Rifampicin-resistant RpoB S522L Vibrio vulnificus exhibits disturbed stress response and hypervirulence traits.

Rifampicin resistance, which is genetically linked to mutations in the RNA polymerase ß-subunit gene rpoB, has a global impact on bacterial transcription and cell physiology. Previously, we identified a substitution of serine 522 in RpoB (i.e., RpoBS522L ) conferring rifampicin resistance to Vibrio vulnificus, a human food-borne and wound-infecting pathogen associated with a high mortality rate. Transcriptional and physiological analysis of V. vulnificus expressing RpoBS522L showed increased basal transcription of stress-related genes and global virulence regulators. Phenotypically these transcriptional changes manifest as disturbed osmo-stress responses and toxin-associated hypervirulence as shown by reduced hypoosmotic-stress resistance and enhanced cytotoxicity of the RpoBS522L strain. These results suggest that RpoB-linked rifampicin resistance has a significant impact on V. vulnificus survival in the environment and during infection.

Venom extraction method influences venom composition and potency in the giant house spider Eratigena atrica (C. L. Koch, 1843).

Extraction is the first step when investigating venom composition and function. In small invertebrates, widely used extraction methods include electrostimulation and venom gland extraction, however, the influence of these methods on composition and toxicology is poorly understood. Using the Giant House Spider Eratigena atrica as a model, we show that electrostimulation and venom gland removal extraction methods produce different protein profiles as assessed by Coomassie-stained SDS-PAGE and significantly different potencies in the cricket Acheta domesticus.

The Vibrio vulnificus stressosome is dispensable in nutrient-rich media.

The stressosome is a protein complex that senses environmental stresses and mediates the stress response in several Gram-positive bacteria through the activation of the alternative sigma factor SigB. The stressosome locus is found in 44 % of Gram-negative Vibrio vulnificus isolates. However, V. vulnificus does not possess SigB. Nonetheless, in nutrient-limited media, the stressosome modulates gene transcription and bacterial behaviour. In this work, the expression of the stressosome genes was proven during stationary phase in nutrient-rich media and co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated. The construction of a stressosome mutant lacking the genes encoding the four proteins constituting the stressosome complex (VvRsbR, VvRsbS, VvRsbT, VvRsbX) allowed us to examine the role of this complex in vivo. Extensive phenotypic characterization of the ΔRSTX mutant in nutrient-rich media showed that the stressosome does not contribute to growth of V. vulnificus . Moreover, the stressosome did not modulate the tolerance or survival response of V. vulnificus to the range of stresses tested, which included ethanol, hyperosmolarity, hypoxia, high temperature, acidity and oxidative stress. Furthermore, the stressosome was dispensable for motility and exoenzyme production of V. vulnificus in nutrient-rich media. Therefore, in conclusion, although stressosome gene transcription occurs in nutrient-rich media, the stressosome neither has an essential role in stress responses of V. vulnificus nor does it seem to modulate these activities in these conditions. We hypothesise that the stressosome is expressed in nutrient-rich conditions as a sensor complex, but that activation of the complex does not occur in this environment.

Inhibition of bacterial biofilms by the snake venom proteome.

Snake venoms possess a range of pharmacological and toxicological activities. Here we evaluated the antibacterial and anti-biofilm activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) of venoms from the Samar spitting cobra Naja samarensis and the Puff adder Bitis arietans. Both venoms prevented biofilm production by pathogenic S. aureus in a growth-independent manner, with the B. arietans venom being most potent. Fractionation showed the active molecule to be heat-labile and >10 kDa in size. Proteomic profiles of N. samarensis venom revealed neurotoxins and cytotoxins, as well as an abundance of serine proteases and three-finger toxins, while serine proteases, metalloproteinases and C-lectin types were abundant in B. arietans venom. These enzymes may have evolved to prevent bacteria colonising the snake venom gland. From a biomedical biotechnology perspective, they have valuable potential for anti-virulence therapy to fight antibiotic resistant microbes.

Sponge-derived fatty acids inhibit biofilm formation of MRSA and MSSA by down-regulating biofilm-related genes specific to each pathogen.

AIM: A promising approach for the development of next-generation antimicrobials is to shift their target from causing bacterial death to inhibiting virulence. Marine sponges are an excellent potential source of bioactive anti-virulence molecules (AVM). We screened fractions prepared from 26 samples of Irish coastal sponges for anti-biofilm activity against clinically relevant pathogens. METHODS AND RESULTS: Fifteen fractions from eight sponge species inhibited biofilm of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and/or Listeria monocytogenes without causing growth inhibition. Gas chromatograph/mass spectroscopy analyses of Mycale contarenii fractions revealed the presence of myristic acid and oleic acid. These fatty acids repressed transcription of the fibronectin-binding protein fnbA and fnbB genes and the polysaccharide intercellular adhesin icaADBC operon, which are required for MRSA and MSSA biofilm formation, respectively. CONCLUSIONS: This study illustrates the potential of AVM from Irish coastal sponges to specifically target bacterial virulence phenotypes, in this case, repression of biofilm formation via decreased transcription of biofilm-associated genes in MSSA and MRSA.

In vivo characterisation of the Vibrio vulnificus stressosome: A complex involved in reshaping glucose metabolism and motility regulation, in nutrient- and iron-limited growth conditions.

Stressosomes are signal-sensing and integration hubs identified in many bacteria. At present, the role of the stressosome has only been investigated in Gram-positive bacteria. This work represents the first in vivo characterisation of the stressosome in a Gram-negative bacterium, Vibrio vulnificus. Previous in vitro characterisation of the complex has led to the hypothesis of a complex involved in iron metabolism and control of c-di-GMP levels. We demonstrate that the stressosome is probably involved in reshaping the glucose metabolism in Fe- and nutrient-limited conditions and mutations of the locus affect the activation of the glyoxylate shunt. Moreover, we show that the stressosome is needed for the transcription of fleQ and to promote motility, consistent with the hypothesis that the stressosome is involved in regulating c-di-GMP. This report highlights the potential role of the stressosome in a Gram-negative bacterium, with implications for the metabolism and motility of this pathogen.

The stressosome is required to transduce low pH signals leading to increased transcription of the amino acid-based acid tolerance mechanisms in Listeria monocytogenes.

Increasing proton concentration in the environment represents a potentially lethal stress for single-celled microorganisms. To survive in an acidifying environment, the foodborne pathogen Listeria monocytogenes quickly activates the alternative sigma factor B (σB), resulting in upregulation of the general stress response (GSR) regulon. Activation of σB is regulated by the stressosome, a multi-protein sensory complex involved in stress detection and signal transduction. In this study, we used L. monocytogenes strains harbouring two stressosome mutants to investigate the role of this complex in triggering expression of known amino acid-based resistance mechanisms in response to low pH. We found that expression of glutamate decarboxylase (gadD3) and arginine and agmatine deiminases (arcA and aguA1, respectively) were upregulated upon acid shock (pH 5 for 15 min) in a stressosome-dependent manner. In contrast, transcription of the arg operons (argGH and argCJBDF), which encode enzymes for the l-arginine biosynthesis pathway, were upregulated upon acid shock in a stressosome-independent manner. Finally, we found that transcription of argR, which encodes a transcriptional regulator of the arc and arg operons, was largely unaffected by acidic shock. Thus, our findings suggest that the stressosome plays a role in activating amino acid-based pH homeostatic mechanisms in L. monocytogenes . Additionally, we show that genes encoding the l-arginine biosynthesis pathway are highly upregulated under acidic conditions, suggesting that intracellular arginine can help withstand environmental acidification in this pathogen.

The Vibrio vulnificus stressosome is an oxygen-sensor involved in regulating iron metabolism.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output - regulation of iron metabolism - for a stressosome complex.

In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures.

The alternative sigma factor B (σB) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σB loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σB or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σB). However, σB becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σB loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σB loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σB in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

Validation of a random Vibrio parahaemolyticus genomic library by selection of quinolone resistance in a heterologous host.

Vibrio parahaemolyticus is a shellfish-borne pathogen that is a highly prevalent causative agent of inflammatory gastroenteritis in humans. Genomic libraries have proven useful for the identification of novel gene functions in many bacterial species. In this study we prepared a library containing 40 kb fragments of randomly sheared V. parahaemolyticus genomic DNA and introduced this into Escherichia coli HB101 using a commercially available low copy cosmid system. In order to estimate coverage and suitability of the library and potentially identify novel antimicrobial resistance determinants, we screened for the acquisition of resistance to the fluoroquinolone norfloxacin - a phenotype exhibited by V. parahaemolyticus but not the heterologous E. coli host. Upon selection on solid medium containing norfloxacin, 0.52% of the library population was resistant, consistent with the selection of a single resistance locus. End-sequencing identified six distinct insert fragments. All clones displayed fourfold increased norfloxacin MIC compared with E. coli HB101 carrying an empty vector. The common locus contained within resistant clones included qnr, a previously described quinolone resistance gene. These results indicate that the library was unbiased, of sufficient coverage and that heterologous expression was possible. While we hope that this library proves useful for identifying the genetic determinants of complex phenotypes such as those related to virulence, not all norfloxacin resistance genes were detected in our screen. As such, we discuss the benefits and limitations of this approach for identifying the genetic basis of uncharacterized bacterial phenotypes.

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes.

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σB), whose role in protection against acid stress is well established. Here, we investigated the involvement of the stressosome, a sensory hub, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σB and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was solely present retained the ability to induce σB activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1-T175A, -T209A, -T241A) and RsbS (rsbS-S56A), or the stressosome kinase RsbT (rsbT-N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes inlAB and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σB in L. monocytogenes. Furthermore, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.

Bis-Indole Alkaloids Isolated from the Sponge Spongosorites calcicola Disrupt Cell Membranes of MRSA.

Antimicrobial resistance (AMR) is a global health challenge with methicillin resistant Staphylococcus aureus (MRSA), a leading cause of nosocomial infection. In the search for novel antibiotics, marine sponges have become model organisms as they produce diverse bioactive compounds. We investigated and compared the antibacterial potential of 3 bis-indole alkaloids-bromodeoxytopsentin, bromotopsentin and spongotine A-isolated from the Northeastern Atlantic sponge Spongosorites calcicola. Antimicrobial activity was determined by MIC and time-kill assays. The mechanism of action of bis-indoles was assessed using bacterial cytological profiling via fluorescence microscopy. Finally, we investigated the ability of bis-indole alkaloids to decrease the cytotoxicity of pathogens upon co-incubation with HeLa cells through the measurement of mammalian cell lysis. The bis-indoles were bactericidal to clinically relevant Gram-positive pathogens including MRSA and to the Gram-negative gastroenteric pathogen Vibrio parahaemolyticus. Furthermore, the alkaloids were synergistic in combination with conventional antibiotics. Antimicrobial activity of the bis-indole alkaloids was due to rapid disruption and permeabilization of the bacterial cell membrane. Significantly, the bis-indoles reduced pathogen cytotoxicity toward mammalian cells, indicating their ability to prevent bacterial virulence. In conclusion, sponge bis-indole alkaloids are membrane-permeabilizing agents that represent good antibiotic candidates because of their potency against Gram-positive and Gram-negative bacterial pathogens.

Metagenomic identification, purification and characterisation of the Bifidobacterium adolescentis BgaC ß-galactosidase.

Members of the human gut microbiota use glycoside hydrolase (GH) enzymes, such as ß-galactosidases, to forage on host mucin glycans and dietary fibres. A human faecal metagenomic fosmid library was constructed and functionally screened to identify novel ß-galactosidases. Out of the 16,000 clones screened, 30 ß-galactosidase-positive clones were identified. The ß-galactosidase gene found in the majority of the clones was BAD_1582 from Bifidobacterium adolescentis, subsequently named bgaC. This gene was cloned with a hexahistidine tag, expressed in Escherichia coli and His-tagged-BgaC was purified using Ni2+-NTA affinity chromatography and size filtration. The enzyme had optimal activity at pH 7.0 and 37 °C, with a wide range of pH (4-10) and temperature (0-40 °C) stability. It required a divalent metal ion co-factor; maximum activity was detected with Mg2+, while Cu2+ and Mn2+ were inhibitory. Kinetic parameters were determined using ortho-nitrophenyl-ß-D-galactopyranoside (ONPG) and lactose substrates. BgaC had a Vmax of 107 µmol/min/mg and a Km of 2.5 mM for ONPG and a Vmax of 22 µmol/min/mg and a Km of 3.7 mM for lactose. It exhibited low product inhibition by galactose with a Ki of 116 mM and high tolerance for glucose (66% activity retained in presence of 700 mM glucose). In addition, BgaC possessed transglycosylation activity to produce galactooligosaccharides (GOS) from lactose, as determined by TLC and HPLC analysis. The enzymatic characteristics of B. adolescentis BgaC make it an ideal candidate for dairy industry applications and prebiotic manufacture.Key points• Bifidobacterium adolescentis BgaC ß-galactosidase was selected from human faecal metagenome.• BgaC possesses sought-after properties for biotechnology, e.g. low product inhibition.• BgaC has transglycosylation activity producing prebiotic oligosaccharides. Graphical Abstract.

Synanthropic spiders, including the global invasive noble false widow Steatoda nobilis, are reservoirs for medically important and antibiotic resistant bacteria.

The false widow spider Steatoda nobilis is associated with bites which develop bacterial infections that are sometimes unresponsive to antibiotics. These could be secondary infections derived from opportunistic bacteria on the skin or infections directly vectored by the spider. In this study, we investigated whether it is plausible for S. nobilis and other synanthropic European spiders to vector bacteria during a bite, by seeking to identify bacteria with pathogenic potential on the spiders. 11 genera of bacteria were identified through 16S rRNA sequencing from the body surfaces and chelicerae of S. nobilis, and two native spiders: Amaurobius similis and Eratigena atrica. Out of 22 bacterial species isolated from S. nobilis, 12 were related to human pathogenicity among which Staphylococcus epidermidis, Kluyvera intermedia, Rothia mucilaginosa and Pseudomonas putida are recognized as class 2 pathogens. The isolates varied in their antibiotic susceptibility: Pseudomonas putida, Staphylococcus capitis and Staphylococcus edaphicus showed the highest extent of resistance, to three antibiotics in total. On the other hand, all bacteria recovered from S. nobilis were susceptible to ciprofloxacin. Our study demonstrates that S. nobilis does carry opportunistic pathogenic bacteria on its body surfaces and chelicerae. Therefore, some post-bite infections could be the result of vector-borne bacterial zoonoses that may be antibiotic resistant.

rpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus.

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the ß subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus, even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml-1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes.

In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype.IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

Brominated Bisindole Alkaloids from the Celtic Sea Sponge Spongosorites calcicola.

As part of an ongoing program to identify new bioactive compounds from Irish marine bioresources, we selected the subtidal sponge Spongosorites calcicola for chemical study, as fractions of this species displayed interesting cytotoxic bioactivities and chemical profiles. The first chemical investigation of this marine species led to the discovery of two new bisindole alkaloids of the topsentin family, together with six other known indole alkaloids. Missing the usual central core featured by the representatives of these marine natural products, the new metabolites may represent key biosynthetic intermediates for other known bisindoles. These compounds were found to exhibit weak cytotoxic activity against HeLa tumour cells, suggesting a specificity towards previously screened carcinoma and leukaemia cells.

In vitro comparative cytotoxicity study of a novel biocidal iodo-thiocyanate complex.

Novel biocides, which avoid the induction of cross-resistance to antibiotics, are an urgent societal requirement. Here, we compared the cytotoxic and bactericidal effects of a new antimicrobial agent, the iodo-thiocyanate complex (ITC), with those of the common antiseptics, hydrogen peroxide (H2O2), povidone iodine (PVP-I) and Lugol's iodine (Lugol). The antimicrobials were co-incubated for 10 min with HeLa and Escherichia coli cells in the presence and absence of organic matter (Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum). The cytotoxic concentrations of ITC were equivalent to its bactericidal concentrations (7.8 µg ml-1). By contrast, cytotoxic effects of H2O2, PVP-I and Lugol were apparent at concentrations lower than their bactericidal concentrations (250, 250 and 125 µg ml-1, respectively). The cellular effects of ITC were not quenched by organic matter, unlike the other antiseptics. ITC, PVP-I and Lugol had hemolytic effect on horse erythrocytes at high concentrations, while H2O2 showed no hemolysis. ITC, at 30 or 300 µg ml-1, did not cause DNA breakage in HeLa cells as assessed by an in vitro comet assay in the absence of S9 metabolic activation, whereas H2O2 caused extensive single-strand DNA breaks. The pronounced antimicrobial potency of ITC and its favorable cytotoxicity profile suggests that ITC should be considered for antiseptic applications.

Manipulation of intestinal epithelial cell function by the cell contact-dependent type III secretion systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus elicits gastroenteritis by deploying Type III Secretion Systems (TTSS) to deliver effector proteins into epithelial cells of the human intestinal tract. The bacteria must adhere to the human cells to allow colonization and operation of the TTSS translocation apparatus bridging the bacterium and the host cell. This article first reviews recent advances in identifying the molecules responsible for intercellular adherence. V. parahaemolyticus possesses two TTSS, each of which delivers an exclusive set of effectors and mediates unique effects on the host cell. TTSS effectors primarily target and alter the activation status of host cell signaling proteins, thereby bringing about changes in the regulation of cellular behavior. TTSS1 is responsible for the cytotoxicity of V. parahaemolyticus, while TTSS2 is necessary for the enterotoxicity of the pathogen. Recent publications have elucidated the function of several TTSS effectors and their importance in the virulence of the bacterium. This review will explore the ability of the TTSS to manipulate activities of human intestinal cells and how this modification of cell function favors bacterial colonization and persistence of V. parahaemolyticus in the host.

Translocation of Vibrio parahaemolyticus across an in vitro M cell model.

Consumption of Vibrio parahaemolyticus via contaminated shellfish results in inflammatory gastroenteritis characterised by severe diarrhoea, nausea and stomach cramps. This study investigated the translocation of V. parahaemolyticus across a Peyer's patch M cell-like Caco-2/Raji B co-culture model system, as M cells represent a primary site of infection for many pathogenic bacteria. Vibrio parahaemolyticus translocated across co-culture monolayers in higher numbers as compared to Caco-2 monolayers. Moreover, the bacteria induced a greater disruption of the transepithelial resistance in M cell-like co-cultures than in Caco-2 monocultures. Virulence factors associated with this pathogen include two type three secretion systems (TTSS-1 and TTSS-2). TTSS-1 had no effect on translocation efficiency, with TTSS-2 exhibiting a modest enhancing effect. ERK activity was required for optimal translocation 1 h postinfection, however, neither ERK nor the JNK and p38 MAPK were required at 2 h pi. Additionally, TER disruption in response to bacterial infection occurred independently of the TTSS and MAPK activation. It was concluded that V. parahaemolyticus causes TER disruption of M cell-like co-cultures and translocates in high numbers across the M cell-like co-culture monolayer. These data implicate M cells as important sites for V. parahaemolyticus invasion across the intestinal epithelium during infection.