Harveyi BB170 Research Articles

Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay

Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe(3+) within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 microM. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of >120 with a standard deviation of approximately 30% in a reliable and reproducible way.

Subinhibitory concentrations of cinnamaldehyde interfere with quorum sensing

To investigate the effect of cinnamaldehyde (CA) on transcription from selected quorum sensing (QS) promoters. The action of CA on QS was assayed using three E. coli green fluorescent protein (GFP) based bioreporters (two inducible and the other constitutive) and two Vibrio harveyi bioluminescent reporter strains. LuxR-mediated transcription from the P(luxI) promoter, which is induced by 3-oxo-C6-homoserine lactone (HSL), was reduced by 70 per cent following exposure to 200 micromol l(-1) CA (26 ppm). The bioluminescence of Vibrio harveyi BB886, which is mediated by 3-hydroxy-C4-HSL, was reduced by 55 per cent after exposure to 60 micromol l(-1) CA (8 ppm), and 100 micromol l(-1) CA (13 ppm) inhibited the bioluminescence of the autoinducer-2 (AI-2) responsive reporter strain V. harveyi BB170 by nearly 60 per cent. CA did not inhibit the growth of the bioreporter strains at these concentrations. CA had a minimal effect on LasR promoter activity, induced by 3-oxo-C12-HSL. Low concentrations of CA were effective at inhibiting two types of acyl homoserine lactone mediated QS, and also autoinducer-2 mediated QS. Because CA is widely used in the food and flavour industries, its potential to affect bacterial QS regulated processes should be recognized.

Haemophilus influenzae luxS mutants form a biofilm and have increased virulence

To gain insight into the role of luxS Hi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxS Hi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5α expressing a cloned luxS Hi contained 350-fold more AI-2 activity per cell than E. coli DH5α containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxS Hi mutants. Two clinical H. influenzae and their luxS Hi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxS Hi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxS Hi mutants tended to be more invasive. The luxS Hi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.

LuxS/autoinducer-2 quorum sensing molecule regulates transcriptional virulence gene expression in Clostridium difficile

Toxigenic Clostridium difficile CCUG19126 produces the autoinducer-2 (AI-2) quorum sensing molecule that induces bioluminescence in Vibrio harveyi BB170 reporter strain. AI-2-containing cell-free supernatants from mid-log phase C. difficile and Escherichia coli DH5α expressing recombinant luxS cd upregulated the transcript levels of tcdA (7–10-fold), tcdB (4–6-fold), and tcdE (2–3-fold) in early-log C. difficile. In contrast, no induction occurred when cells were exposed to sterile medium or to cell-free supernatant from E. coli DH5α. AI-2 did not significantly increase the level of toxin A in early-log C. difficile. These results suggest that LuxS cd-dependent signaling regulates virulence gene expression at the transcriptional level in C. difficile.

Methods for Analysis of Bacterial Autoinducer‐2 Production

The quorum-sensing signal molecule autoinducer-2 (AI-2) is produced by over 50 diverse bacterial species and controls many different processes, including antibiotic production, biofilm formation, and virulence. AI-2 production often varies according to growth phase, media conditions, and the presence of specific factors. This unit describes a biological assay for AI-2 activity produced by a bacterial strain of interest. The assay employs an AI-2 reporter strain, Vibrio harveyi BB170, which produces light in response to AI-2. In the first stage of the assay, culture fluids of the bacterial strain of interest are collected over a time course of growth and filtered to remove cells. In the next stage, these culture fluids are mixed with BB170, and the light produced in response to AI-2 in the culture fluids is measured using a luminometer. BB170 is exquisitely sensitive to AI-2, and therefore, even low amounts of AI-2 can be detected using this bioassay.

Autoinducer-2–like Activity Associated with Foods and Its Interaction with Food Additives

The autoinducer-2 (AI-2) molecule produced by bacteria as part of quorum sensing is considered to be a universal inducer signal in bacteria because it reportedly influences gene expression in a variety of both gram-negative and gram-positive bacteria. The objective of this study was to determine whether selected fresh produce and processed foods have AI-2–like activity and whether specific food additives can act as AI-2 mimics and result in AI-2–like activity. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used as the basis for determining AI-2 activity in the selected foods and food ingredients. Maximum AI-2 activity was seen on the frozen fish sample (203-fold, compared with the negative control) followed by tomato, cantaloupe, carrots, tofu, and milk samples. Interestingly, some samples were capable of inhibiting AI-2 activity. Turkey patties showed the highest inhibition (99.8% compared with the positive control) followed by chicken breast (97.5%), homemade cheeses (93.7%), beef steak (90.6%), and beef patties (84.4%). AI-2 activity was almost totally inhibited by sodium propionate, whereas sodium benzoate caused 93.3% inhibition, compared with 75% inhibition by sodium acetate. Sodium nitrate did not have any appreciable effect, even at 200 ppm. Understanding the relationships that exist between AI-2 activity on foods and the ecology of pathogens and food spoilage bacteria on foods could yield clues about factors controlling food spoilage and pathogen virulence.

Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen.

Quorum sensing in Campylobacter jejuni: detection of a luxS encoded signalling molecule.

The expression of a wide variety of physiological functions in many bacterial species is modulated by quorum sensing, a population-dependent signalling mechanism that involves the production and detection of extracellular signalling molecules. The genome sequence of Campylobacter jejuni NCTC 11168 contains a gene encoding an orthologue of LuxS, which is required for autoinducer-2 (AI-2) production in other bacterial species, but does not contain genes predicted to encode any known acyl-homoserine lactone synthetase. This study demonstrates that C. jejuni produces functional AI-2 activity through the ability of cell-free extracts to specifically induce bioluminescence in Vibrio harveyi BB170, a reporter strain for quorum-sensing system 2. Production of this signalling compound was shown to be dependent upon the product of the C. jejuni luxS gene (Cj1198). While the luxS mutant showed comparable growth rate, resistance to oxidative stress and ability to invade Caco-2 cell monolayers to the parental strain, it exhibited decreased motility haloes in semisolid media, suggesting a role for quorum sensing in the regulation of motility.

LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence.

Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5alpha. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the mid-exponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5alpha transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45% less Rgp and 30% less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.