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virulent characteristics of emerging pathogens virulent characteristics of emerging pathogens Presentation Transcript

  • Major advisorParampal SahotaVainy GargM.Sc MicrobiologyL-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, Neisseriameningitidis, and Pseudomonasaeruginosa. differences in capsular polysaccharidechemical structure determine themeningococcal serogroups. CELL WALL : contain toxic components involved inbacterial septic shock, collapse of thecirculatory system and multiple organsystem failure. Acts via the initiation of an inflammatoryresponse through the stimulation ofmonocytes and macrophages.
  •  ADHESINS : Adherence of the pathogen to host surfaces.MECHANISMS OF ADHERENCE TO CELL ORTISSUE SURFACES Possible interactions and forces involvedare: hydrophobic interactions electrostatic attractions atomic and molecular vibrations resulting fromfluctuating dipoles of similar frequencies Brownian movement recruitment and trapping by biofilm polymersinteracting with the bacterial glycocalyx (capsule)
  •  Receptor-ligandinteractions promotesadherence involves protein-protein and protein-carbohydrate interactions. molecules serve as hostreceptors for microbesinclude membrane-spanningproteins, surfaceimmunoglobulin, glycolipids,glycoproteins, andextracellular matrix proteins.
  • Enterotoxigenic E.coliType-I fimbriaeIntestinalepitheliumDiarrheaUropathogenicE. coliType I fimbriaeUrethralepitheliumUrethritisUropathogenicE. coliP-pili (pap) Upper urinary tract PyelonephritisBordetella pertussisFimbriae (filamentoushemagglutinin)RespiratoryepitheliumWhoopingcoughV. choleraeN-methylphenyl-alanine piliIntestinalepitheliumCholeraTreponemapallidumPeptide in outermembraneMucosalepitheliumSyphilis
  •  INVASION : Pathogens gain deeper access into the host to perpetuate the infectioncycle. Extracellular invasion : allows pathogens to proliferate tissues,disseminate to other sites in the body, express toxins, and initiateinflammatory responses. E.g. : β hemolytic streptococcus and S. aureus- secrets hyaluronidasestreptokinase and staphylokinase, lipase, and nuclease . Intracellular invasion : microbe penetrates the cells of host tissue andsurvives within this environment. E.g. : Chlamydia spp, Rickettsia spp and Mycobacterium leprae : obligateintracellular lifecycle that requires mammalian cell for growth.
  • Invasin ActivityHyaluronidase Degrades hyaluronic of connective tissueCollagenase Dissolves collagen framework of musclesNeuraminidase Degrades neuraminic acid of intestinal mucosaCoagulase Converts fibrinogen to fibrin which causes clottingKinases Converts plasminogen to plasmin which digests fibrinLeukocidinDisrupts neutrophil membranes and causes discharge oflysosomal granulesStreptolysinRepels phagocytes and disrupts phagocyte membrane andcauses discharge of lysosomal granulesHemolysinsPhospholipases or lecithinases that destroy red blood cells(and other cells) by lysisLecithinases Destroy lecithin in cell membranesPhospholipases Destroy phospholipids in cell membraneAnthrax EFOne component (EF) is an adenylate cyclase which causesincreased levels of intracellular cyclic AMPPertussis ACOne toxin component is an adenylate cyclase that actslocally producing an increase in intracellular cyclic AMPExtracellular bacterial proteins that are considered invasins
  •  Toxigenicity : Exotoxins and Endotoxins Classification of exotoxins:a. Membrane damaging toxins:destroy the host membranes byinducing pore formation or bydestabilising cytoplasmicmembranes.b. Superantigens: bind non-specifically, activate large numbersof T cells leading to shock.c. A-B component toxins : The Bcomponent binds to specific hostcell receptors and A isenzymaticcally active portion of thetoxin.
  •  PLASMIDS: Gene coding for virulent characteristics can beplasmidborne. E.g. surface antigens responsible for colonisation of intestinalmucosa by E. coli and enterotoxin production by E. coli and S.aureus. BACTERIOPHAGES : In diphtheria bacilli, gene for toxicproduction is present in beta or other toxic corynephages. COMMUNICABILITY : Ability of parasite to spread fromone host to another. Highly virulent parasite may not exhibit high degree ofcommunicability due to rapid lethal effect on host. E.g.respiratory and intestinal diseases. BACTERIAL APPENDAGES: Bacterial surface antigenssuch as Vi antigen of S typhi and K antigens of E coli : preventsbacteria from phagocytosis and lytic activity of compliments.
  •  INFECTING DOSE :Dosage estimated asminimum infecting dose(MID) or minimum lethaldose (MLD) Route of infection : Modesby which different bacteriaable to initiate tissuedamage. Cholera vibrios infect orallyand unable to cause infectionsubcutaneously.
  • • SECRETED OR SURFACE-EXPOSEDBACTERIAL PROTEINS IN BACTERIAL-HOST INTERACTIONS.• Proteins secreted by the Type I system crossdirectly from the cytoplasm to the cell surface,bypassing the general secretory pathwaycompletely.• Type II-secreted proteins use the generalsecretory pathway to reach the periplasm andtraverse the outer membrane through distinctchannel proteins.• Type III system : effector molecules move to theexternal surface of the bacterium, facilitate thepathogen’s ability to survive and replicate.
  •  colonization of theintestinal tract, andpenetrate M cells ofPeyer’s patches. replicate in extracellularform within micro-abscesses. form microcolonies andresistant to phagocytosisby macrophages andneutrophils (Fabrega et al2011).
  •  six biotypes differentiated by physiochemical andbiochemical tests (1A, 1B, 2, 3, 4, and 5) more than 50 serotypes differentiated by antigenicvariation in cell wall lipopolysaccharide. virulent biotypes 1B and 2–5 has highly conserved70-kb virulence plasmid, termed pYV/pCD andcertain chromosomal genes.
  •  The biotype 1A lack pYV plasmid encodes virulencefactors {Yersinia adhesin A (YadA) and Ysc-Yop typeIII secretion system (TTSS)} and chromosomallyborne virulence genes {ail, myfA, ystA, ysa, highpathogenicity island- (HPI-)} Biotype 1B carry high-pathogenicity island(HPI),facilitates the uptake and utilization of iron bybacterial cells, promote growth under iron-limitingconditions in host tissues.
  •  Virulence-associated determinants of pYV-negativestrains includes: cell surface lipopolysaccharide SodA (a superoxide dismutase) : bacterial survivalin tissues. urease, enhances bacterial resistance to stomachacid and in nitrogen assimilation.
  •  pYV, antihost plasmid, resistphagocytosis and complement-mediated lysis, to proliferateextracellularly in tissues. virulence factors, outermembrane protein adhesin,YadA, and type III proteinsecretory apparatustranslocates effector proteins(Ysc-Yops) from bacterial cellto the cytoplasm of susceptiblehost cells.
  •  Adaptation : Yersinia adapt surface antigenic structures(outer membrane proteins) to colonize in the intestines ofhumans at temperature of 37◦C through 70-kb virulenceplasmid (pYV). Adhesion : allow intimate attachment to the epithelialcells. YadA, a pYV plasmid-encoded protein, adhesion forattachment, induction of disease (e.g., inflammation andnecrosis in the liver). mediates adherence to epithelial cells, phagocytes andextracellular matrix components, and protects thebacterium killed by neutrophils.
  •  Fimbriae present in biotype1A : MR/Y-HA : 8 nm in diameter, agglutinateserythrocytes in the presence of mannose andexpressed in vitro at low temperature. MR/K like HA : 4 nm in diameter and mediatesmannose resistant hemagglutination of chickenerythrocytes. Y. enterocolitica produces Myf (for mucoid Yersiniafibrillae), bestows mucoid appearance on bacterialcolonies. (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--thephagocytic 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 adhesionevents to numerous signaling pathways, and the bacterium is takenup by zipper mechanism.
  •  Mechanisms of bacterialepithelial cell internalization: “zippering” process : tightenclosing of the bacterial cellby the mammalian cellmembrane. involving surface boundbacterial protein invasin (Inv)binding an integrins of the β1family of mammalian cellsurface.
  •  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 ofthe bacteria in the host via yersiniabactin.
  •  LOCAL AND SYSTEMIC DISSEMINATION Cross the intestinal epithelium through FAE (follicle associatedepithelial cell), in the Peyer’s patches of the ileum. Invasin (Inv), a 103 kDa outer membrane protein of Yersinia bindsβ1 integrins that are expressed apically on M cells. Yersinia surface proteins (Ail, PsaA, and YadA ) account forresidual invasion of inv mutants. Yersinia defend attack by resident macrophages by expressing anantiphagocytic strategy. mediated by plasmid encoded type III secretion, of three proteineffectors, YopH, T, and E, disrupt cytoskeletal assembly requiredfor phagocytosis process. Extracellular in infected Peyer’s patches and mesenteric lymphnodes and disseminate to cause local and systemic infection.
  •  When bacteria bind to tissueculture cells, 10 differenteffector molecules secretedand three injected into cells. YopE and YopH, modifymacrophage proteins todestroy the cells abilities toengulf and kill bacteria. immune cells neutralized byeffector molecules, enablesYersinia spp to flourish in thereticuloendothelialenvironment.
  • • capture the iron molecules for systemic disseminationin the host.• Yeriniabactin, sub-group of phenolate siderophores andhas affinity for ferric iron.•FyuA/Psn-Irp system uses yersiniabactin, a siderophorethat remove iron from mammalian proteins.• YbtA, AraC-like regulator required for transcriptionof fyuA/psn, irp2 and ybtP, downregulate its owntranscription.• In the presence of iron, Fur, a cytosolic protein, bindferrous iron, changes conformation and binds DNA atspecific site called Fur box, preventing transcription,downregulates transcription of fyuA/psn, irp2 and otheriron-regulated genes.
  •  classified into 96 serogroups and the O-antigenLPS of A. hydrophila 0:34 strains in adhesion toHEp-2 cells. attaches and enters into host cells throughproduction of flagella, pili and adhesins. multiplication in host tissue by production ofsiderophores and outer membrane proteins enterotoxins, proteases, phospholipases, andhemolysins cause damage to host cells leading tocell death.
  •  directed locomotion attachment togastrointestinal epithelium biofilm formation colonization elaboration of virulencefactors infection (Janda et al
  •  Enterotoxins—cytotonic and cytotoxic. Cytotonic enterotoxins (heat-labile (Alt) and heat-stable (Ast)), donot degenerate crypts and villi ofsmall intestine. Cytotoxic enterotoxin (Act) result in extensivedamage to epithelium. Aerolysin, extracellular, soluble, hydrophilic proteinexhibiting hemolytic and cytolytic properties. The mature form of Act is 49 kDa in size andinvolved in hemolytic, cytotoxic, enterotoxic andlethality 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 transportgenes region II : five genes. type IV pili (bundle-forming pili (Bfp) and Tap (typeIV 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 distinctfrom the polar flagellum and involved in swarmingmotility
  •  Entry of water from the external milieu intoerythrocytes through the pores, resulting in cellswelling and subsequent lysis. Preincubation of the toxin with cholesterol result in dose-dependent reduction in hemoglobin release fromerythrocytes. Act interact with cholesterol on the membranes oferythrocytes aggregation occurred resulting in transmembrane pore formation and cytolysisof 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 theircarboxy-terminal end (removal of 4–5 kDapeptide) to form a mature, biologically activetoxin. their characteristics of punching holes in themembranes.
  •  divert some of the metal ions tomicrobial metabolism. produce siderophores,enterobactin or amonabactin. amonabactin producers-siderophore-dependent andindependent means for ironacquisition. enterobactin producers-nonsiderophore hemeutilization.
  • The ligand exchange step occurs at the cell surface and involves the exchange of ironfrom a ferric siderophore to an iron-free siderophore bound to the receptor.
  •  CRYSTAL VIOLET BINDINGTEST Virulent plasmid bearingcolonies (P+) : dark violet bybinding of crystal violet dye Plasmidless (P-) coloniesremained white as they could notbind the crystal violet dye. CONGO-RED BINDING TEST Plasmid bearing strains : redcolonies Plasmidless strains : colourlessto 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 precipitationring and a clear zone. AUTOAGGLUTINATIONTEST Test organisms grown in MR-VPtubes. Agglutination positive (Agg+) :Flocculation of irregularly edgedlayer of a agglutinated bacteria atthe bottom of the tube with clearsupernatant fluid. Agg-: smooth round pellet atbottom.Autoagglutination testLaird and Cavanaugh (1989).
  •  DEOXYRIBONUCLEASE (DNase) TESTThe test and positivecontrol organism(Staphylococcus aureus)were examined forappearance of clear zonearound the colonies within5 min of adding HClwhich indicated positivetest.
  •  PROTEASE PRODUCTION The protease production was estimated by the formation of aclear zone caused by casein degradation. HEMOLYSIN PRODUCTION Young test culture (3-4 hour old) was streaked on to the sheepblood agar plates (5%) and observe for hemolysis zone. ESCULIN HYDROLYSISProtease production Esculin producion
  •  PYRAZINAMIDASE ACTIVITY Pink colour indicate the presence ofpyrazinoic acid and werepyrazinamidase positive (Pyz+). Pyz-strains were positive forvirulence. SIDEROPHORE PRODUCTION Succinate media with CAB dye wasprepared and yellow to orange colorzone showed positive forsiderophore production. Yellow colored zones showingsiderophore productionKandola and Wauters (1985).
  • Plasmid based phenotypictestsAeromonas YersiniaCrystal violet binding test Violet coloured colonies Violet coloured coloniesCongo red binding test Red coloured colonies Red coloured coloniesLipase test Clear zone formed aroundcoloniesClear zone formed aroundcoloniesAutoagglutination test An irregular edged layerof aggglutinated bacteriawhich formed flocculatecovering at the bottom oftube with clearsupernatant fluid.An irregular edged layer ofaggglutinated bacteria whichformed flocculate covering atthe bottom of tube with clearsupernatant fluid.Deoxyribonuclease(Dnase) testNo clear zone formed. No clear zone formed.
  • Aeromonas YersiniaCapsular polysaccharide Positive PositiveCell surfacehydrophobicityPositive PositiveProtease production Positive PositiveHemolysin production Positive PositiveLipopolysaccharideproductionPositive PositiveSiderophore production Positive PositiveEsculin hydrolysis Positive Positive