The Campylobacter jejuni response regulator, CbrR, modulates sodium deoxycholate resistance and chicken colonization

Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of Campylobacter jejuni strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the C. jejuni F38011 wild-type strain. Complementation of the C. jejuni cbrR mutant in trans restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as cbrR (Campylobacter bile resistance regulator). While the MICs of a variety of bile salts and other detergents for the C. jejuni cbrR mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the C. jejuni cbrR mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that bile resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates resistance to bile salts in C. jejuni.     

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 ( C ampylobacter p lanktonic growth r egulation). While the response regulator was essential, a cprS sensor kinase mutant was viable. The Δ cprS mutant displayed an apparent growth defect and formed dramatically enhanced and accelerated biofilms independent of upregulation of previously characterized surface polysaccharides. Δ 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 Δ 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.     

Cellular response of Campylobacter jejuni to trisodium phosphate

The highly alkaline compound trisodium phosphate (TSP) is used as an intervention to reduce the load of Campylobacter on poultry meat in U.S. poultry slaughter plants. The aim of the present study was to investigate the cellular responses of Campylobacter jejuni NCTC11168 when exposed to sublethal concentrations of TSP. Preexposure of C. jejuni to TSP resulted in a significant increase in heat sensitivity, suggesting that a combined heat and TSP treatment may increase reduction of C. jejuni. A microarray analysis identified a limited number of genes that were differently expressed after sublethal TSP exposure; however, the response was mainly associated with ion transport processes. C. jejuni NCTC11168 nhaA1 (Cj1655c) and nhaA2 (Cj1654c), which encode orthologues to the Escherichia coli NhaA cation/proton antiporter, were able to partially restore TSP, alkaline, and sodium resistance phenotypes to an E. coli cation/proton antiporter mutant. In addition, inhibition of resistance-nodulation-cell division (RND) multidrug efflux pumps by the inhibitor PaβN (Phe-Arg β-naphthylamide dihydrochloride) decreased tolerance to sublethal TSP. Therefore, we propose that NhaA1/NhaA2 cation/proton antiporters and RND multidrug efflux pumps function in tolerance to sublethal TSP exposure in C. jejuni.     

The Campylobacter jejuni dccRS two-component system is required for optimal in vivo colonization but is dispensable for in vitro growth

A Campylobacter jejuni two-component signal transduction system (TCSTS), designated dccR-dccS (diminished capacity to colonize; Cj1223c-Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A DeltadccR response regulator mutant generated using the virulent strain 81-176 background exhibited significantly reduced colonization of immunocompetent limited flora (I-LF) mice, severe combined immunodeficient limited flora (SCID-LF) mice, and 1-day-old chicks. A DeltadccS sensor kinase mutant was likewise defective for colonization in the I-LF mouse model. DeltadccR-infected SCID-LF mice also exhibited dramatically reduced inflammation relative to wild type-infected SCID-LF mice. Despite this diminished colonization capacity, the DeltadccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two-component system; binding of purified His-tagged DccR to the promoter region of two of these genes supports a direct protein-DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two-component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.     

Regulation of oxidative stress response by CosR, an essential response regulator in Campylobacter jejuni

CosR (Campylobacter oxidative stress regulator; Cj0355c) is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a leading foodborne pathogen causing human gastroenteritis worldwide. Despite importance, the function of CosR remains completely unknown mainly because of cell death caused by its knockout mutation. To overcome this technical limitation, in this study, antisense technology was used to investigate the regulatory function of CosR by modulating the level of CosR expression. Two-dimensional gel electrophoresis (2DGE) was performed to identify the CosR regulon either by suppressing CosR expression with antisense peptide nucleic acid (PNA) or by overexpressing CosR in C. jejuni. According to the results of 2DGE, CosR regulated 32 proteins involved in various cellular processes. Notably, CosR negatively regulated a few key proteins of the oxidative stress response of C. jejuni, such as SodB, Dps, Rrc and LuxS, whereas CosR positively controlled AhpC. Electrophoretic mobility shift assay showed that CosR directly bound to the promoter region of the oxidative stress genes. DNase I footprinting assays identified 21-bp CosR binding sequences in the sodB and ahpC promoters, suggesting CosR specifically recognizes and binds to the regulated genes. Interestingly, the level of CosR protein was significantly reduced by paraquat (a superoxide generator) but not by hydrogen peroxide. Consistent with the overall negative regulation of oxidative stress defense proteins by CosR, the CosR knockdown by antisense rendered C. jejuni more resistant to oxidative stress compared to the wild type. Overall, this study reveals the important role played by the essential response regulator CosR in the oxidative stress defense of C. jejuni.     

Heme utilization in Campylobacter jejuni

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.     

Identification of a Campylobacter jejuni-secreted protein required for maximal invasion of host cells

The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter ( yplA ). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242 , and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.     

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.     

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein

Campylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain-Barré syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two-domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a K(d) of approximately 10(-7) M consistent with a role as a receptor for a high-affinity transporter. These data imply that Cj0982 is the binding protein component of an ABC-type cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni.     

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.