Vibrio cholerae adhesins having a role in the marine environment and pathogenicity for humans. Carla Pruzzo

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1 Vibrio cholerae adhesins having a role in the marine environment and pathogenicity for humans Carla Pruzzo

2 All vibrios are aquatic bacteria and can be found in a wide range of niches. Some Vibrio species are pathogenic for humans (e.g., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus) Niche specialization of the Vibrionaceae. Jerry Reen et al., 2006, Nature Rev Microbiol 4:

3 V. cholerae in the aquatic environment interacts with a variety of surfaces Vezzulli L, Pruzzo C. Huq A. and Colwell R.R. (2010) Environ Microbiol. Reports 2 : 27 33

4 Flexible physiology Thermo-dependance Capability to survive under adverse conditions for long time Genetic plasticity Capability to interact with human and environmental substrates Survival strategies of non spore-forming bacteria Marine bacteria response to environmental and climate-linked changes Evolution of pathogenicity traits

5 Vibrio ecology in the Mediterranean Sea distribution in different aquatic matrices relationships with environmental variables long-term relationships between Vibrio occurrence and climatic variables through retrospective molecular analysis of Continous Plankton Recorder samples. Vezzulli et al Microb. Ecol. 58: works in progress in Genova on Vibrio ecology interactions with marine organisms: coral Paramuricea clavata interactions with marine organisms: zooplankton Vezzulli et al Environ Microbiol. 12:2007 zooplankton

6 Vibrio cholerae interactions with chitin containing zooplankton organisms Tigriopus fulvus copepod V. cholerae connection to chitin is a widely documented phenomenon and one of the best known examples of a successful bacteria-substrate interaction

7 V. cholerae binding to chitin affects cell physiology (multiplication, competence) biofilm formation diffusion association with chitinous organisms Carbon and Nitrogen cycling pathogenicity for humans Pruzzo etal Global impact of Vibriocholerae interactions with chitin. Env. Microbiol. 10:

8 CHITIN BINDING PROTEINS 53 V. alginolyticus - Sarkosyl resistant MPs (gel a) - Chitin binding proteins (gel b) Lanes 1-5: different environental isolates Carli et al 1993 Appl.Environ.Microbiol.59:1960 Pruzzo et al Microbiology 142:2181 V. cholerae O1 - Sarkosyl resistant MPs (lane A) -Chitin binding proteins Lanes B C D E: in the absence of GlcNAc Lanes F, G, H, I, L, M: in the presence of increasing amounts of GlcNAc (ATCC 14034, classic) Tarsi and Pruzzo Appl. Env. Microbiol. 65:1348 Montanari et al FEMS Microbiol. Ecol. 29:241

9 Effect of membrane proteins (MP) and GlcNAc on the attachment of representative vibrios to chitin particles Tigriopus fulvus copepods adapted from: Vezzulli et al Letters in Applied Microbiology 46:

10 ROLE OF p53 PROTEIN: analysis of TnphoA and gbpa deletion mutants Adhesive properties MPs wt bacteria per mg chitin (x 10 5 ) copepod (x 10 5 ) wild-type delta gbpa TnphoA mutant TnphoA mutant chitin copepod Adapted from: Zampini et al FEMS Microbiol. Lett. 244:

11 Stauder et al Env. Microbiol. Reports 2:

12 GbpA and msha gene expression under different environmental conditions. The expression of gbpa and msha by V. cholerae N16961 strain was determined relative to the expression of the endogenous control gene gyra using the comparative Ct (DDCt) method of RT-PCR. Stauder et al Env. Microbiol. Reports 2:

13 Relationship between sea surface temperature and the percentage of (A) culturable V. cholerae and (B) culturable Vibrio spp. attached to zooplankton versus total (adhering plus free-living in seawater) in the NW Mediterranean Sea. Stauder et al Env. Microbiol. Reports 2:

14 53 kda protein mediates V. cholerae association with chitin particles crustacea (copepds, cladocera, egg sacs) in the aquatic environment may it have a role in pathogenicity for humans?

15 Vibrio cholerae interactions with chitin and infection Chitin-containing zooplankton organisms (mainly copepods) represent an environmental reservoir of such bacteria V. cholerae biofilms that develop on a single chitin particle or chitin-containing plankton organism may rise to the level of an infectious dose Bacterial association with chitin increases resistance to acids such as those secreted in the stomach.

16 Chitin is not present on the epithelial surfaces but the chitin monomer GlcNAc is a common modification of glycoproteins and lipids present in the intestinal epithelium Is it possible that the same ligand involved in chitin binding mediates interactions with human cells?

17 Adherence to cultured cell monolayer Bacteria per monolayer (x 10 6 ) CD81 T33 T87 T21 CD81+GlcNAc T33+GlcNAc T87+GlcNAc T21+GlcNAc 0 Caco-2 cell Intestine 407 Adapted from: Zampini et al FEMS Microbiol. Lett. 244:

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19 V. cholerae, evolving as a marine bacterium able to interact with chitin-containing substrates present in sea water, adapted its adhesive properties to the human host. GBPA (p53) DUAL ROLE COLONIZATION FACTOR - connects the two V. cholerae s lifestyles (in humans and in the aquatic environments) - and provides evidence that some virulence factors used by pathogens during infection may derive from their natural habitat

20 Vibrio cholerae interactions with marine organisms Mytilus galloproncialis

21 BIVALVES CONCENTRATE A HUGE AMOUNT OF BACTERIA PRESENT IN THE WATER BACTERIA HAVE DIFFERENT CAPABILITY TO PERSIST INSIDE BIVALVES IF PATHOGENIC BACTERIA SURVIVE INSIDE BIVALVES, THEY CAN REACH CONCENTRATIONS DANGEROUS FOR HUMAN HEALTH

22 Persistence of bacteria in bivalves also depends on their sensitivity to the hemolymph bactericidal activity Hemolymph CELLS Granular and agranular hemocytes SERUM soluble lectins hydrolytic enzymes antimicrobial peptides Chemiotaxis Binding/ internalization Oxidative burst Lysosomal enzymes Antimicrobial peptides BACTERIAL KILLING

23 N. bacteria/monolayer x ASW Wild-type MSHA-mutant Serum ASW+ Mann Serum+Mann Serum ads.mshamutant Serum ads. MSHAmutant+Mann MSHA is a mannosesensitive adhesin of V. cholerae Adapted from: Zampini et al Appl Environ Microbiol.. 69: hemocyte Opsonin(s) in M. galloprovincialis serum V. cholerae MSHA References: Zampini et al Appl Environ Microbiol.. 69: Canesi et al Cell. Microbiol.. 7: Pruzzo et al Environ. Microbiology 7: Canesi et al 2007 Ciesm Workshop Monographs 31:

24 Role of type 1 fimbriae in E. coli interactions with bivalves unfimbriated mutant wild type strain Survival of fimbriated strain MG1655 and unfimbriated strain AAEC072 of E. coli in hemocyte monolayers in mussel hemolymph in vivo. Zero time corresponded to 30 postinjection., Adapted from: Canesi et al 2001 Appl. Env. Microbiol. 67:464

25 Although bivalves have a simple defence system, the presence of broad-range opsonins in hemolymph allows these marine organisms to control invasion of a variety of bacteria.

26 V. cholerae adhesins GBPA It binds N-acetyl glucosamine, and mediates adherence to -chitin containing substrates in sea water - intestinal cells in humans Zampini et al FEMS Microbiol. Lett.. 244: Vezzulli et al Lett Appl Microbiol,, 46: Vezzulli et al Curr. Opin. Biotechnol.. 19: Pruzzo C. et al Environ Microbiol. 10: Vezzulli et al Environ Microbiol. Reports 2 : Stauder et al Env. Microbiol. Reports 2: MSHA It binds D- mannose containing opsonins of M. galloprovincialis hemolymph, and makes bacteria -more sensitive to phagocytosis and killing by hemocytes inside bivalves -more adhesive towards cultured human intestinal cells hemocyte Canesi et al 2001 Appl. Env. Microbiol. 67:464 Canesi et al Cell. Microbiol.. 7: Pruzzo et al Environ. Microbiology 7: Canesi et al 2007 Ciesm Workshop Monographs 31: Vezzulli et al 2008 Microbial Ecol. Health Dis. 20:193

27 Some virulence factors used by Vibrio cholerae during infection are acquired/selected outside the human host Pathogenicity of Vibrio cholerae is a complex phenomenon and a combination of cell properties and environment-derived factors Pathogenicity of environmental bacteria is a reflection of their multiple life styles.