Examination of nanoparticle inactivation of Campylobacter jejuni biofilms using infrared and Raman spectroscopies.

AIMS: To investigate inactivation effect and mechanism of zinc oxide nanoparticles (ZnO NPs) activity against Campylobacter jejuni biofilms. METHODS AND RESULTS: ZnO NPs with concentrations of 0, 0·6, 1·2 and 6 mmol l(-1) were employed in antimicrobial tests against Camp. jejuni planktonic cells and biofilms. Campylobacter jejuni sessile cells in biofilms were more resistant to a low concentration of ZnO NPs when compared to planktonic cells. The ZnO NPs penetrated the extracellular polymeric substance (EPS) without damage to the EPS and directly interacted with the sessile bacterial cells, as determined using infrared spectroscopy and scanning electron microscopy. Raman spectroscopy shows alterations in quinone structures and damage to nucleic acids following Camp. jejuni treatment with ZnO NPs. The mechanism of DNA damage is most likely due to the generation of reactive oxygen species (ROS). Spectroscopic-based partial least squares regression (PLSR) models could predict the number of surviving sessile cell numbers within a bacterial biofilm (≥log 4 CFU, root mean square error of estimation <0·36) from Fourier transform infrared (FT-IR) spectral measurements. CONCLUSIONS: ZnO NPs were found to have antimicrobial activity against Camp. jejuni biofilms. ZnO NPs penetrated the biofilm EPS within 1 h without damaging it and interacted directly with sessile cells in biofilms. Alterations in the DNA/RNA bases, which are owing to the generation of ROS, appear to result in Camp. jejuni cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: ZnO NPs may offer a realistic strategy to eliminate Camp. jejuni biofilms in the environment.

Formation and movement of myosin-containing structures in living fibroblasts.

Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, the fluorescent myosin showed a distribution identical to that revealed by immunofluorescence with antimyosin antibodies. Injected myosin became localized in small beads, which were found along large stress fibers, along fine fibers, and in a poorly organized form near the lamellipodia. De novo assembly of beads was observed continuously within or near the lamellipodia, suggesting that myosin molecules may undergo a constant cycling between polymerized and unpolymerized states. The nascent structures then moved away from lamellipodia and became organized into linear arrays. Similar movement was also observed for beads already associated with linear structures, and may represent a continuous flux of myosin structures. The dynamic reorganization of myosin may play an important role in cell movement and polarity.

Identification of a functional homolog of the Escherichia coli and Salmonella typhimurium cysM gene encoding O-acetylserine sulfhydrylase B in Campylobacter jejuni.

The final step of L-cysteine biosynthesis in Escherichia coli and Salmonella typhimurium consists of the formation of L-cysteine from O-acetylserine and sulfide. This reaction can be catalyzed by two enzymes, O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B, the former of which has been more rigorously characterized. In contrast to O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B is preferentially used for cysteine biosynthesis during anaerobic growth and is able to utilize thiosulfate as a substrate. Campylobacter jejuni is a micro-aerophilic, Gram-negative bacterium, and a member of the epsilon subdivision of eubacteria. We have cloned, sequenced, and expressed a gene from C. jejuni that encodes a protein of 299 aa with a calculated molecular mass of 32,367 Da. Complementation analysis of an E. coli cysteine auxotroph with the pMEK34-14 recombinant plasmid containing a 1.2-kb insert of chromosomal DNA from C. jejuni revealed that transformants were capable of growth in medium containing either sulfide or thiosulfate as sole sulfur sources. These data indicate that the cloned C. jejuni gene is a functional homolog of the cysM gene that codes for O-acetylserine sulfhydrylase B in E. coli and S. typhimurium.

Cloning, sequencing, and characterization of the lipopolysaccharide biosynthetic enzyme heptosyltransferase I gene (waaC) from Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are common causes of gastrointestinal disease and a proportion of C. jejuni infections have been shown to be associated with the Guillain-Barré syndrome. The waaC gene from Campylobacter coli, involved in lipopolysaccharide core biosynthesis, was cloned by complementation of a heptose-deficient strain of Salmonella typhimurium, as judged by novobiocin sensitivity, lipopolysaccharide (LPS)-specific phage sensitivity, and polyacrylamide-resolved lipopolysaccharide profiles. The C. jejuni waaC gene was subsequently cloned using the waaC gene isolated from C. coli as a probe. The C. jejuni and C. coli waaC genes are capable of encoding proteins of 342 amino acids with calculated molecular masses of 39381Da and 39317Da, respectively. Sequence and in-vitro analyses suggested that the C. coli waaC gene may be transcribed from its own promoter. Translation of the C. coli waaC gene in a cell-free system yielded a protein with a Mr of 39000. The waaC gene was detected in every C. jejuni and C. coli isolate tested as judged by dot-blot hybridization analysis. Southern hybridization analysis indicated that both Campylobacter species contain a single copy of the waaC gene. Unlike Escherichia coli and S. typhimurium isolates, the waaC gene in C. jejuni and C. coli isolates does not appear to be linked to the waaF (rfaF) gene.

Cloning and expression of the hup encoding a histone-like protein of Campylobacter jejuni.

A Campylobacter jejuni gene, designated hup, that appears to encode a homolog of the histone-like DNA-binding protein, HU, has been cloned, sequenced and expressed in Escherichia coli. Immunoblotting and in vitro transcription/translation analyses revealed a 11-kDa protein that was produced by recombinant plasmids containing hup. The gene contains an open reading frame (ORF) sufficient to encode a protein of 98 amino acids (aa) with a calculated molecular mass of 10,267 Da and a predicted isoelectric point of 10.1. The deduced aa sequence of the protein, designated HCj, exhibits considerable sequence identity with members of the HU family of proteins from other eubacterial species. The transcription start point was identified by primer extension analysis and appropriately spaced promoter sequences were found which exhibit considerable similarity to E. coli and Bacillus promoters. Southern hybridization analyses indicate that C. jejuni has a single copy of hup.

Temperature-regulated expression of bacterial virulence genes.

Virulence gene expression in most bacteria is a highly regulated phenomenon, affected by a variety of parameters including osmolarity, pH, ion concentration, iron levels, growth phase, and population density. Virulence genes are also regulated by temperature, which acts as an 'on-off' switch in a manner distinct from the more general heat-shock response. Here, we review temperature-responsive expression of virulence genes in four diverse pathogens.

Factors that influence the interaction of Campylobacter jejuni with cultured mammalian cells.

Although Campylobacter jejuni is now recognised as a common enteric pathogen, the mechanisms by which this organism produces enteritis remain ill-defined. It has been proposed that its abilities to adhere to and enter epithelial cells represent properties essential to virulence. However, the characteristics of these interactions and factors that may influence the association of C. jejuni with epithelial cells are incompletely described. We have determined that the ability of C. jejuni to bind to epithelial cell lines in vitro is significantly affected by the growth temperature and growth stage of the bacteria, but not by growth-medium composition. Binding of C. jejuni to cultured cells is not affected by temperature or phylogenetic origin of the target cell, and exhibits a non-uniform or patchy distribution. In contrast, internalisation is markedly diminished at low temperature, appears to involve active invagination of the target cell membrane via pseudopod formation, and is maximal when cells of human origin are employed.

The absence of cecal colonization of chicks by a mutant of Campylobacter jejuni not expressing bacterial fibronectin-binding protein.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.

Role of Campylobacter jejuni potential virulence genes in cecal colonization.

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.

Secretion of Campylobacter jejuni Cia proteins is contact dependent.

Campylobacter jejuni is a common cause of human gastrointestinal disease worldwide. Despite the prevalence of C. jejuni infections, the mechanisms of C. jejuni pathogenesis remain ill-defined. Invasion of the cells lining the intestinal tract is hypothesized to be essential for the development of C. jejuni-mediated enteritis. Recent studies in our laboratory have revealed that C. jejuni secrete proteins, termed Cia for Campylobacter invasion antigens, upon incubation with human intestinal cells. A mutation in one of the genes encoding a secreted protein resulted in an invasion-deficient phenotype. The purpose of this study was to identify a component capable of stimulating the synthesis and secretion of the Cia proteins from C. jejuni. Here, we report that these processes can be induced upon incubating C. jejuni in medium supplemented with fetal bovine serum. The synthesis and secretion of the Cia proteins were not affected by heat-treatment of the fetal bovine serum, indicating that the stimulating molecule in serum is heat stable. The stimulatory molecule was not unique to fetal bovine serum as sera from other sources including human, pig, sheep, goat, rabbit, mouse, and chicken also induced the synthesis and release of the Cia proteins. These findings indicate that the synthesis and secretion of the Cia proteins can be induced in a cell-free system by incubating C. jejuni in serum-supplemented tissue culture medium.

Codon usage in the A/T-rich bacterium Campylobacter jejuni.

Campylobacter jejuni is a Gram negative, microaerophilic pathogen that causes gastroenteritis in humans. The genome of C. jejuni is AT-rich, with a mol% G + C of 30.4. This high AT content was hypothesized to result in unique codon usage. In the present study, we analyzed the codon usage of sixty-seven C. jejuni genes and generated a codon frequency table. As predicted, the codon usage of C. jejuni revealed a strong bias towards codons ending in A or U. In addition to determining codon usage frequencies, the relative synonymous codon usage values were calculated to identify rare and optimal codons. Seventeen codons were identified as optimal and twelve codons as rare. Thirty-two codons exhibited little or no bias. A plot of the effective number of codons versus the third position %G + C values for the sixty-seven genes revealed that C. jejuni uses an average of 39 of the 61 codons to encode proteins. These data will be useful for various molecular analyses including selection of degenerate primers to screen C. jejuni-genomic DNA libraries.

Identification of proteins required for the internalization of Campylobacter jejuni into cultured mammalian cells.

Clinical and in vitro experimental data suggest that invasion of intestinal epithelial cells is an essential step in the pathogenesis of Campylobacter jejuni-mediated enteritis. However, the molecular mechanism of C. jejuni internalization remains poorly defined. The goal of this study was to identify a gene that encodes a protein required for the internalization of C. jejuni into host cells. A C. jejuni gene, designated ciaB, was identified upon immunoscreening C. jejuni genomic DNA-phage libraries with an antiserum generated against C. jejuni co-cultivated with INT 407 cells. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73,154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins, associated with invasion of host cells, from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Immunoblot analysis using an anti-CiaB specific antibody revealed that CiaB is secreted into the supernatant fluids upon co-cultivation of C. jejuni with INT 407 cell conditioned medium. Metabolic labeling experiments revealed that at least eight C. jejuni proteins, ranging in size from 12.8 to 108 kDa, are secreted into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all proteins, indicating that CiaB is required for the secretion process. Identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization.

Capsaicin-sensitive vagal afferent neurons contribute to the detection of pathogenic bacterial colonization in the gut.

Vagal activation can reduce inflammation and disease activity in various animal models of intestinal inflammation via the cholinergic anti-inflammatory pathway. In the current model of this pathway, activation of descending vagal efferents is dependent on a signal initiated by stimulation of vagal afferents. However, little is known about how vagal afferents are activated, especially in the context of subclinical or clinical pathogenic bacterial infection. To address this question, we first determined if selective lesions of capsaicin-sensitive vagal afferents altered c-Fos expression in the nucleus of the solitary tract (nTS) after mice were inoculated with either Campylobacter jejuni or Salmonella typhimurium. Our results demonstrate that the activation of nTS neurons by intraluminal pathogenic bacteria is dependent on intact, capsaicin sensitive vagal afferents. We next determined if inflammatory mediators could cause the observed increase in c-Fos expression in the nTS by a direct action on vagal afferents. This was tested by the use of single-cell calcium measurements in cultured vagal afferent neurons. We found that tumor necrosis factor alpha (TNFα) and lipopolysaccharide (LPS) directly activate cultured vagal afferent neurons and that almost all TNFα and LPS responsive neurons were sensitive to capsaicin. We conclude that activation of the afferent arm of the parasympathetic neuroimmune reflex by pathogenic bacteria in the gut is dependent on capsaicin sensitive vagal afferent neurons and that the release of inflammatory mediators into intestinal tissue can be directly sensed by these neurons.

Adhesion to and invasion of HEp-2 cells by Campylobacter spp.

Twenty-one isolates were tested for their ability to adhere to and invade HEp-2 cells in vitro. Of the 21 organisms tested, 2 did not invade the HEp-2 cells, and 1 of these did not adhere to the epithelial cells. Campylobacter jejuni clinical isolates were more invasive than the nonclinical strains that were tested. When HEp-2 cells were treated with cytochalasin B, the invasiveness of C. jejuni was reduced, indicating active participation of the host cell in the uptake of these organisms. The number of intracellular C. jejuni isolates decreased when Campylobacter whole-cell lysates were absorbed onto HEp-2 cell monolayers. Experiments were also conducted to identify the functional sites of the antigens responsible for expression of Campylobacter invasion. Oxidation of lysates with sodium meta-periodate significantly affected its inhibitory capacity. This implies that the Campylobacter invasive ligand appears to be dependent upon an intact carbohydrate moiety.

Flow cytometric detection of host cell apoptosis induced by bacterial infection.

We detail two methods for detection of cell death induced by infection of a human monocytic cell line with invasive Campylobacter bacteria. Staining with a natural ligand for exposed phosphatidylserine residues coupled with propidum iodide discriminated between apoptosis and necrosis. Additionally, cells infected with a bacterial strain expressing green fluorescent protein stained with dye sensitive to mitochondrial membrane potential demonstrated a direct association of bacteria with dying cells. Analyses of cells stained by these methods employing flow cytometry enumerated proportions of cell populations undergoing either apoptosis or necrosis after bacterial infection in vitro.

Effect of enteroviruses on adherence to and invasion of HEp-2 cells by Campylobacter isolates.

Coinfection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, poliovirus (LSc type 1), porcine enterovirus, and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of Campylobacter jejuni ATCC 33560 was significantly increased for HEp-2 cells preinfected with echovirus 7, coxsackievirus B3, and UV-inactivated (noninfectious) coxsackievirus B3 particles. Additionally, the invasiveness of C. jejuni M96, a clinical isolate, was significantly increased for HEp-2 cells preinfected with coxsackievirus B3. Poliovirus and porcine enterovirus had no effect on C. jejuni ATCC 33560 adherence and invasiveness. Furthermore, poliovirus had no effect on the ability of C. jejuni M96 to adhere to and invade HEp-2 cells. Campylobacter hyointestinalis and Campylobacter mucosalis, two noninvasive isolates, did not invade virus-infected HEp-2 cells. The increase in the invasiveness of C. jejuni appeared to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter spp. is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter spp. but are not sufficient to allow internalization of noninvasive bacteria.

Altered synthetic response of Campylobacter jejuni to cocultivation with human epithelial cells is associated with enhanced internalization.

Campylobacter jejuni has been shown to bind to and enter epithelial cells in culture. The interaction of C. jejuni with INT 407 epithelial cells was examined to determine whether bacterial protein synthesis is required for either binding or internalization. Chloramphenicol, a selective inhibitor of bacterial protein synthesis, significantly reduced the internalization, but not binding, of C. jejuni compared with untreated controls as determined by protection from gentamicin. Electrophoretic analysis of metabolically labeled proteins revealed that C. jejuni cultured with INT 407 cells synthesized 14 proteins that were not detected in organisms cultured in medium alone. The inhibitory effect of chloramphenicol on internalization was reduced by preincubation of C. jejuni with INT 407 cells. The results indicate that C. jejuni, like some other enteric pathogens, engages in a directed response to cocultivation with epithelial cells by synthesizing one or more proteins that facilitate internalization and suggest that this phenomenon is relevant to the pathogenesis of enteritis caused by C. jejuni.

Molecular characterization of a Campylobacter jejuni 29-kilodalton periplasmic binding protein.

Campylobacter jejuni, a gram-negative, microaerophilic, spiral bacterium, is a common cause of human gastrointestinal disease. Although investigators commonly use C. jejuni glycine-hydrochloride extracts in assays to determine the products that promote the binding of the organism to eukaryotic cells, the proteins contained within these extracts remain ill defined. Characterization of these proteins will provide a better understanding of C. jejuni gene regulation and organization. An antiserum was raised against a C. jejuni 29-kDa gel-purified protein detected in glycine-hydrochloride extracts. This antiserum was used to screen an expression library of C. jejuni. A reactive clone that contained an open reading frame of 256 amino acids was identified. The cloned gene was transcribed and translated, and the product was exported to the periplasmic space in Escherichia coli XL1-Blue. The translated C. jejuni product, designated P29, exhibited significant similarity to the histidine and lysine-arginine-ornithine periplasmic binding proteins (HisJ and LAO, respectively) of Salmonella typhimurium. The C. jejuni gene encoding the P29 protein complemented an S. typhimurium HisJ mutant but not a LAO mutant when provided in trans. These data suggest that the C. jejuni gene encoding the P29 protein is a homolog of the S. typhimurium hisJ gene.

Invasion-related antigens of Campylobacter jejuni.

A HEp-2 cell culture model was used to investigate the antigens required for epithelial cell penetration by Campylobacter jejuni. Penetration of HEp-2 epithelial cells by C. jejuni was significantly inhibited (P less than .05) with C. jejuni lysate and a monoclonal antibody (MAb 1B4) in competitive inhibition assays. Immunogold electron microscopy revealed that MAb 1B4 bound to the flagella and cell surface of low-passage (invasive) C. jejuni M 96, whereas only the flagella of high-passage (noninvasive) C. jejuni M 96 were labeled. Western blot analysis revealed that MAb 1B4 identified an epitope on antigens of 64-44 kDa in lysates prepared from invasive and noninvasive isolates. In addition, antigens of 42-38 kDa were recognized in lysates prepared from only invasive C. jejuni strains. Proteinase K and sodium meta-periodate chemical treatment of C. jejuni M 96 lysate changed the mobility of antigens recognized by MAb 1B4. The increase in mobility demonstrated a decrease in size of molecules and suggested that antigens required for HEp-2 cell invasion by Campylobacter species may be glycoprotein in nature.