Campylobacter jejuni FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence

Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.Members of the genus Campylobacter are gram-negative, asaccharolytic, motile bacteria, which grow optimally in the laboratory at temperatures between 37 and 42°C under microaerophilic conditions. Although members of Campylobacter spp. were initially recognized to cause disease in sheep and cattle, Campylobacter jejuni was not recognized as a human pathogen until much later (25). Infection of humans with C. jejuni is characterized by a rapid onset of fever, abdominal cramps, and diarrhea. C. jejuni is now recognized as one of the leading bacterial causes of gastroenteritis in the world. In spite of the incidence of campylobacteriosis, relatively few C. jejuni virulence genes have been characterized, and our understanding of the virulence properties of C. jejuni is limited compared with that of other enteric pathogens, including Salmonella, Shigella, and Yersinia spp.The ability of C. jejuni to cause disease is a complex, multifactorial process. Virulence factors that contribute to the pathogenesis of C. jejuni are associated with motility, host (target) cell adherence, host cell invasion, protein secretion, alteration of host cell signaling pathways, induction of host cell death, evasion of host immune defenses, iron acquisition, and drug/detergent resistance (14, 18). The binding of C. jejuni to specific host cell ligands is hypothesized to play a fundamental role in host colonization and disease progression, since it prevents the organism''s clearance from the intestine by peristalsis and fluid flow. Fauchere et al. (5) reported that C. jejuni isolates recovered from individuals with fever and diarrhea adhered to cultured cells in greater numbers than isolates recovered from asymptomatic individuals. While there is no evidence indicating that C. jejuni produces fimbriae that assist in host colonization (7), a number of constitutively synthesized proteins have been proposed to act as adhesins. Bacterial adhesins are surface-exposed macromolecules that facilitate an organism''s binding to the host cell receptors. Known and putative C. jejuni adhesins include CadF, CapA, FlpA, and PorA (MOMP) (6).An emerging theme among pathogenic microorganisms is their ability to utilize host cell molecules during the infectious process to facilitate their binding and entry into host cells (27). More specifically, many bacterial pathogens have been found to bind to fibronectin (Fn), which in turn modifies host cell signaling pathways to the pathogen''s advantage. Fn exists as a dimer of nearly identical 250-kDa subunits that are linked by a pair of disulfide bonds near their C termini. Each Fn monomer is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids) (22). In total, each monomer contains 12 type I repeats, two type II repeats, and 15 to 17 type III repeats. Fn participates in many cellular interactions, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. Plasma Fn, which is synthesized by hepatocytes, is soluble (22). In contrast, Fn involved in host cell-extracellular matrix (ECM) interaction, which is synthesized by chondrocytes, fibroblasts, endothelial cells, macrophages, and certain epithelial cells, is present in an insoluble form (22). Fn serves as an adhesion molecule that anchors cells to ECM components, including collagen and other proteoglycan substrates.The bacterial proteins that bind to ECM components have been termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (23). The C. jejuni CadF protein is a member of the MSCRAMM family and one of the most extensively characterized C. jejuni virulence determinants (10-12, 15, 16, 19-21, 24, 28). CadF mediates the binding of C. jejuni to Fn, promotes bacterium-host cell interactions, and facilitates the organism''s colonization of chickens (10, 11, 15, 16, 20, 21, 28). In addition to CadF, we recently reported that a mutation in Cj1279c resulted in a C. jejuni mutant that poorly colonized broiler chickens compared with a C. jejuni wild-type strain. The product encoded by the Cj1279c gene was termed Fibronectin-like protein A (FlpA) because the protein harbors Fn type III domains (6). The goal of this study was to characterize the binding properties of FlpA and to determine if this protein is a member of the MSCRAMM family. Here we provide experimental evidence that C. jejuni FlpA is surface exposed, promotes the bacterium''s attachment to host epithelial cells, and has Fn binding activity. Assays were also performed to determine if CadF and FlpA act cooperatively to promote binding of C. jejuni to host cells and Fn. We submit that the identification of a second MSCRAMM in C. jejuni highlights the importance of Fn binding in host colonization and disease.     

Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization

Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.     

The Carboxin-Binding Site on Paracoccus denitrificans Succinate:Quinone Reductase Identified by Mutations

Succinate:quinone reductase catalyzes electron transfer from succinate to quinone in aerobic respiration. Carboxin is a specific inhibitor of this enzyme from several different organisms. We have isolated mutant strains of the bacterium Paracoccus denitrificans that are resistant to carboxin due to mutations in the succinate:quinone reductase. The mutations identify two amino acid residues, His228 in SdhB and Asp89 in SdhD, that most likely constitute part of a carboxin-binding site. This site is in the same region of the enzyme as the proposed active site for ubiquinone reduction. From the combined mutant data and structural information derived from Escherichia coli and Wolinella succinogenes quinol:fumarate reductase, we suggest that carboxin acts by blocking binding of ubiquinone to the active site. The block would be either by direct exclusion of ubiquinone from the active site or by occlusion of a pore that leads to the active site.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

The Campylobacter jejuni Cj0268c Protein Is Required for Adhesion and Invasion In Vitro

Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.     

The Intestinal Microbiota Influences Campylobacter jejuni Colonization and Extraintestinal Dissemination in Mice

Campylobacter jejuni is a leading cause of human foodborne gastroenteritis worldwide. The interactions between this pathogen and the intestinal microbiome within a host are of interest as endogenous intestinal microbiota mediates a form of resistance to the pathogen. This resistance, termed colonization resistance, is the ability of commensal microbiota to prevent colonization by exogenous pathogens or opportunistic commensals. Although mice normally demonstrate colonization resistance to C. jejuni, we found that mice treated with ampicillin are colonized by C. jejuni, with recovery of Campylobacter from the colon, mesenteric lymph nodes, and spleen. Furthermore, there was a significant reduction in recovery of C. jejuni from ampicillin-treated mice inoculated with a C. jejuni virulence mutant (ΔflgL strain) compared to recovery of mice inoculated with the C. jejuni wild-type strain or the C. jejuni complemented isolate (ΔflgL/flgL). Comparative analysis of the microbiota from nontreated and ampicillin-treated CBA/J mice led to the identification of a lactic acid-fermenting isolate of Enterococcus faecalis that prevented C. jejuni growth in vitro and limited C. jejuni colonization of mice. Next-generation sequencing of DNA from fecal pellets that were collected from ampicillin-treated CBA/J mice revealed a significant decrease in diversity of operational taxonomic units (OTUs) compared to that in control (nontreated) mice. Taken together, we have demonstrated that treatment of mice with ampicillin alters the intestinal microbiota and permits C. jejuni colonization. These findings provide valuable insights for researchers using mice to investigate C. jejuni colonization factors, virulence determinants, or the mechanistic basis of probiotics.     

Pentavalent Single-Domain Antibodies Reduce Campylobacter jejuni Motility and Colonization in Chickens

Campylobacter jejuni is the leading cause of bacterial foodborne illness in the world, with symptoms ranging from acute diarrhea to severe neurological disorders. Contaminated poultry meat is a major source of C. jejuni infection, and therefore, strategies to reduce this organism in poultry, are expected to reduce the incidence of Campylobacter-associated diseases. We have investigated whether oral administration of C. jejuni-specific single-domain antibodies would reduce bacterial colonization levels in chickens. Llama single-domain antibodies specific for C. jejuni were isolated from a phage display library generated from the heavy chain IgG variable domain repertoire of a llama immunized with C. jejuni flagella. Two flagella-specific single-domain antibodies were pentamerized to yield high avidity antibodies capable of multivalent binding to the target antigen. When administered orally to C. jejuni-infected two-day old chicks, the pentabodies significantly reduced C. jejuni colonization in the ceca. In vitro, the motility of the bacteria was also reduced in the presence of the flagella-specific pentabodies, suggesting the mechanism of action is through either direct interference with flagellar motility or antibody-mediated aggregation. Fluorescent microscopy and Western blot analyses revealed specific binding of the anti-flagella pentabodies to the C. jejuni flagellin.     

A Novel Campylobacter jejuni Two-Component Regulatory System Important for Temperature-Dependent Growth and Colonization

Campylobacter jejuni colonizes the intestines of domestic and wild animals and is a common cause of human diarrheal disease. We identified a two-component regulatory system, designated the RacR-RacS (reduced ability to colonize) system, that is involved in a temperature-dependent signalling pathway. A mutation of the response regulator gene racR reduced the organism's ability to colonize the chicken intestinal tract and resulted in temperature-dependent changes in its protein profile and growth characteristics.     

Campylobacter jejuni Type VI Secretion System: Roles in Adaptation to Deoxycholic Acid, Host Cell Adherence, Invasion, and In Vivo Colonization

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes - survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%-0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10(tm1Cgn) mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to DCA, providing new insights into the role of T6SS in C. jejuni pathogenesis.     

Campylobacter jejuni Colonization in Wild Birds: Results from an Infection Experiment

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.     

Anaerobic Expression of Escherichia coli Succinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the P_FRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.     

Important Role for the Murid Herpesvirus 4 Ribonucleotide Reductase Large Subunit in Host Colonization via the Respiratory Tract

Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint—the replicative potential of spontaneous enzyme mutants—has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.Cellular deoxyribonucleotide synthesis is strongly cell cycle dependent. DNA viruses replicating in noncycling cells must therefore either induce cellular enzymes or supply their own. Most herpesviruses encode multiple homologs of nucleotide metabolism enzymes, including both subunits of the cellular ribonucleotide reductase (RNR) (4). While most in vivo cells are resting, most in vitro cell lines divide continuously (29). The importance of viral RNRs may therefore only be apparent in vivo (14). In contrast to alpha- and betaherpesviruses, gammaherpesviruses cause disease mainly through latency-associated cell proliferation. However, gamma-2 herpesviruses show lytic gene expression in sites of latency (9, 17), and lytic reactivation could potentially alleviate some gammaherpesvirus-infected cancers (7, 8). Therefore, it is important also to understand the pathogenetic roles of gammaherpesvirus lytic cycle enzymes, such as RNR.The known human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) have narrow species tropisms that preclude most pathogenesis studies. In contrast, murid herpesvirus 4 (MuHV-4) (21, 26) allows gammaherpesvirus host colonization to be studied in vivo. After intranasal (i.n.) inoculation, MuHV-4 replicates lytically in lung epithelial cells before seeding to lymphoid tissue (27). Long-term virus loads are independent of extensive primary lytic spread (25). However, whether persistence requires some lytic gene expression remains unclear. Replication-deficient viral DNA reached the spleen after intraperitoneal (i.p.) but not i.n. virus inoculation (15, 20, 28), suggesting that virus dissemination from the lung to lymphoid tissue requires lytic replication. In addition, less invasive inoculations may increase further the viral functions required to establish a persistent infection. Thymidine kinase (TK)-deficient MuHV-4 given i.n. without general anesthesia, in which method the wild-type virus infects the upper respiratory tract and reaches lymphoid tissue without infecting the lungs (18), fails to colonize in mice at all (12). The implication is that virions using a likely physiological route of host entry must replicate in terminally differentiated cells to establish a significant infection. However, some unusual features of gammaherpesvirus TKs (11) suggest that they have functions besides thymidine phosphorylation. We therefore targeted here another enzyme linked to viral DNA replication, the MuHV-4 RNR. We aimed to define the in vivo importance of a potential therapeutic target and to advance generally our understanding of gammaherpesvirus pathogenesis.Transposon insertions in the MuHV-4 RNR small (ORF60) and large (ORF61) RNR subunit genes have been described as either attenuating or not for lytic replication in vitro (19, 23). We disrupted ORF61 (RNR⁻) by inserting stop codons close to its 5′ end (Fig. ​(Fig.11 a). An EcoRI-L genomic clone (coordinates 80644 to 84996) in pUC19 (6) was digested with AleI to remove nucleotides 82320 to 82534 of ORF61 (82865 to 80514). An oligonucleotide encoding multiple stop codons and an EcoRI restriction site (5′-CTAGCATGCTAGAATTCTAGCATGCATG-3′) was ligated in place. Nucleotides 81365 to 83883 were then PCR amplified, including a BamHI site in the 81365 primer, cloned as a BglII/BamHI fragment into the BamHI site of pST76K-SR, and recombined into a MuHV-4 bacterial artificial chromosome (BAC) (1). A revertant virus was made by reconstituting the corresponding, unmutated genomic fragment. Southern blots (5) of viral DNA (Fig. ​(Fig.1b)1b) confirmed the expected genomic structures, and immunoblots (5) of infected cell lysates (Fig. ​(Fig.1c)1c) established that mutant viruses no longer expressed the RNR large subunit.Open in a separate windowFIG. 1.Disruption of the MuHV-4 ORF61. (a) Schematic diagram of the ORF61 (RNR large) locus, showing the mutation introduced and relevant restriction sites. (b) Viral DNA was digested with EcoRI and probed for ORF61. Oligonucleotide insertion into ORF61 changes a 4,352-bp wild-type band to 2,462 bp plus 1,676 bp. The 2,462-bp fragment is not visible because it overlaps the probe by only 331 nucleotides (nt) and comigrates with a background band of unknown origin. WT, wild type; REV, revertant; RNR⁻, mutant; RNR⁻ ind, independent mutant. WT luc⁺ is MuHV-4 expressing luciferase from an ORF57/ORF58 intergenic cassette. RNR⁻ luc⁺ and RNR⁻ luc⁺ind have ORF61 disrupted on this background. (c) Infected cell lysates were immunoblotted for gp150 (virion envelope glycoprotein, monoclonal antibody [MAb] T1A1), ORF17 (capsid component, MAb 150-7D1), TK (tegument component, MAb CS-4A5), and ORF61 (MAb PS-8A7). (d) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (0.01 eGFP units/cell, 2 h, 37°C), washed two times with phosphate-buffered saline (PBS) to remove unbound virions, and cultured at 37°C to allow virus spread. Infectivity (in eGFP units) at each time point was determined on fresh BHK-21 cells in the presence of phosphonoacetic acid to prevent further viral spread, with the number of eGFP-postive cells counted 18 h later by flow cytometry. (e) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (2 eGFP units/cell, 2 h, 37°C), washed in medium (pH 3) to inactivate nonendocytosed virions, and cultured at 37°C to allow virus replication. The infectivity of replicate cultures was then assayed as described in the legend of panel d. (f) BHK-21 cells were incubated with RNR⁺ or RNR⁻ viruses (0.3 eGFP units/cell, 37°C) for the times indicated, and the numbers of eGFP-positive cells in the cultures were then determined by flow cytometry.RNR⁻ viruses were noticeably slower than RNR⁺ viruses when spreading through BHK-21 cell monolayers after BAC DNA transfection. Normalizing by immunoblot signal, RNR⁻ virus stocks had titers similar to that of the wild type by viral enhanced green fluorescent protein (eGFP) expression but 10- to 100-fold lower plaque titers. Using eGFP expression as a readout, RNR⁻ virion production after a low multiplicity of infection lagged 1 day behind that of the wild type (Fig. ​(Fig.1d).1d). Maximum infectivity yields were also reduced, but once BHK-21 cells become confluent, they support MuHV-4 lytic infection poorly, so this was probably a consequence of the slower lytic spread. After a high multiplicity of infection (Fig. ​(Fig.1e),1e), RNR⁻ mutants showed a 10-h lag in virion production and no difference in the final yield. They showed no defect in single-cycle eGFP expression (Fig. ​(Fig.1f),1f), implying normal virion entry. Therefore, the main RNR⁻ defect lay in infectious virion production.For in vivo experiments, the loxP-flanked viral BAC-eGFP cassette must be removed (1). Therefore, to monitor infection in vivo without having to rely on new virion production as a readout, we transferred the RNR mutation onto a luciferase-positive (luc⁺) background (18). Viral luciferase expression (from an early lytic promoter) by in vitro luminometry (18) was independent of either viral DNA replication or RNR expression (Fig. ​(Fig.22 a). After i.n. inoculation of anesthetized mice, RNR⁻ luciferase signals measured in vivo by i.p. luciferin injection and IVIS Lumina charge-coupled-device (CCD) camera scanning (18) were visible in lungs (Fig. ​(Fig.2b)2b) but were 100-fold lower than those of the RNR⁺ controls (Fig. ​(Fig.2c).2c). A severe impairment of RNR⁻ lytic replication was confirmed by plaque assay (18) (Fig. ​(Fig.2d);2d); the difference between RNR⁻ and RNR⁺ plaque titers greatly exceeded any difference in plaquing efficiency.Open in a separate windowFIG. 2.Host colonization by RNR⁻ MuHV-4 mutants. (a) BHK-21 cells were left uninfected or infected overnight with RNR⁺ or RNR⁻ luc⁺ MuHV-4 and then assayed for luciferase expression by luminometry. Phosphonoacetic acid (PAA; 100 μg/ml) was either added or not to cultures to block viral late gene expression. Each point shows the mean ± standard deviation from triplicate cultures. (b) BALB/c mice were infected i.n. under general anesthesia with RNR⁻ or RNR⁺ luc⁺ MuHV-4 (5 × 10³ PFU) and then assayed for luciferase expression by luciferin injection and CCD camera scanning. The images are from 5 days postinfection. Note that the RNR⁺ and RNR⁻ images have different sensitivity scales. (c) For quantitation, dorsal and ventral luciferase signals were summed. Each point shows 1 mouse. The dashed lines show detection thresholds. The RNR⁺ signal was significantly greater than the RNR⁻ signal for all sites and time points (P < 0.001 by Student''s t test). (d) C57BL/6 mice were infected i.n. under anesthesia with RNR⁻ or RNR⁺ MuHV-4 (5 × 10³ PFU). Five days later, infectious virus loads in noses and lungs were measured by plaque assay. Each point shows 1 mouse. RNR⁻ infections yielded no plaques and therefore are shown at the sensitivity limits of each assay. (e) BALB/c mice were infected i.n. with RNR⁻ or RNR⁺ MuHV-4 without anesthesia and then monitored by luciferin injection and CCD camera scanning. Each point shows the summed ventral and dorsal signals of the relevant region for 1 mouse. Neck signals correspond to the superficial cervical lymph nodes (SCLN). The dashed lines show detection thresholds. RNR⁻ luciferase signals were undetectable at all time points.No RNR⁻ luciferase signals were visible in noses, nor did RNR⁻ MuHV-4 give signals in the superficial cervical lymph nodes (SCLN), which drain the nose (Fig. ​(Fig.2c).2c). This lack of live imaging signals from the upper respiratory tract was confirmed by ex vivo imaging of SCLN at day 14 postinfection. We examined upper respiratory tract infection further with an independently derived luc⁺ RNR⁻ mutant, inoculating i.n. without anesthesia so as to avoid virus aspiration into the lungs. No RNR⁻ luciferase signals were detected, while wild-type signals were readily observed in the nose and superficial cervical lymph nodes (Fig. ​(Fig.2e2e).Like RNR⁻ MuHV-4, TK⁻ mutants are severely attenuated for lytic replication in the lower respiratory tract. However, they eventually establish a reactivatable latent infection and induce virus-specific antibody (3). Latent virus titers in spleens peak at 1 month postinoculation. Infectious center assays showed no RNR⁻ infection of spleens at that time (Fig. ​(Fig.33 a). We also looked for viral DNA in spleens by quantitative PCR (Fig. ​(Fig.3b).3b). Genomic coordinates 4166 to 4252 were amplified and hybridized to a probe with coordinates 4218 to 4189. Viral genome copies, relative to the cellular adenosine phosphoribosyl transferase copy number, were calculated from standard curves of cloned plasmid DNA (10). No RNR⁻ viral DNA was detected. ELISA for MuHV-4-specific serum IgG (24) detected an antibody response after lung infection but not upper respiratory tract infection of BALB/c mice with RNR⁻ MuHV-4 (Fig. ​(Fig.3c).3c). There was a similar lack of antibody 1 month after upper respiratory tract infection of C57BL/6 mice with independently derived RNR⁻ mutants (Fig. ​(Fig.3d)3d) and 3 months after exposure of 6 BALB/c mice to RNR⁻ luc⁺ MuHV-4. In contrast, i.p. RNR⁻ luc⁺ MuHV-4 gave lower luciferase signals than RNR⁺ luc⁺ MuHV-4 (Fig. ​(Fig.44 a), but RNR⁻ infectious centers (Fig. ​(Fig.4b)4b) and viral genomes (Fig. ​(Fig.4c)4c) were detected in spleens, and enzyme-linked immunosorbent assays (ELISAs) (Fig. ​(Fig.4d)4d) showed MuHV-4-specific serum IgG.Open in a separate windowFIG. 3.Spleen colonization by RNR⁻ MuHV-4. (a) BALB/c or C57BL/6 mice were infected i.n. either with general anesthesia (lung infection) or without (nose infection). One month later, spleens were assayed for recoverable latent virus by infectious center assay. Lower detection limit, 10 infectious centers per spleen. (b) The spleens described in the legend of panel a were further analyzed for viral DNA by quantitative PCR. Copy numbers are expressed relative to the cellular adenosine phosphoribosyl transferase copy number in each sample. The dashed lines show lower detection limits (1 viral copy/10,000 cellular copies). (c) Sera from BALB/c mice after i.n. infection either with (lung infection) or without (nose infection) general anesthesia were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance curve for 1 mouse. The dashed lines show naive serum. (d) Sera from C57BL/6 mice 1 month after infection with independent RNR mutants were analyzed for MuHV-4-specific IgG, as described in the legend to panel c.Open in a separate windowFIG. 4.Intraperitoneal infection with RNR⁺ and RNR⁻ MuHV-4. (a) Mice were infected i.p. with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 and then monitored for luciferase expression. Each point shows the total abdominal signal of 1 mouse. The x axis is at the lower limit of signal detection above the background. (b) Spleens were assayed for recoverable virus by infectious center assay 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4. Each point shows the titer of 1 mouse. One log₁₀ infectious center per mouse corresponds to the lower limit of detection. (c) Spleen DNA was analyzed for viral genome content by quantitative PCR. Each point shows viral copy/cellular copy for the mean of triplicate reactions for 1 mouse. (d) Sera taken 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance values for the serum of 1 mouse. “Naive” represents age-matched, uninfected controls.The failure of both the RNR large subunit (ORF61) and TK⁻ MuHV-4 mutants to infect via the upper respiratory tract argues that this requires viral replication in a nucleotide-poor cell. The additional lack of lymphoid RNR⁻ infection after inoculation into the lungs seemed likely to reflect a defect in virus transport, as RNR⁻ MuHV-4 did colonize the spleen after i.p. inoculation. It is also possible that the first cells infected simply produced no infectious virions, although this seemed a more likely explanation for upper respiratory tract infection being undetectable; lung infection progressed sufficiently to give detectable luciferase expression and to induce an antiviral antibody response. How transport from lung to lymphoid tissue occurs is unknown, but likely scenarios include latently infected dendritic cells (22) carrying MuHV-4 along afferent lymphatics to germinal centers and cell-free virions being captured in lymph nodes by subcapsular sinus macrophages (13). Therefore, RNR may be important for MuHV-4 to spread from myeloid cells to B cells.The difference between RNR⁻ and TK⁻ mutants in host colonization via the lung—TK⁻ mutants reached lymphoid tissue whereas RNR⁻ mutants did not—could reflect additional ORF61 functions, as precedent exists for functional drift (2, 16). Alternatively, RNR may be needed more than TK for MuHV-4 replication in some cell types. Formidable hurdles to RNR-based therapies remain: human gammaherpesvirus infections rarely present until latency is well established, so blocking virus spread to lymphoid tissue may have a limited impact, and no drugs sufficiently selective to target viral RNRs in a clinical setting have yet emerged. Nevertheless, the severe in vivo attenuation of RNR⁻ MuHV-4 suggested that RNR may be a viable target for limiting gammaherpesvirus lytic spread.     

A Novel Serine/Threonine Protein Kinase Homologue of Pseudomonas aeruginosa Is Specifically Inducible within the Host Infection Site and Is Required for Full Virulence in Neutropenic Mice

A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.     

PCR-ELISAs for the detection of Campylobacter jejuni and Campylobacter coli in poultry samples

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on Nucleo-Link wells and hybridized to PCR products modified with a 5' biotin moiety. The limit of detection of the PCR-ELISA was 100-300 fg (40-120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.     

FlhF and Its GTPase Activity Are Required for Distinct Processes in Flagellar Gene Regulation and Biosynthesis in Campylobacter jejuni

FlhF proteins are putative GTPases that are often necessary for one or more steps in flagellar organelle development in polarly flagellated bacteria. In Campylobacter jejuni, FlhF is required for σ⁵⁴-dependent flagellar gene expression and flagellar biosynthesis, but how FlhF influences these processes is unknown. Furthermore, the GTPase activity of any FlhF protein and the requirement of this speculated activity for steps in flagellar biosynthesis remain uncharacterized. We show here that C. jejuni FlhF hydrolyzes GTP, indicating that these proteins are GTPases. C. jejuni mutants producing FlhF proteins with reduced GTPase activity were not severely defective for σ⁵⁴-dependent flagellar gene expression, unlike a mutant lacking FlhF. Instead, these mutants had a propensity to lack flagella or produce flagella in improper numbers or at nonpolar locations, indicating that GTP hydrolysis by FlhF is required for proper flagellar biosynthesis. Additional studies focused on elucidating a possible role for FlhF in σ⁵⁴-dependent flagellar gene expression were conducted. These studies revealed that FlhF does not influence production of or signaling between the flagellar export apparatus and the FlgSR two-component regulatory system to activate σ⁵⁴. Instead, our data suggest that FlhF functions in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway to influence σ⁵⁴-dependent gene expression. This study provides corroborative biochemical and genetic analyses suggesting that different activities of the C. jejuni FlhF GTPase are required for distinct steps in flagellar gene expression and biosynthesis. Our findings are likely applicable to many polarly flagellated bacteria that utilize FlhF in flagellar biosynthesis processes.Flagellar biosynthesis in bacteria is a complex process that requires expression of more than 50 genes in a sequential manner to ensure that the encoded proteins are secreted and interact in a proper order to construct a flagellar organelle (8). Formation of a flagellum to impart swimming motility is often an essential determinant for many bacteria to infect hosts or reside in an environmental niche. As such, flagella and flagellar motility are required for Campylobacter jejuni to initiate and maintain a harmless intestinal colonization in many wild and agriculturally important animals (16, 17, 19, 35, 47, 49), which leads to large reservoirs of the bacterium in the environment and the human food supply (13). In addition, flagellar motility is essential for the bacterium to infect human hosts to cause a diarrheal disease, which can range from a mild, watery enteritis to a severe, bloody diarrheal syndrome (4). Due to its prevalence in nature and in the food supply, C. jejuni is a leading cause of enteritis in humans throughout the world (7).C. jejuni belongs to a subset of motile bacteria that produce polarly localized flagella, which includes important pathogens of humans, such as Helicobacter, Vibrio, and Pseudomonas species. These bacteria have some commonalities in mechanisms for flagellar gene expression and biosynthesis, such as using both alternative σ factors, σ²⁸ and σ⁵⁴, for expression of distinct sets of flagellar genes (1, 6, 9, 11, 18, 20-22, 26, 36, 40, 44, 45, 49). In addition, these bacteria produce the putative FlhF GTPase, which is required in each bacterium for at least one of the following: expression of a subset of flagellar genes, biosynthesis of flagella, or the polar placement of the flagella. For instance, FlhF is required for expression of some σ⁵⁴- and σ²⁸-dependent flagellar genes and for production of flagella in the classical biotype of Vibrio cholerae (10). However, V. cholerae flhF mutants of another biotype can produce a flagellum in a minority of cells, but the flagellum is at a lateral site (14). Similar lateral flagella were found in flhF mutants of Pseudomonas aeruginosa and Pseudomonas putida (34, 37). FlhF of Vibrio alginolyticus may also be involved in the polar formation of flagella and may possibly influence the number of flagella produced (28, 29). Demonstration that FlhF is polarly localized in some of these species and the fact that FlhF has been observed to assist the early flagellar MS ring protein, FliF, in localizing to the old pole in one biotype of V. cholerae give credence that FlhF may be involved in the polar placement of flagella in the respective organisms (14, 29, 34).Bioinformatic analysis indicates that the FlhF proteins belong to the SIMIBI class of NTP-binding proteins (30). More specifically, the GTPase domains of FlhF proteins are most similar to those of the signal recognition particle (SRP) pathway GTPases, such as Ffh and FtsY. Because of the homology of the GTPase domains, these three proteins may form a unique subset within the SIMIBI proteins. Whereas the GTPase activities of the interacting Ffh and FtsY proteins have been extensively characterized (32, 38, 39, 42), little is known about the GTP hydrolysis activity of FlhF. Structural determination of FlhF of Bacillus subtilis indicates that the potential GTPase activity of FlhF is likely varied relative to those of Ffh and FtsY (2). However, no biochemical analysis has been performed to verify or characterize the ability of an FlhF protein to hydrolyze GTP. As such, no studies have correlated the biochemical activity of FlhF in relation to GTP hydrolysis with the role that FlhF performs in flagellar gene expression or biosynthesis.Through previous work, we have delineated the regulatory cascades governing flagellar gene expression in C. jejuni. We have found that formation of the flagellar export apparatus (FEA), a multiprotein inner membrane complex (consisting of the proteins FlhA, FlhB, FliF, FliO, FliP, FliQ, and FliR) that secretes most of the flagellar proteins out of the cytoplasm to form the flagellum, is required to activate the FlgS sensor kinase to begin a phosphorelay to the cognate FlgR response regulator (23, 24). Once activated by phosphorylation, FlgR likely interacts with σ⁵⁴ in RNA polymerase to initiate expression of many flagellar genes encoding components of the flagellar basal body, rod, and hook (20, 24). After formation of the hook, flaA, encoding the major flagellin, is expressed via σ²⁸ and RNA polymerase to generate the flagellar filament and complete flagellar biosynthesis (6, 18, 20, 21, 49). In two separate genetic analyses, we found that flhF mutants of C. jejuni are nonmotile and show a more than 10-fold reduction in expression of σ⁵⁴-dependent flagellar genes, indicating that FlhF is required for both flagellar gene expression and biosynthesis (20). However, it is unclear how FlhF influences expression of σ⁵⁴-dependent flagellar genes. Furthermore, it is unknown if the GTPase activity of FlhF is required for flagellar gene expression or biosynthesis in C. jejuni.We have performed experiments to determine that C. jejuni FlhF specifically hydrolyzes GTP, confirming that FlhF is a GTPase. Whereas the FlhF protein is required for motility, flagellar biosynthesis, and expression of σ⁵⁴-dependent flagellar genes, the GTPase activity of the protein significantly influences only proper biosynthesis of flagella. These results suggest that multiple biochemical activities of FlhF (including GTPase activity and likely other, as yet uncharacterized activities mediated by other domains) are required at distinct steps in flagellar gene expression and biosynthesis. In addition, we provide biochemical and genetic evidence that FlhF likely functions in a pathway separate from the FEA-FlgSR pathway in C. jejuni to influence expression of σ⁵⁴-dependent flagellar genes. This study provides corroborative genetic and biochemical analysis of FlhF to indicate that FlhF has multiple inherent activities that function at different steps in development of the flagellar organelle, which may be applicable to many polarly flagellated bacteria.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.