Look who s s talking now communication among foodborne microbes.

Transcription

1 Look who s s talking now communication among foodborne microbes. STATE UNIVERSITY OF NEW JERSEY RUTGERS Thomas J. Montville Rutgers, the State University of New Jersey New Brunswick, NJ, USA

2 Cells make and respond to all kinds of chemicals, how is cell signaling different? STATE UNIVERSITY OF NEW JERSEY RUTGERS

3 Extracellular signal Cells make and respond to all kinds of chemicals, how is cell signaling different? Intracellular response STATE UNIVERSITY OF NEW JERSEY RUTGERS

4 Types of Signals Signal Transduction Quorum Sensing See excellent review: Smith, Fratamico and Novak, 2004 J. Food Prot. 67:

5 Signal Transduction 2 Component Histidine kinase: response regulator. 3 Component Gene product is auto-inducer.

6 STATE UNIVERSITY OF NEW JERSEY RUTGERS Lactic Acid Bacteria signal transduction w/ autoinduction Widespread production of antimicrobial proteins - Nisin Important for listeria control Auto-inducible Class I (lantibiotic) and Class IIa (pediocin-like)

7 Autoregulation of Antimicrobial Peptides P* P P* reg1 =Histidine kinase reg2 =Response regulator

8 Food Processing Implications Nisin-controlled expression system (NICE, Kuipers et al, 1998) Stimulate in situ and in vitro bacteriocin production Production of autolysins and proteases for cheese ripening STATE UNIVERSITY OF NEW JERSEY RUTGERS

9 Listeria monocytogenes- Signal Transduction and Virulence Ubiquitous Grows at 0 C Grows at high salt Intracellular spread High fatality rate in susceptible hosts Regulatory challenge inl act

10 Listeria monocytogenes Two Component Systems Virulence control Van Langendonck, 1998 L.m. inlab dependent pathway activates host protein tyrosine kinases srcfr proteins activated, they act on actin if phosphorylation blocked, no entry

11 Staphylococcus aureus STATE UNIVERSITY OF NEW JERSEY RUTGERS (Koreim,, et al. 2003) produces RAP, a ribosmal activating protein that, when excreted, acts as extracellular regulator Induces virulence genes. Nonvirulent staph make, but don t excrete RAP

12 Quorum Sensing per se STATE UNIVERSITY OF NEW JERSEY RUTGERS

13 Salmonella Vibrio reporter E. coli avirulent Dr. Bonnie Bassler

14 Quorum Sensing Type Function AI-1 Intra- species talk AI-2 Inter- species talk Compound AHL (Acyl homoserine lactone) Furanosyl bromide diester Gene lux I lux S? Protein ALH synthase STATE UNIVERSITY OF NEW JERSEY RUTGERS

15 Campylobacter, Salmonella,, and E. coli O157:H7 Produce AI-2 Cloak, et al., 2002, Appl. Environ Microbiol. 68: Production parameters: Broth> chicken soup > milk 37 >24 > 4 C 24 > 6 > 48 hours at 24 and 37 C at 4 C, only observed at 3 hours STATE UNIVERSITY OF NEW JERSEY RUTGERS

16 Sequence for Quorum Sensing Speculative: observation hypothesis Presumptive: detection of AI-1 1 or AI-2 2 like activity in V. harveyi system Linkage of signal to response Confirmation: use of mutants to demonstrate causality STATE UNIVERSITY OF NEW JERSEY RUTGERS

17 Germination Influenced by Population Density- B. megaterium Caipo, Duffy, Zhao & Schaffner J. Appl. Microbiol. 92: Inoculum size influences global germination rate. Distance to nearest neighbor influences local germination rate: The nearer the neighbor, the faster the germination. 15 min. 75 min. 220 min. 10 µm

18 Simulation Rejects No Interaction Hypothesis in C. botulinum Relative Frequency Observed Simulated 10, ,000 1 A B Zhao, et al., Appl. Environ. Microbiol. 69: Ln (TTD) (h) C

19 C. botulinum AI-2 Activity Supernatant Source Bioluminescence detected using strain BB120 BB170 BB886 AI-1 and 2 a AI-1 b AI-2 c V. harveyi C. botulium 8 hour 14 hour 20 hour

20 Implications for Food Processing High inoculum lab experiments may give results fundamentally different from the low inoculum real world. Perhaps a threshold number is more important than log reduction. STATE UNIVERSITY OF NEW JERSEY RUTGERS

21 Organism Signal System Phenotype Genetic involvement Reference Salmonella Typhimurium Quorum sensing, AI-2 fitness in chickens Pleiotropic effect of LuxS Brandl, 2005 Enteropath. E. coli Quorum sensing, AI-2 Flagella, formation of attachment & effacement lesions Pleiotropic (?) effect of LuxS, qse, ee loci Sircili, 2004 Otto, 2001 Vibrio cholerae Quorum sensing, AI-1, AI-2, other Virulence Activates virulence regulon by repressing hapr Miller, 2002

22 Spoilage Organisms Gram et al., 2002, Int. J. Food Microbiol.78: Liu et al., 2004, J. Food Prot. 67: AHL activity detected in bean sprouts, vacuum packed beef, fish fillet, turkey, produce, etc. AHL activity inhibition caused by Na propionate, benzoate Enterobacteriacea, Pesudomonas, Aeromonas, Shewanella, and Photobacterium among the microbiota Phenotypes regulated as yet unknown

23 Stringent Meat Study Finds No Link Bruhn et al., Appl. Environ. Microbiol. 70: AHLs from vacuum packed meat identified by TLC and Mass Spec 96 species of AHL producing bacteria found but Meat inoculated with wild-type and AHL- mutant spoil at same rate Addition of QS inhibitor (halogenated( furones) ) has no affect on spoilage.

24 Confirmed AI-1 1 & Sprout Spoilage Rasch et al., Appl. Environ. Microbiol. 70: Suggestive data -AHL activity extracted from soft rot sprouts. -No activity in sound sprouts (in spite of high population levels). -Enterobacterieae and pseudomonas that produce AHL activity, proteases, and pectinases isolated from soft rot sprouts. - Only AHL, prot+, pect+ + strains produce spoilage.

25 AI-1, Sprout Spoilage (con t con t.) Slam-dunk data - AHL identified by TLC and Mass spec as N-3-oxo-hexanoyl-L-homoserine homoserine lactone. - lux I gene from Pectobacterium cloned, and sequenced. - lux I - (AHL negative) Pectobacterium had delayed spoilage, reduced protease & pectinase, and were negative for iron chelation. - Addition of AHL to AHL negative Pectobacterium restores spoilage phenotype.

26 Is Anybody Listening? Bacterial cell-to to-cell communication: sorry, can t talk now gone to lunch Winzer et al., 2002, Current Opinion in Microbiol. 5:216 Communication requires signal and response. AI-2 2 production may serve as a detoxification pathway. STATE UNIVERSITY OF NEW JERSEY RUTGERS

27 The Activated Methyl Cycle AI-2 RH cleavage enzyme LuxS? Ribosyl homosysteine Homocysteine SAH Methionine SAM Winzer et al., 2002, Current Opinion in Microbiology 5:216 STATE UNIVERSITY OF NEW JERSEY RUTGERS

28 Oh, be quiet already! STATE UNIVERSITY OF NEW JERSEY RUTGERS

29 Identification of Quorum-Quenching Quenching N-Acyl Homoserine Lactonases from Bacillus (Dong et al, Appl. Environ. Microbiol. 68: ) An autoinducer inactivation gene, aiia, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. More than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources were identified.

30 Closing Thoughts Cells do communicate with each other. Quorum sensing may provide scientific basis for changing pathogen control and regulations. Potential to reduce virulence by altering food environment.

31 Closing Word Who will have the final word in cellular communication? STATE UNIVERSITY OF NEW JERSEY RUTGERS

32 C est les microbes qui auront le dernier mot. (It s the microbes who will have the last word.) STATE UNIVERSITY OF NEW JERSEY RUTGERS