assimilation.

pYV, antihost plasmid, resist phagocytosis and complement-mediated lysis, to proliferate extracellularly in tissues.

virulence factors, outer membrane protein adhesin, YadA, and type III protein secretory apparatus translocates effector proteins (Ysc-Yops) from bacterial cell to the cytoplasm of susceptible host cells.

Adaptation : Yersinia adapt surface antigenic structures (outer membrane proteins) to colonize in the intestines of humans at temperature of 37◦C through 70-kb virulence plasmid (pYV).

Adhesion : allow intimate attachment to the epithelial cells.

YadA, a pYV plasmid-encoded protein, adhesion for attachment, induction of disease (e.g., inflammation and necrosis in the liver).

mediates adherence to epithelial cells, phagocytes and extracellular matrix components, and protects the bacterium killed by neutrophils.

Fimbriae present in biotype1A : MR/Y-HA : 8 nm in diameter, agglutinates

erythrocytes in the presence of mannose and expressed in vitro at low temperature.

MR/K like HA : 4 nm in diameter and mediates mannose resistant hemagglutination of chicken erythrocytes.

Y. enterocolitica produces Myf (for mucoid Yersinia fibrillae), bestows mucoid appearance on bacterial colonies. (Sabina et al 2011)

Invasion :

Ail (attachment-invasion locus) : Localized in the OM. Eight transmembrane β-sheets and four cell surface-exposed loops,

and the extracellular loop for Ail-mediated binding to host cells. Promotes Yop delivery into the primary target of T3SS--the

phagocytic cells and into epithelial cells.

Invasin : Located in middle of gene cluster encoding the flagella proteins ~100 kDa, anchored by its amino-terminal region Receptor is β1 integrin, intergrins couple extracellular adhesion

events to numerous signaling pathways, and the bacterium is taken up by zipper mechanism.

Mechanisms of bacterial epithelial cell internalization :

“zippering” process : tight enclosing of the bacterial cell by the mammalian cell membrane.

involving surface bound bacterial protein invasin (Inv) binding an integrins of the β1 family of mammalian cell surface.

TOXINS : ystA, ystB : heat-stable enterotoxin. causes Yersinia associated diarrhea. stimulate cGMP synthesis in the intestinal brush border,

leading to fluid loss and lack of fluid absorption.

Pathogenicity islands in Yersinia : HPI capture the iron molecules for systemic dissemination of

the bacteria in the host via yersiniabactin.

LOCAL AND SYSTEMIC DISSEMINATION

Cross the intestinal epithelium through FAE (follicle associated epithelial cell), in the Peyer’s patches of the ileum.

Invasin (Inv), a 103 kDa outer membrane protein of Yersinia binds

β1 integrins that are expressed apically on M cells.

Yersinia surface proteins (Ail, PsaA, and YadA ) account for residual invasion of inv mutants.

Yersinia defend attack by resident macrophages by expressing an antiphagocytic strategy.

mediated by plasmid encoded type III secretion, of three protein effectors, YopH, T, and E, disrupt cytoskeletal assembly required for phagocytosis process.

Extracellular in infected Peyer’s patches and mesenteric lymph nodes and disseminate to cause local and systemic infection.

When bacteria bind to tissue culture cells, 10 different effector molecules secreted and three injected into cells.

YopE and YopH, modify macrophage proteins to destroy the cells abilities to engulf and kill bacteria.

immune cells neutralized by effector molecules, enables Yersinia spp to flourish in the reticuloendothelial environment.

• capture the iron molecules for systemic dissemination in the host.

• Yeriniabactin, sub-group of phenolate siderophores and has affinity for ferric iron.

•FyuA/Psn-Irp system uses yersiniabactin, a siderophore that remove iron from mammalian proteins.

• YbtA, AraC-like regulator required for transcription of fyuA/psn, irp2 and ybtP, downregulate its own transcription.

• In the presence of iron, Fur, a cytosolic protein, bind ferrous iron, changes conformation and binds DNA at specific site called Fur box, preventing transcription, downregulates transcription of fyuA/psn, irp2 and other iron-regulated genes.

classified into 96 serogroups and the O-antigen LPS of A. hydrophila 0:34 strains in adhesion to HEp-2 cells.

attaches and enters into host cells through production of flagella, pili and adhesins.

multiplication in host tissue by production of siderophores and outer membrane proteins

enterotoxins, proteases, phospholipases, and hemolysins cause damage to host cells leading to cell death.

directed locomotion attachment to

gastrointestinal epithelium

biofilm formation colonization elaboration of virulence

factors infection (Janda et al

2010).

Enterotoxins—cytotonic and cytotoxic. Cytotonic enterotoxins (heat-labile (Alt) and heat-

stable (Ast)), donot degenerate crypts and villi of small intestine.

Cytotoxic enterotoxin (Act) result in extensive damage to epithelium.

Aerolysin, extracellular, soluble, hydrophilic protein exhibiting hemolytic and cytolytic properties.

The mature form of Act is 49 kDa in size and involved in hemolytic, cytotoxic, enterotoxic and lethality of mice.

The capsular gene cluster 17,562 bp long. include 13 genes assembled into three distinct regions. Regions I and III: four- and two-capsule transport

genes region II : five genes.

type IV pili (bundle-forming pili (Bfp) and Tap (type IV Aeromonas pili)) associated with gastroenteritis.

Bfp promote colonization by forming bacterium-to-bacterium linkages.

The Tap biogenesis gene cluster : four genes (tapABCD) -

tapA gene encode subunit protein tapB and tapC genes involved in pilus biogenesis tapD gene encoded type IV prepilin peptidase/N-

methyltransferase.

Nine lateral flagellar genes (lafA,B,C,E,F,S,T,U,X) : lateral flagella distinct from the polar flagellum and involved in swarming motility

Entry of water from the external milieu into erythrocytes through the pores, resulting in cell swelling and subsequent lysis.

Preincubation of the toxin with cholesterol result in dose-dependent reduction in hemoglobin release from erythrocytes.

Act interact with cholesterol on the membranes of erythrocytes

aggregation occurred resulting in transmembrane pore formation and cytolysis

of erythrocytes.

production in two precursor forms (pre-protoxin)

conversion to an active toxin by removal of a 23-aa-long NH2-terminal signal peptide (protoxin)

proteolytic cleavage of the protoxins at their carboxy-terminal end (removal of 4–5 kDa peptide) to form a mature, biologically active toxin.

their characteristics of punching holes in the membranes.

divert some of the metal ions to microbial metabolism.

produce siderophores, enterobactin or amonabactin.

amonabactin producers -siderophore-dependent and independent means for iron acquisition.

enterobactin producers- nonsiderophore heme utilization.

The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore bound to the receptor.

CRYSTAL VIOLET BINDING TEST

Virulent plasmid bearing colonies (P+) : dark violet by binding of crystal violet dye

Plasmidless (P-) colonies remained white as they could not bind the crystal violet dye.

CONGO-RED BINDING TEST

Plasmid bearing strains : red colonies

Plasmidless strains : colourless to pale pink colonies.

Bhaduri et al (1987).

Riley and Toma (1989).

LIPASE TEST egg yolk agar medium. Colonies iridescent and pearl like surrounded by a precipitation

ring and a clear zone.

AUTOAGGLUTINATION TEST

Test organisms grown in MR-VP tubes.

Agglutination positive (Agg+) : Flocculation of irregularly edged layer of a agglutinated bacteria at the bottom of the tube with clear supernatant fluid.

Agg- : smooth round pellet at bottom.

Autoagglutination test

Laird and Cavanaugh (1989).

DEOXYRIBONUCLEASE (DNase) TEST

The test and positive control organism (Staphylococcus aureus) were examined for appearance of clear zone around the colonies within 5 min of adding HCl which indicated positive test.

PROTEASE PRODUCTION The protease production was estimated by the formation of a

clear zone caused by casein degradation. HEMOLYSIN PRODUCTION Young test culture (3-4 hour old) was streaked on to the sheep

blood agar plates (5%) and observe for hemolysis zone. ESCULIN HYDROLYSIS

Protease production

Esculin producion

PYRAZINAMIDASE ACTIVITY

Pink colour indicate the presence of pyrazinoic acid and were pyrazinamidase positive (Pyz+).

Pyz- strains were positive for virulence.

SIDEROPHORE PRODUCTION Succinate media with CAB dye was

prepared and yellow to orange color zone showed positive for siderophore production. Yellow colored zones showing

siderophore production

Kandola and Wauters (1985).

Plasmid based phenotypic tests

Aeromonas Yersinia

Crystal violet binding test Violet coloured colonies Violet coloured colonies

Congo red binding test Red coloured colonies Red coloured colonies

Lipase test Clear zone formed around colonies

Clear zone formed around colonies

Autoagglutination test An irregular edged layer of aggglutinated bacteria which formed flocculate covering at the bottom of tube with clear supernatant fluid.

An irregular edged layer of aggglutinated bacteria which formed flocculate covering at the bottom of tube with clear supernatant fluid.

Deoxyribonuclease (Dnase) test

No clear zone formed. No clear zone formed.

Aeromonas Yersinia

Capsular polysaccharide Positive Positive

Cell surface hydrophobicity

Positive Positive

Protease production Positive Positive

Hemolysin production Positive Positive

Lipopolysaccharide production

Positive Positive

Siderophore production Positive Positive

Esculin hydrolysis Positive Positive