L7 - Adherence and Colonisation slides 2024-1.pdf

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COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 Warning This material has been reproduced and communicated to you by or on behalf of the University of Melbourne pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further copying or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice  Bacterial Pathogenesis: Adherence and Colonisation MIIM30011 Dr Aimee Tan [email protected] 2  Learning Objectives By the end of this lecture, students should be able to: Give examples of major adhesin classes that bacteria use to establish colonisation in the human host Explain the significance of differences in adhesin type, sequence and expression, and dicusss how this can contribute to pathogenesis, using examples from E. coli 3  Roadmap to Disease To cause disease, most pathogens must: 1) Enter the body 2) Colonise the host 3) Evade host defences 4) Multiply and disseminate 5) Cause damage to the host 4  Roadmap to Disease To cause disease, most pathogens must: 1) Enter the body 2) Colonise the host 3) Evade host defences Colonisation: process whereby pathogens establish 4) themselves Multiply within the host and disseminate 5) Cause damage Adherence: processtowhereby the host pathogens attach to cells or receptors within the host 5  Why are adherence and colonisation important? - You come into contact with millions of bacteria every day – most encounters are transient tears mucus - The body is a hostile environment: Skin - - Environmental conditions: skin; intestine desiccation shedding - Mechanical factors: cells replacement (shedding), general movement of surface layers (mucus, peristalsis... ) pH, bile salts peristalsis - Normal flora – you are 50% bacteria - Immune system 6 - If bacteria can’t stay, they can’t cause disease  Today: - What is the molecular basis of bacteria adhesion? - How do differences in adhesins affect disease? 7 What is the molecular basis of bacterial adhesion? Pili/Fimbrial Afimbrial Common features ? ? G-ve ? ? G+ve ? ?  Gram negative Gram positive Different cell wall types Secretion mechanisms differ 9 Wilson et al. (2011) Bacterial pathogenesis  Pili/fimbrial adhesins Fig 2. Kaper JB, et al. (2004). Nat Rev Micro Hair/thread like adhesins, protrude out from cell Complex multigene assemblies Generally: repeating subunit that is assembled by secretion mechanism, usually tip protein, secretion system Pili attach to more things than the host! For purposes here we are virulence focussed 10 Pili and fimbriae G-ve Chaperone-usher Chaperone-usher secretion Eg. type I pili (fim), pap, afa/Dr Type IV Analogous to T2SS, retractable! Additional functions. Eg. pil, bfp, tcp, CFA/III curli Conjugative pili Type IV SS Cell-cell conjugation NOT host adhesion Eg. F-pili CS1 (type V) Alternative chaperone-usher secretion Eg. CFA/1 G+ve Sortase secretion GAS M1 pili Type IV Eg. competence pili 11  Example 1: Gram negative pili Chaperone-usher pili – Type I fimbriae Use the chaperone (C)-usher (D) secretion method to assemble pili on surface SEC pathway in PP, then pili specific genes Major repeating subunit (A) Tip complex (FGH): binding specificity Examples: Type I fimbriae Pap Fig 2A. Type I fimbriae (fim). 12 Proft T, Baker EN. (2008). Cellular and Molecular Life Sciences  Example 2: Gram negative/positive pili Type IV pili Widespread: Found in G-ve, G+ve and cyanobacteria Assembly by T2SS/(archaea) flagella like secretion system (BGQ) Major repeating subunit (E) Tip protein (C): binding specificity Accessory and other structural proteins Examples: Fig 2B. Type IV pilus (Neissseria) Type IVa: Neissseria Proft T, Baker EN. (2008). Cellular and Molecular Life Sciences Type IVb: BFP, TCP, CFA/III 13  Example 3: Gram positive pili Sortase mediated M1 pili Eg. M1 pilus Assembly uses class C sortase enzyme to covalently link proteins, then Sortase A to link into cell wall PG Repeat unit (Spy0128) and minor pilins (Cpa, Spy0130) Fig 2C. M1 pilus (S. pyogenes) Proft T, Baker EN. (2008). Cellular and Molecular Life Sciences 14  Afimbrial adhesins Proteins that mediate adherence, that do not share the repeating subunit and tip structure Usually encoded by single genes with multiple domains (often multiple functions) Examples of G-ve and G+ve classes, but not exhaustive 15 Wilson et al. (2011) Bacterial pathogenesis  Example 4: Gram negative afimbrial adhesins TAAs Trimeric autotransporter adhesins (or Oligomeric coiled coil adhesins) Type 5 mediated secretion – Autotransporters Eg. NadA (Neisseria spp), YadA (Yersinia spp), BadA (Bartonella spp). Single protein, forming a trimer Head, stalk and anchor domains 16 Linke D et al. (2006) Trends in Microbiology  Example 4: Gram negative afimbrial adhesins TAAs Structurally similar key domains, but huge sequence variation and additional domains >3000 aa 350 aa 17 Linke D et al. (2006) Trends in Microbiology  Example 5: Gram positive afimbrial adhesins MSCRAMMs Microbial Surface Molecules Recognising Adhesive Matrix Molecules Eg/ S. aureus ClfA Two Ig-like folds (‘A domain’) at the tip of a flexible stalk Cell wall linked by sortase enzyme Binding of ligand induces conformational change that reorients ‘A’ and allows binding of receptor: ‘Door-Lock- Latch/Collagen hug mechanism’ 18 Foster TJ. (2019). Trends in Microbiology 27:927-941.  Example 5: Gram positive afimbrial adhesins MSCRAMMs Structurally similar key domains, but huge sequence variation and additional domains 19 Foster TJ. (2019). Trends in Microbiology 27:927-941. What is the molecular basis of bacterial adhesion? Pili/Fimbrial Afimbrial Common features ? ? G-ve ? ? G+ve ? ? What is the molecular basis of bacterial adhesion? Pili/Fimbrial Afimbrial Common Multi-subunit gene complexes, Single genes, varied sequence and repeating main pilin and tip protein structure features G-ve Chaperone-usher; associated Trimeric Autotransporters (T5SS) secretion Eg. YadA, NadA, BadA Eg. type I pili (fim), pap, afa/Dr Others! Type IV pili Opa/Opc, intimin... Analogous to T2SS Eg. pil, bfp, tcp, CFA/III Others! Curli, F-pili... G+ve Sortase mediated MSCRAMMs Eg. GAS M1 pili Eg. S. aureus ClfA Why are there so many types? Adhesin diversity: How do differences in adhesins affect disease? Tropism Immune evasion Timing 22  Adhesin diversity: Tropism Different adhesins attach to different things Host receptors (mimic host molecules) Glycoproteins/sugars Other cells Plastic/other surfaces The site of infection is dictated by receptor specificity: tropism 23  Adhesin diversity: Tropism Tropism can be mediated - By different adhesins - By diversity wihin adhesins: Type I Fimbriae (Usher-Chaperone) FimH tip protein binds mannose: – Host tropism/specificity: in Salmonella, different alleles for different hosts – Tissue/organ tropism: Some SNPs alter affinity for mono vs tri-mannose; higher affinity Yue M et al. (2015) Nat Comms, 6:8754 24  Adhesin diversity: Immune evasion Adhesins are surface exposed Exposed to immune system Loss of function correlates with loss of virulence – attractive target for antimicrobials 25 Masignani V et al. (2019) Frontiers in Immunology, 10 Spaulding, C.N et al. (2016) Pathogens 5:30  Adhesin diversity: Immune evasion Binding: oropharynx and LRT epithelia Pathogens don’t like this: evolutionary arms race Alternate colonisation factors Additional domains/functions: bind host factors; encode anti-immune mechanisms Su Y-C, Singh B, Riesbeck K. (2012). Future Microbiology, 7: 1073 26  Adhesin diversity: Immune evasion Pathogens don’t like this: evolutionary arms race Alternate colonisation factors Additional domains/functions: bind host factors; encode anti-immune mechanisms Genetic variation in non-binding domains of the protein Expression variation (regulator, phase variation) Su Y-C, Singh B, Riesbeck K. (2012). Future Microbiology, 7: 1073 27 Brooks MJ et al. (2008) Infection and Immunity, 76:5330-5340  Adhesin diversity: Timing Adhesins are NOT ALWAYS EXPRESSED Random: Phase variation 28 Brooks MJ et al. (2008) Infection and Immunity, 76:5330-5340  Adhesin diversity: Timing Adhesins are NOT ALWAYS EXPRESSED Random: Phase variation Tightly regulated Signals ongoing processes (toxins etc) – Reduces exposure to immune system – Not wasting energy producing proteins when not needed 29 – Does not interfere with opposing actions – i.e. motility Escherichia coli and adherence Kaper JB, et al. (2004). Nat Rev Micro 30  Escherichia coli Gram-negative bacilli, best studied bacteria Laboratory workhorse Pathogen – diarrheal disease Croxen MA, Finlay BB. (2010). Nature Reviews Microbiology 31 Pathotypes adhere differently EPEC EAEC DAEC 32 Robins-Browne RM, Hartland EL. 2002. J Gastroenterol Hepatol 17:467-475.  Pathotypes have different adhesin complements Pathotype Infection, site Adhesins (K12) Non pathogenic E. coli (F-pili?), Fim Enteroaggregative (EAEC) Persistent diarhea, small and large bowel Fim, AAF, dispersin epithelia Enterohaemorrhagic (EHEC) HUS, colon Fim, LEE, Paa, ToxB, Efa-1/LifA, LPF Enteropathogenic (EPEC) Diarrhoea (childhood); small bowel Fim, Bfp, LEE, Paa, LPF enterocytes Enterotoxigenic (ETEC) Watery diarrhoea (travellers), small Fim, TCP, CFAs bowel enterocytes Enteroinvasive (EIEC) Shigella - invades colonic epithelia Fim Diffusely adhering (DAEC) Fim, Dr adhsins Uropathogenic (UPEC) UTI Fim, Pap, F1C, Dr Meningitis asssocited Meninges; invasive Fim, S fimbriae, OmpA (MNEC) 33 Avian pathogenic (APEC) Birds fim  ETEC vs EAEC: alternate colonisation factors Enterotoxigenic E. coli Enteroaggregative E. coli Colonise intestinal mucosa Colonise intestinal mucosa Over 20 fimbrial Aggregative adherence colonisation factors fimbriae (AAF): 4 alleles, identified; major types in dispersin, AggR regulated humans CFAI, CFAII, CFAIV Toxin release (mild) Toxin release (mild to severe) Persistent (mucoidal) Watery diarrhoea in diarrhoea in developing children (developing and developed countries; countries), travellers adults and children diarrhoea 34 Kaper JB, et al. (2004). Nat Rev Micro  UPEC: tropism Uropathogenic E. coli NOT intestinal – urinary tract Has both Fim and Pap (usher- chaperone pili): tropism Fim – mannose specificity : bladder Pap – digalactoside specificity: kidney epithelium. 35 Kaper JB, et al. (2004). Nat Rev Micro  A/E Pathogens: Complex regulation A/E = attaching and effacing EPEC, EHEC, C. rodentium (mouse) Characterised by intimate adherence and pedestal formation (host cytoskeletal rearrangement) TWO STAGE process, best characterised in EPEC pEAF: encoding fimbrial BFP LEE PAI: encoding afimbrial intimin and Tir Kaper JB, et al. (2004). Nat Rev Micro 36  A/E example: EPEC Entry into host: fecal oral route Cell migrate to the intestine Loose adherence: bundle-forming pili, activated by the PerA regulator triggered by pH and nutrition 37 Lee JB, Kim SK, Yoon JW. (2022). J Vet Sci 23.  A/E example: EPEC PerA also then triggers This encodes a T3SS that is activation of the LEE used to inject it’s own pathogenicity island (Ler), receptor – the Translocated encoding the afimbrial Intimin Receptor adhesin Intimin Gaytán MO, Martínez-Santos VI, Soto E, González- 38 Pedrajo B. (2016). Front Cell Infect Microbiol 6. Lee JB, Kim SK, Yoon JW. (2022). J Vet Sci 23.  A/E example: EPEC Attachment induced cytoskeletal changes in the host – actin polymerisation leading to pedestal formation 39 Lee JB, Kim SK, Yoon JW. (2022). J Vet Sci 23.  A/E example: EPEC 40 Lee JB, Kim SK, Yoon JW. 2022. J Vet Sci 23.  Summary: Colonisation: The process whereby pathogens establish themselves within the host 1. Key stop in the Roadmap to Disease: colonisation precedes damage 2. The molecular basis of adhesion: Many types of adhesin exist, broadly classified by secretion pathway 3. Differences in adhesin expression: a) Tropism: Diversity can cause differences in binding specificity b) Immune evasion: Diversity can be driven by evasion of host factors i. Expression: timing of expression can be tightly regulated This alters the course of disease! 41  Resources and references Textbook: Wilson BA, Salyers AA, Whitt DD and Proft T, Baker EN. 2008. Pili in Gram-negative and Winkler ME. 2011. Bacterial pathogenesis: A Gram-positive bacteria — structure, assembly and Molecular Approach (3rd edition). ASM Press their role in disease. Cellular and Molecular Life Sciences 66:613. Kaper JB, Nataro JP, Mobley HLT. 2004. Pathogenic Escherichia coli. Nature Reviews Microbiology Linke D, Riess T, Autenrieth IB, Lupas A, Kempf VAJ. 2:123-140. 2006. Trimeric autotransporter adhesins: variable structure, common function. Trends in Microbiology Croxen MA, Finlay BB. 2010. Molecular mechanisms 14:264-270. of Escherichia coli pathogenicity. Nature Reviews Microbiology 8:26-38. Foster TJ. 2019. The MSCRAMM Family of Cell-Wall- Anchored Surface Proteins of Gram-Positive Cocci. Pizarro-Cerdá J, Cossart P. 2006. Bacterial Adhesion Trends in Microbiology 27:927-941. and Entry into Host Cells. Cell 124:715-727. 42