INT-407 Cells Research Articles

Involvement of capsular polysaccharide via a TLR2/NF-κB pathway in Vibrio vulnificus-induced IL-8 secretion of human intestinal epithelial cells

In a previous study, we reported that a wbpP gene mutation in Vibrio vulnificus was significantly impaired in its ability to synthesize surface capsular polysaccharide (CPS). In this study, we evaluated the functions of the V. vulnificus capsular polysaccharide on interleukin (IL)-8 production, as well as its underlying mechanisms in human intestinal epithelial cells. The CPS-defective wbpP mutant induced significantly lower levels of IL-8 production, IL-8 gene promoter activation and NF-kappaB activity in INT-407 cells than was noted with the wild-type or wbpP-complemented V. vulnificus. The expression levels of Toll-like receptor (TLR)2 mRNA and protein were also found to be lower in INT-407 cells infected with the CPS-defective wbpP mutant than in those cells infected with the wild-type or the wbpP-complemented strains. Additionally, the treatment of INT-407 cells with anti-TLR2 antibody proved to significantly block IL-8 production and NF-kappaB minimal promoter activity induced by the wild-type or the wbpP-complemented strains. Furthermore, purified V. vulnificus CPS was found to significantly induce IL-8 production and NF-kappaB activation, both of which were inhibited upon the addition of the anti-TLR2 antibody. Taken together, these results demonstrate that V. vulnificus capsular polysaccharide is involved in the induction of IL-8 production of human intestinal epithelial cells via a TLR2/NF-kappaB-dependent pathway.

The absence of functional dectin-1 on enterocytes may serve to prevent intestinal damage

Enterocytes are exposed to antigens present in the intestinal lumen, like beta-glucans that are carbohydrate structures present not only in the cell wall of yeast and fungi but also in cereals. Beta-glucans are known for their immune modulating properties and we have earlier reported an increased immune response by enterocytes after addition of fecal water prepared from ileostomic contents obtained from participants consuming an oat beta-glucan diet versus a placebo diet. We hypothesized that our observation of immune stimulating effects by oat beta-glucan in enterocytes was mediated through the beta-glucan receptor dectin-1. Presence of dectin-1 in enterocytes was examined by reverse transcriptase PCR, western blot, and flow cytometry followed by an evaluation of the functional involvement of dectin-1 by using dectin-1 inhibitors during fecal water incubations. Reverse transcriptase PCR and western blot analysis showed dectin-1 presence in the INT407 and Caco-2 NF-kappaB reporter enterocyte cell lines. Moreover, human enterocytes isolated from ileum or colon biopsies also contained dectin-1 protein. However, dectin-1 expression could not be confirmed by flow cytometry in INT407 cells, suggesting that in these cell lines dectin-1 is not expressed at the extracellular membrane. Furthermore, dectin-1 inhibitors did not suppress the beta-glucan containing fecal water-induced IL-8 production by INT407 cells and NF-kappaB transactivation by Caco-2 NF-kappaB reporter cells. INT407 and Caco-2 NF-kappaB reporter cells seem to express no functional dectin-1. The absence of this pattern recognition receptor may function to protect the intestine against inflammatory damage, as the dectin-1 ligand beta-glucan is largely present in the intestinal lumen.

Adhesion and immunomodulatory effects ofBifidobacterium lactisHN019 on intestinal epithelial cells INT-407

To elucidate the adherence and immunomodulatory properties of a probiotic strain Bifidobacterium lactis (B. lactis) HN019. Adhesion assays of B. lactis HN019 and Salmonella typhimurium (S. typhimurium) ATCC 14028 to INT-407 cells were carried out by detecting copies of species-specific genes with real-time polymerase chain reaction. Morphological study was further conducted by transmission electron microscopy. Interleukin-1beta (IL-1beta), interleukin-8, and tumor necrosis factor-alpha (TNF-alpha) gene expression were assessed while enzyme linked immunosorbent assay was used to detect IL-8 protein secretion. The attachment of S. typhimurium ATCC 14028 to INT407 intestinal epithelial cells was inhibited significantly by B. lactis HN019. B. lactis HN019 could be internalized into the INT-407 cells and attenuated IL-8 mRNA level at both baseline and S. typhimurium-induced pro-inflammatory responses. IL-8 secretion was reduced while IL-1beta and TNF-alpha mRNA expression level remained unchanged at baseline after treated with B. lactis HN019. B. lactis HN019 does not up-regulate the intestinal epithelium expressed pro-inflammatory cytokine, it showed the potential to protect enterocytes from an acute inflammatory response induced by enteropathogen.

Activation of proinflammatory response in human intestinal epithelial cells followingVibrio choleraeinfection through PI3K/Akt pathway

Vibrio cholerae activates proinflammatory response in cultured intestinal epithelial cells. In this study, we demonstrate that V. cholerae O395 infection of intestinal epithelial cells results in the activation of Akt. Inhibition of Akt significantly decreases IL-1alpha, IL-6, and TNF-alpha production in V. cholerae infected Int407 cells. Analysis of the mechanisms of Akt influences on cytokine response demonstrates that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with virulence, adherence, or motility. Insertion mutants in the virulence genes coding for CtxA, ToxT, and OmpU of V. cholerae modulate the activation of PI3K/Akt signaling pathway, whereas an aflagellate non-motile mutant (O395FLAN) and a adherent and less motile mutant (O395Y3N/O395Y4N) of V. cholerae both show very significant down-regulation of Akt activity in Int407 cells. Together, these observations indicate that Akt promotes proinflammatory cytokine production by V. cholerae infected human intestinal epithelial cells through its influences on NF-kappaB.

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-κB and AP-1 in INT-407 cells

Enteroaggregative Escherichia coli (EAEC) is emerging as a cause of acute and persistent diarrhea in developing countries. An important feature of EAEC pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. In this article, we have shown that EAEC-induced activation of mitogen-activated protein kinases (MAPK) (ERK-1/2, JNK and p38MAPK) in cultured human intestinal epithelial cells (INT-407) leads to the induction of DNA-binding activity of NF-kappaB and AP-1, resulting in IL-8 production. Plasmid-cured EAEC could also activate the MAPK and the transcription factors leading to IL-8 secretion, but to a lesser extent than that of wild-type EAEC. Further, pretreatment of these cells with the highly specific MEK inhibitor (PD 098059), the JNK inhibitor (SP 600125), and the p38MAPK inhibitor (SB 203580) resulted in inhibition of the IL-8 secretion by EAEC (wild type as well as plasmid cured)-infected INT-407 cells. These findings demonstrate that the inflammatory response induced by EAEC may be due to the specific stimulation of MAPK signaling pathways leading to nuclear responses. To our knowledge, this is the first article regarding the detailed mechanism of IL-8 secretion from the EAEC-infected human intestinal epithelial cell line.

Identification of the Vibrio vulnificus ahpCl gene and its influence on survival under oxidative stress and virulence

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H(2)O(2). In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H(2)O(2) and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H(2)O(2) in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.

OxyR , a LysR-Type Regulator Involved in Klebsiella pneumoniae Mucosal and Abiotic Colonization

Colonization of the gastrointestinal tract is the first event in Klebsiella pneumoniae nosocomial infections, followed by colonization of the bladder or respiratory tract or entry into the bloodstream. To survive in the host, bacteria must harbor specific traits and overcome multiple stresses. OxyR is a conserved bacterial transcription factor with a key role both in the upregulation of defense mechanisms against oxidative stress and in pathogenesis by enhancing biofilm formation, fimbrial expression, and mucosal colonization. A homolog of oxyR was detected in silico in the K. pneumoniae sequenced genome and amplified from the LM21 wild-type strain. To determine the role of oxyR in K. pneumoniae host-interaction processes, an oxyR isogenic mutant was constructed, and its behavior was assessed. At concentrations lower than 10(7) ml(-1), oxyR-deficient organisms were easily killed by micromolar concentrations of H(2)O(2) and exhibited typical aerobic phenotypes. The oxyR mutant was impaired in biofilm formation and types 1 and 3 fimbrial gene expression. In addition, the oxyR mutant was unable to colonize the murine gastrointestinal tract, and in vitro assays showed that it was defective in adhesion to Int-407 and HT-29 intestinal epithelial cells. The behavior of the oxyR mutant was also determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment: deletion of oxyR resulted in higher sensitivity to bile and acid stresses but not to osmotic stress. These results show the pleiotropic role of oxyR in K. pneumoniae gastrointestinal colonization.

Proteinaceous Cytotoxic Component of Allium sativum Induces Apoptosis of INT-407 Intestinal Cells

Garlic has long been known for its wide array of therapeutic effects, including hypolipidemic, antihypertensive, antimicrobial, and possibly anticancer effects; conversely, some adverse effects of garlic, such as acute pain and neurogenic inflammation, have also been reported. However, information detailing the toxicological significance of garlic is scarce. In this study, the cytotoxicities of fresh garlic extract (FGE) and boiled garlic extract (BGE) and their underlying toxic mechanisms were investigated using INT-407 intestinal epithelial cells. A brief exposure (20 minutes) to FGE induced a concentration-dependent increase in cell death (37 +/- 2% at 300 microg/mL), but no cytotoxic effects were induced after exposure to BGE. For FGE, only the high-molecular-mass (>10-kDa) proteins were associated with cytotoxic effects. FGE-treated cells showed morphological changes such as increased cell rounding and fragmentation, suggesting programmed cell death (apoptosis). Apoptosis of FGE-treated cells was evaluated by observing the fragmented multinuclei stained with Hoechst 33342. From the cell cycle analysis, the increase in hypodiploidic cells and in the G2/M phase cell population suggested not only apoptosis but also cell cycle arrest of FGE-treated cells. Pretreatment with N-acetyl-l-cysteine almost completely prevented FGE-induced cell death, suggesting that reactive oxygen species (ROS) may play a key role in FGE-associated cytotoxicity. Consumption of fresh garlic may be linked to potential cytotoxicity of intestinal cells when ROS scavengers are not present.

In vitro and in vivo antimicrobial activity of water-soluble chitosan oligosaccharides against Vibrio vulnificus

Vibrio vulnificus is a Gram-negative bacterium that induces severely rapid pathological progress. In this study, we evaluated the antibacterial activity of two water-soluble chitosan oligosaccharides, COS A (MW, 10,000 Da) and COS B (MW, 1,000 Da), from 90-95% deacetylated chitosan, against V. vulnificus in vitro and in vivo. Treatment with COS A resulted in significantly higher suppressive effects on the growth of V. vulnificus than treatment with COS B. The growth of V. vulnificus was inhibited within 1 h of treatment with water-soluble COS A in concentrations ranging from 0.5 to 10 mg/ml. Additionally, treatment with COS A completely inhibited V. vulnificus-induced cytotoxicity in human intestinal epithelial INT-407 cells, while COS B did not. Furthermore, the administration of COS A (0.1-0.5 mg per mouse) significantly increased the survival period of V. vulnificus-infected mice. The number of viable V. vulnificus in the spleen, liver, small intestine, and blood was significantly lower in COS A-treated mice than in untreated mice. Here, we clearly demonstrate that COS A is a potential agent for the prevention and treatment of infection with V. vulnificus.

The Capability of Catabolic Utilization of N -Acetylneuraminic Acid, a Sialic Acid, Is Essential for Vibrio vulnificus Pathogenesis

N-acetylneuraminic acid (Neu5Ac, sialic acid) could provide a good substrate for enteropathogenic bacteria in the intestine, when the bacteria invade and colonize in human gut. In order to analyze the role of Neu5Ac catabolism in Vibrio vulnificus pathogenesis, a mutant with disruption of the nanA gene encoding Neu5Ac lyase was constructed by allelic exchanges. The nanA mutant was not able to utilize Neu5Ac as a sole carbon source and revealed an altered colony morphotype with reduced opacity in the presence of Neu5Ac. Compared to the wild type, the nanA mutant exhibited a low level of cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. The disruption of nanA also resulted in a substantial decrease in histopathological damage in jejunum and colon tissues from the mouse intestine. These results indicated that NanA plays an important role in V. vulnificus pathogenesis. In addition, the nanA mutant was significantly diminished in growth with and adherence to INT-407 epithelial cells in vitro, and was defective for intestinal colonization, reflecting the impaired ability of the mutant to grow and survive with, persist in, and adhere to the intestine in vivo. Consequently, the combined results suggest that NanA and the capability of catabolic utilization of Neu5Ac contribute to V. vulnificus virulence by ensuring growth, adhesion, and survival during infection.

Differential modulation of NF-κB-mediated pro-inflammatory response in human intestinal epithelial cells by cheY homologues of Vibrio cholerae

Vibrio cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial surface. Chemotaxis and motility greatly influence the infectivity of V. cholerae although the role of chemotaxis genes in V. cholerae pathogenesis is less well understood. Four cheY genes are present in three clusters in the complete genome sequence of V. cholerae. A less motile and less adherent mutant was generated by inactivation of cheY-3 (O395Y3N) or cheY-4 (O395Y4N) whereas alterations in motility or adherence were not observed for cheY-1 (O395Y1N) or cheY-2 (O395Y2N) insertional mutants. In contrast to O395Y1N and O395Y2N, O395Y3N and O395Y4N showed reduced cholera toxin production compared to wild-type in vitro. Infection of the human intestinal epithelial cell line Int407 with O395Y3N and O395Y4N caused reduced secretion of interleukin (IL)-1a, IL-6, tumor necrosis factor (TNF-a) and monocyte chemotactic protein-1 (MCP-1) compared to wild-type and was associated with delayed activation of nuclear factor kappa B (NF-kappaB) p65 and its co-activator cAMP response element binding protein (CREB). Further, the absence of nuclear translocation of NF-kappaB p50 subunit upon infection with O395Y3N or O395Y4N and its reversal upon complementation indicates the involvement of cheY-3 and cheY-4 in V. cholerae-induced pro-inflammatory response in the INT407 cell line.

Cellular response induced by a galactose-specific adhesin of enteroaggregativeEscherichia coliin INT-407 cells

In the present study, the role of a fimbrial galactose-specific adhesin of the T7 strain of enteroaggregative Escherichia coli (EAEC-T7) in the signal transduction pathways in human small intestinal epithelial cells (INT-407) was explored. The adhesin was purified by anion exchange chromatography using a Mono Q HR5/5 column in the AKTA purifier system. The characteristic stacked brick pattern of aggregative adherence of EAEC-T7 to INT-407 cells was found to be inhibited in the presence of immunoglobulin G against the purified adhesin as well as d-galactose. The adhesin induced a significant increase in the intracellular calcium concentration [Ca(2+)](i) in INT-407 cells, which was reduced in the presence of dantrolene (inhibitor of intracellular calcium stores), verapamil, calciseptin (calcium channel blockers) as well as neomycin [inhibitor of phospholipase C (PLC)]. Further, an increased level of PLCgamma1 and inositol 1,4,5-tri phosphate as well as enhanced activity of protein kinase C (PKC) in the adhesin-stimulated cells were found to be downregulated in the presence of neomycin and U73122 (inhibitors of PLC) and H-7 (inhibitor of PKC), respectively. The adhesin could also induce interleukin-8 secretion from INT-407 cells, which was inhibited in the presence of dantrolene as well as staurosporin (inhibitor of PKC). Collectively, our results have suggested that the galactose-specific adhesin-induced signal transduction pathway might play a crucial role in the EAEC-induced pathogenesis.

PCR Detection of Four Virulence-Associated Genes of Campylobacter jejuni Isolates from Thai Broilers and Their Abilities of Adhesion to and Invasion of INT-407 Cells

Campylobacter jejuni is a major cause of food borne pathogens in humans and a major reservoir for this pathogen is poultry. The C. jejuni in broilers was investigated from in the caeca of broilers. Twenty broiler/flock samples from 7 flocks were assessed. The average prevalence of C. jejuni was 65% in the broiler flocks. The adhesion and invasion ability of 48 strains of C. jejuni on INT 407 were studied. The adhesion and invasion ability of 48 Campylobacter isolates from caecal contents were analyzed with Human embryonic intestine (INT-407) cells being used as a gentamicin resistance assay. The caecal isolates exhibited a wide range of adherence and invasion ability. There was a significant correlation (p<0.01) between the adherence and the invasion ability of the Campylobacter isolates. Each of the virulence-associated genes: dnaJ, cadF, pldA and ciaB was detected by polymerase chain reaction from 100, 76, 31 and 41% of the Campylobacter strains, respectively. All of four virulence-associated genes were detected in 11 isolates. However, there was unclear association between the invasion ability and the presence of virulence-associated genes in this experiment, suggesting that more genes may be involved in the invasion process.

Boswellic acid inhibits expression of acid sphingomyelinase in intestinal cells

BackgroundBoswellic acid is a type of triterpenoids with antiinflammatory and antiproliferative properties. Sphingomyelin metabolism generates multiple lipid signals affecting cell proliferation, inflammation, and apoptosis. Upregulation of acid sphingomyelinase (SMase) has been found in several inflammation-related diseases such as inflammatory bowel diseases, atherosclerosis, and diabetes.MethodsThe present study is to examine the effect of 3-acetyl-11-keto-β-boswellic acids (AKBA), a potent boswellic acid, on acid SMase activity and expression in intestinal cells. Both transformed Caco-2 cells and non-transformed Int407 cells were incubated with AKBA. After incubation, the change of acid SMase activity was assayed biochemically, the enzyme protein was examined by Western blot, and acid SMase mRNA was quantified by qPCR.ResultsWe found that AKBA decreased acid SMase activity in both intestinal cell lines in dose and time dependent manners without affecting the secretion of the enzyme to the cell culture medium. The effect of AKBA was more effective in the fetal bovine serum-free culture medium. Among different types of boswellic acid, AKBA was the most potent one. The inhibitory effect on acid SMase activity occurred only in the intact cells but not in cell-free extract in the test tubes. At low concentration, AKBA only decreased the acid SMase activity but not the quantity of the enzyme protein. However, at high concentration, AKBA decreased both the mass of acid SMase protein and the mRNA levels of acid SMase in the cells, as demonstrated by Western blot and qPCR, respectively. Under the concentrations decreasing acid SMase activity, AKBA significantly inhibited cell proliferation.ConclusionWe identified a novel inhibitory effect of boswellic acids on acid SMase expression, which may have implications in human diseases and health.

The CprS sensor kinase of the zoonotic pathogen Campylobacter jejuni influences biofilm formation and is required for optimal chick colonization.

Campylobacter jejuni, a prevalent cause of bacterial gastroenteritis, must adapt to different environments to be a successful pathogen. We previously identified a C. jejuni two-component regulatory system (Cj1226/7c) as upregulated during cell infections. Analyses described herein led us to designate the system CprRS (Campylobacter planktonic growth regulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Delta cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Delta cprS also displayed a striking dose-dependent defect for colonization of chicks and was modestly enhanced for intracellular survival in INT407 cells. Proteomics analyses identified changes consistent with modulation of essential metabolic genes, upregulation of stress tolerance proteins, and increased expression of MOMP and FlaA. Consistent with expression profiling, we observed enhanced motility and secretion in Delta cprS, and decreased osmotolerance and oxidative stress tolerance. We also found that C. jejuni biofilms contain a DNase I-sensitive component and that biofilm formation is influenced by deoxycholate and the metabolic substrate fumarate. These results suggest that CprRS influences expression of factors important for biofilm formation, colonization and stress tolerance, and also add to our understanding of C. jejuni biofilm physiology.

Involvement of intracellular signaling cascades in inflammatory responses in human intestinal epithelial cells following Vibrio cholerae infection

Vibrio cholerae, the etiological agent of cholera, leads to the induction of host cell nuclear responses and the activation of proinflammatory cytokines in the cultured intestinal epithelial cells. However, the host cell signaling pathway leading to proinflammatory response is not explored. In this study, we demonstrated that V. cholerae infection on intestinal epithelial cells results in the activation of extracellular signal-regulated kinases1/2(ERK1/2) and p38 of the mitogen activated protein kinase (MAPK) family. V. cholerae induced intracellular pathways in Int407 cells leading to the activation of protein kinase A (PKA) and protein tyrosin kinase (PTK) in upstream of MAPK and nuclear factor-kappaB (NF-κB) pathway. Inhibitor study of Ca2+ and phospholipase-gamma (PLC-γ) pathway suggested the possible involvement of Ca2+ signaling in the V. cholerae pathogenesis. V. cholerae culture supernatants as also insertional mutants of ctxA, toxR and toxT genes modulate the activation of MAPK and NF-κB signaling pathways. MAPK and NF-κB signaling pathway activation were also modulated by adherence and motility of V. cholerae. Studies with inhibitor of NF-κB, MAPK, PTK, PKA, PKC, Ca2+ and PLC pathways showed differential cytokine secretion in Int407 following V. cholerae infection. Therefore V. cholerae mediated induction of nuclear responses through signal transduction pathway and subsequent activation of proinflammatory cytokines in Int407 modulated by V. cholerae secretory factors, virulence, adhesion/motility which might explain some of its reactogenic mechanisms.

Enhanced Microscopic Definition of Campylobacter jejuni 81-176 Adherence to, Invasion of, Translocation across, and Exocytosis from Polarized Human Intestinal Caco-2 Cells

Campylobacter jejuni-mediated pathogenesis involves gut adherence and translocation across intestinal cells. The current study was undertaken to examine the C. jejuni interaction with and translocation across differentiated Caco-2 cells to better understand Campylobacter's pathogenesis. The efficiency of C. jejuni 81-176 invasion of Caco-2 cells was two- to threefold less than the efficiency of invasion of INT407 cells. Adherence-invasion analyses indicated that C. jejuni 81-176 adhered to most INT407 cells but invaded only about two-thirds of the host cells over 2 h (two bacteria/cell). In contrast, only 11 to 17% of differentiated Caco-2 cells were observed to bind and internalize either C. jejuni strain 81-176 or NCTC 11168, and a small percentage of infected Caco-2 cells contained 5 to 20 internalized bacteria per cell after 2 h. Electron microscopy revealed that individual C. jejuni cells adhered to the tips of host cell microvilli via intimate flagellar contacts and by lateral bacterial binding to the sides of microvilli. Next, bacteria were observed to bind at the apical host membrane surface via presumed interactions at one pole of the bacterium and with host membrane protrusions located near intercellular junctions. The latter contacts apparently resulted in coordinated, localized plasma membrane invagination, causing simultaneous internalization of bacteria into an endosome. Passage of this Campylobacter endosome intracellularly from the apical surface to the basolateral surface occurred over time, and bacterial release apparently resulted from endosome-basolateral membrane fusion (i.e., exocytosis). Bacteria were found intercellularly below tight junctions at 60 min postinfection, but not at earlier times. This study revealed unique host cell adherence contacts, early endocytosis-specific structures, and a presumptive exocytosis component of the transcellular transcytosis route.

Role of the Campylobacter jejuni Cj1461 DNA Methyltransferase in Regulating Virulence Characteristics

Mutation of the cj1461 predicted methyltransferase gene reduced the motility of Campylobacter jejuni 81-176. Electron microscopy revealed that the mutant strain had flagella but with aberrant structure. The Deltacj1461 mutant was sevenfold more adherent to but 50-fold less invasive of INT-407 human epithelial cells than the wild type.

Molecular typing, serotyping and cytotoxicity testing ofCampylobacter jejunistrains isolated from commercial broilers in Puerto Rico

Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 microg ml(-1)) and trimethoprim (MIC of >32 microg ml(-1)); few were resistant to clindamycin (MIC(90) 4 microg ml(-1)), erythromycin (MIC(90) 8 microg ml(-1)) and tetracycline (MIC(90) 8 microg ml(-1)); but none was resistant to azithromycin (MIC(90) 4 microg ml(-1)), ciprofloxacin (MIC(90) 1 microg ml(-1)) or gentamycin (MIC(90) 4 microg ml(-1)). Most strains restricted with SmaI, but a combination of SmaI-KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST-2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco-2 cells, medium cytotoxicity with INT-407 and Hep-2 cells and high cytotoxicity with CHO cells. A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.

Vibrio vulnificus rtxE Is Important for Virulence, and Its Expression Is Induced by Exposure to Host Cells

Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity to INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. A transposon-tagging method was used to identify and clone an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from Vibrio cholerae. Functions of the rtxE gene in virulence were assessed by constructing an isogenic mutant whose rtxE gene was inactivated by allelic exchanges and by evaluating the differences between its virulence phenotype and that of the wild type in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity against epithelial cells in vitro. Also, the intraperitoneal 50% lethal dose of the rtxE mutant was 10(4) to 10(5) times higher than that of the parental wild type, indicating that RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter, P(rtxBDE). The activity of V. vulnificus P(rtxBDE) is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria with the host cells.