Ozan Gundogdu

Re-annotation and re-analysis of the Campylobacter jejuni NCTC11168 genome sequence

BackgroundCampylobacter jejuni is the leading bacterial cause of human gastroenteritis in the developed world. To improve our understanding of this important human pathogen, the C. jejuni NCTC11168 genome was sequenced and published in 2000. The original annotation was a milestone in Campylobacter research, but is outdated. We now describe the complete re-annotation and re-analysis of the C. jejuni NCTC11168 genome using current database information, novel tools and annotation techniques not used during the original annotation.ResultsRe-annotation was carried out using sequence database searches such as FASTA, along with programs such as TMHMM for additional support. The re-annotation also utilises sequence data from additional Campylobacter strains and species not available during the original annotation. Re-annotation was accompanied by a full literature search that was incorporated into the updated EMBL file [EMBL: AL111168]. The C. jejuni NCTC11168 re-annotation reduced the total number of coding sequences from 1654 to 1643, of which 90.0% have additional information regarding the identification of new motifs and/or relevant literature. Re-annotation has led to 18.2% of coding sequence product functions being revised.ConclusionsMajor updates were made to genes involved in the biosynthesis of important surface structures such as lipooligosaccharide, capsule and both O- and N-linked glycosylation. This re-annotation will be a key resource for Campylobacter research and will also provide a prototype for the re-annotation and re-interpretation of other bacterial genomes.

Campylobacter jejuni Outer Membrane Vesicles Play an Important Role in Bacterial Interactions with Human Intestinal Epithelial Cells

ABSTRACT Campylobacter jejuni is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However, C. jejuni lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of C. jejuni proteins into host cells. Proteomic analysis of C. jejuni 11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT). C. jejuni OMVs contained 16 N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells. C. jejuni OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with C. jejuni OMVs. OMVs isolated from a C. jejuni 11168H cdtA mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

The Campylobacter jejuni Transcriptional Regulator Cj1556 Plays a Role in the Oxidative and Aerobic Stress Response and Is Important for Bacterial Survival In Vivo

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide. Despite stringent microaerobic growth requirements, C. jejuni is ubiquitous in the aerobic environment and so must possess regulatory systems to sense and adapt to external stimuli, such as oxidative and aerobic (O(2)) stress. Reannotation of the C. jejuni NCTC11168 genome sequence identified Cj1556 (originally annotated as a hypothetical protein) as a MarR family transcriptional regulator, and further analysis indicated a potential role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic stress, decreased ability for intracellular survival in Caco-2 human intestinal epithelial cells and J774A.1 mouse macrophages, and a reduction in virulence in the Galleria mellonella infection model. Microarray analysis of gene expression changes in the Cj1556 mutant indicated negative autoregulation of Cj1556 expression and downregulation of genes associated with oxidative and aerobic stress responses, such as katA, perR, and hspR. Electrophoretic mobility shift assays confirmed the binding of recombinant Cj1556 to the promoter region upstream of the Cj1556 gene. cprS, which encodes a sensor kinase involved in regulation of biofilm formation, was also upregulated in the Cj1556 mutant, and subsequent studies showed that the mutant had a reduced ability to form biofilms. This study identified a novel C. jejuni transcriptional regulator, Cj1556, that is involved in oxidative and aerobic stress responses and is important for the survival of C. jejuni in the natural environment and in vivo.

Campylobacter jejuni Lipooligosaccharide Sialylation, Phosphorylation, and Amide/Ester Linkage Modifications Fine-tune Human Toll-like Receptor 4 Activation

Background: Campylobacter jejuni lipooligosaccharide (LOS) is a critical determinant of host innate immunity. Results: Three structural features of the LOS moiety vary significantly between strains and in combination impact monocyte activation. Conclusion: Variation of LOS structure suggests that LOS-TLR4 engagement during C. jejuni infection is strain-specific. Significance: Source and variation of LOS structure among C. jejuni strains may impact host proinflammatory responses. Campylobacter jejuni is a leading cause of acute gastroenteritis. C. jejuni lipooligosaccharide (LOS) is a potent activator of Toll-like receptor (TLR) 4-mediated innate immunity. Structural variations of the LOS have been previously reported in the oligosaccharide (OS) moiety, the disaccharide lipid A (LA) backbone, and the phosphorylation of the LA. Here, we studied LOS structural variation between C. jejuni strains associated with different ecological sources and analyzed their ability to activate TLR4 function. MALDI-TOF MS was performed to characterize structural variation in both the OS and LA among 15 different C. jejuni isolates. Cytokine induction in THP-1 cells and primary monocytes was correlated with LOS structural variation in each strain. Additionally, structural variation was correlated with the source of each strain. OS sialylation, increasing abundance of LA d-glucosamine versus 2,3-diamino-2,3-dideoxy-d-glucose, and phosphorylation status all correlated with TLR4 activation as measured in THP-1 cells and monocytes. Importantly, LOS-induced inflammatory responses were similar to those elicited by live bacteria, highlighting the prominent contribution of the LOS component in driving host immunity. OS sialylation status but not LA structure showed significant association with strains clustering with livestock sources. Our study highlights how variations in three structural components of C. jejuni LOS alter TLR4 activation and consequent monocyte activation.

Campylobacter jejuni outer membrane vesicle‐associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E‐cadherin and occludin

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram‐negative bacteria. Campylobacter jejuni produces OMVs that trigger IL‐8, IL‐6, hBD‐3 and TNF‐α responses from T84 intestinal epithelial cells and are cytotoxic to Caco‐2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E‐cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co‐incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E‐cadherin and occludin. The addition of 11168H OMVs to the co‐culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time‐dependent and dose‐dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells.

Increase in Campylobacter jejuni Invasion of Intestinal Epithelial Cells under Low-Oxygen Coculture Conditions That Reflect the In Vivo Environment

ABSTRACT Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses.

The ability of the human intestinal pathogen Campylobacter jejuni to respond to oxidative stress is central to bacterial survival both in vivo during infection and in the environment. Re-annotation of the C. jejuni NCTC11168 genome revealed the presence of two MarR-type transcriptional regulators Cj1546 and Cj1556, originally annotated as hypothetical proteins, which we have designated RrpA and RrpB (regulator of response to peroxide) respectively. Previously we demonstrated a role for RrpB in both oxidative and aerobic (O2) stress and that RrpB was a DNA binding protein with auto-regulatory activity, typical of MarR-type transcriptional regulators. In this study, we show that RrpA is also a DNA binding protein and that a rrpA mutant in strain 11168H exhibits increased sensitivity to hydrogen peroxide oxidative stress. Mutation of either rrpA or rrpB reduces catalase (KatA) expression. However, a rrpAB double mutant exhibits higher levels of resistance to hydrogen peroxide oxidative stress, with levels of KatA expression similar to the wild-type strain. Mutation of either rrpA or rrpB also results in a reduction in the level of katA expression, but this reduction was not observed in the rrpAB double mutant. Neither the rrpA nor rrpB mutant exhibits any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibit a reduced ability to survive aerobic (O2) stress, enhanced biofilm formation and reduced virulence in the Galleria mellonella infection model. The rrpAB double mutant exhibits wild-type levels of biofilm formation and wild-type levels of virulence in the G mellonella infection model. Together these data indicate a role for both RrpA and RrpB in the C. jejuni peroxide oxidative and aerobic (O2) stress responses, enhancing bacterial survival in vivo and in the environment.

Prevalence of Type VI Secretion System in Spanish Campylobacter jejuni Isolates

Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole‐genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS‐positive C. jejuni strains.

Assessment of the influence of intrinsic environmental and geographical factors on the bacterial ecology of pit latrines

Improving the rate and extent of faecal decomposition in basic forms of sanitation such as pit latrines would benefit around 1.7 billion users worldwide, but to do so requires a major advance in our understanding of the biology of these systems. As a critical first step, bacterial diversity and composition was studied in 30 latrines in Tanzania and Vietnam using pyrosequencing of 16S rRNA genes, and correlated with a number of intrinsic environmental factors such as pH, temperature, organic matter content/composition and geographical factors. Clear differences were observed at the operational taxonomic unit, family and phylum level in terms of richness and community composition between latrines in Tanzania and Vietnam. The results also clearly show that environmental variables, particularly substrate type and availability, can exert a strong structuring influence on bacterial communities in latrines from both countries. The origins and significance of these environmental differences are discussed. This work describes the bacterial ecology of pit latrines in combination with inherent latrine characteristics at an unprecedented level of detail. As such, it provides useful baseline information for future studies that aim to understand the factors that affect decomposition rates in pit latrines.

The Campylobacter jejuni Oxidative Stress Regulator RrpB Is Associated with a Genomic Hypervariable Region and Altered Oxidative Stress Resistance

Campylobacter jejuni is the leading cause of bacterial foodborne diarrhoeal disease worldwide. Despite the microaerophilic nature of the bacterium, C. jejuni can survive the atmospheric oxygen conditions in the environment. Bacteria that can survive either within a host or in the environment like C. jejuni require variable responses to survive the stresses associated with exposure to different levels of reactive oxygen species. The MarR-type transcriptional regulators RrpA and RrpB have recently been shown to play a role in controlling both the C. jejuni oxidative and aerobic stress responses. Analysis of 3,746 C. jejuni and 486 C. coli genome sequences showed that whilst rrpA is present in over 99% of C. jejuni strains, the presence of rrpB is restricted and appears to correlate with specific MLST clonal complexes (predominantly ST-21 and ST-61). C. coli strains in contrast lack both rrpA and rrpB. In C. jejuni rrpB+ strains, the rrpB gene is located within a variable genomic region containing the IF subtype of the type I Restriction-Modification (hsd) system, whilst this variable genomic region in C. jejuni rrpB- strains contains the IAB subtype hsd system and not the rrpB gene. C. jejuni rrpB- strains exhibit greater resistance to peroxide and aerobic stress than C. jejuni rrpB+ strains. Inactivation of rrpA resulted in increased sensitivity to peroxide stress in rrpB+ strains, but not in rrpB- strains. Mutation of rrpA resulted in reduced killing of Galleria mellonella larvae and enhanced biofilm formation independent of rrpB status. The oxidative and aerobic stress responses of rrpB- and rrpB+ strains suggest adaptation of C. jejuni within different hosts and niches that can be linked to specific MLST clonal complexes.