Re-annotation of the Campylobacter jejuni NCTC11168 genome and functional characterisation of selected genes involved in strain pathogenesis

Campylobacter jejuni is the leading bacterial cause of foodborne human gastroenteritis worldwide. The first C. jejuni genome (strain NCTC11168) was sequenced in 2000. This original annotation was a milestone in Campylobacter research, but soon became outdated. A re-annotation and re-analysis of this genome sequence was performed resulting in updates to over 90% of coding sequences (CDSs) and modification of 18.2% of CDS product functions (Gundogdu et al., 2007). Following this re-annotation, 15 uncharacterised CDSs with revised functions relating to virulence, signal transduction or regulation of gene expression were selected for further investigation. Defined isogenic C. jejuni 11168H mutants were constructed and after preliminary analysis, the Cj1556 and Cj0248 mutants were selected for further characterisation. Cj1556 was originally annotated as a hypothetical protein and was updated to a MarR family transcriptional regulator. Further bioinformatic analysis indicated a putative role in regulating the oxidative stress response. A C. jejuni 11168H Cj1556 mutant exhibited increased sensitivity to oxidative and aerobic (O2) stress, decreased ability for intracellular survival in both Caco-2 intestinal epithelial cells (IECs) 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 compared to the wild-type strain indicated negative autoregulation of Cj1556 expression and down-regulation of genes associated with oxidative and aerobic (O2) stress responses. Cj0248 was originally annotated as a hypothetical protein however the re-annotation identified a HD domain linked to a superfamily of metal-dependent phosphohydrolases with roles in signal transduction in bacteria. Previously a C. jejuni 81-176 Cj0248 mutant was shown to be deficient for motility and chick colonisation, however the exact function of Cj0248 was not investigated. The C. jejuni 11168H Cj0248 mutant also possessed a reduced motility phenotype and exhibited reduced interaction and invasion when co-cultured with Caco-2 IECs compared to the wild-type strain. However the Cj0248 mutant showed no difference in autoagglutination compared to the wild-type strain and TEM analysis indicated the mutant possessed intact flagella. Higher magnification TEM indicated the possibility of an altered flagella basal body region in the Cj0248 mutant. Secretion profile analysis identified no differences in the protein profile of the Cj0248 mutant compared to the wild-type strain. The exact function of Cj0248 remains unclear

Foodborne Pathogen Campylobacter

Publication history: Accepted - 3 June 2021; Published online - 8 June 2021

Prevalence and persistence of Listeria monocytogenes in premises and products of small food business operators in Northern Ireland

peer-reviewedListeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the elderly. Under European Union legislation, EC 2073/2005, food business operators are encouraged to undertake sampling to ensure that the food processing environment, and required to ensure that food products, are free of Listeria monocytogenes. To determine the prevalence of L. monocytogenes in smaller food processing facilities in Northern Ireland, 24 companies submitted six processing environment swabs and two food samples every two months for eighteen months (July 2015 to November 2016) for L. monocytogenes examination. The prevalence of L. monocytogenes was 4.6% in food samples, and 6.3% in processing environment swabs. Over the duration of the study, 96 isolates of L. monocytogenes were obtained, one from each positive sample, except for two meat samples that had >100 cfu/g, where two isolates were obtained from each sample. No seasonality in occurrence of L. monocytogenes was seen for food isolates but significantly higher numbers of positive processing environment swabs were found in the warmer months of May, July and September (p = .007). Pulsed Field Gel Electrophoresis (PFGE) analysis revealed the presence of 27 pulsotypes; 9 pulsotypes were shared between different facilities and 9 were persistent. Based on a Combase predictive growth model, 77.5% (n = 130) of the foods tested were predicted to support the growth of L. monocytogenes. All of the isolates carried the pathogenicity genes inlA and actA and 71.4% carried qacH, which confers resistance to quaternary ammonium compounds which are frequently used in sanitizers. Whole genome sequencing of the isolates allowed multi-locus sequence typing to be undertaken. The data indicated that the sequence types identified included those with disease-causing ability, highlighting the disease-causing potential of the isolates

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

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

BACKGROUND: Campylobacter 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. RESULTS: Re-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. CONCLUSIONS: Major 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

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

Funding Information: This research received financial support from the Bill and Melinda Gates Foundation (grant number OPP52641). AWW and JP were supported by the Wellcome Trust [grant number 098051]. AWW and the Rowett Institute of Nutrition and Health, University of Aberdeen, receive core funding support from the Scottish Government Rural and Environmental Science and Analysis Service (RESAS). UZ is funded by Natural Environment Research Council (NERC) Independent Research Fellowship (NE/L011956/1). CQ is funded through an Medical Research Council fellowship (MR/M50161X/1) as part of the MRC Cloud Infrastructure for Microbial Bioinformatics consortium (MR/L015080/1).Peer reviewedPublisher PD

Revisiting Campylobacter jejuni Virulence and Fitness Factors: Role in Sensing, Adapting, and Competing.

Campylobacter jejuni is the leading cause of bacterial foodborne gastroenteritis world wide and represents a major public health concern. Over the past two decades, significant progress in functional genomics, proteomics, enzymatic-based virulence profiling (EBVP), and the cellular biology of C. jejuni have improved our basic understanding of this important pathogen. We review key advances in our understanding of the multitude of emerging virulence factors that influence the outcome of C. jejuni-mediated infections. We highlight, the spatial and temporal dynamics of factors that promote C. jejuni to sense, adapt and survive in multiple hosts. Finally, we propose cohesive research directions to obtain a comprehensive understanding of C. jejuni virulence mechanisms

Investigating the Role of FlhF Identifies Novel Interactions With Genes Involved in Flagellar Synthesis in Campylobacter jejuni.

FlhF is a key protein required for complete flagellar synthesis, and its deletion results in the complete absence of a flagella and thus motility in Campylobacter jejuni. However, the specific mechanism still remains unknown. In this study, RNA-Seq, EMSAs, ChIP-qPCR and β-Galactosidase assays were performed to elucidate the novel interactions between FlhF and genes involved in flagellar synthesis. Results showed that FlhF has an overall influence on the transcription of flagellar genes with an flhF mutant displaying down-regulation of most flagellar related genes. FlhF can directly bind to the flgI promoter to regulate its expression, which has significant expression change in an flhF mutant. The possible binding site of FlhF to the flgI promoter was explored by continuously narrowing the flgI promoter region and performing further point mutations. Meanwhile, FlhF can directly bind to the promoters of rpoD, flgS, and fliA encoding early flagellin regulators, thereby directly or indirectly regulating the synthesis of class I, II, and III flagellar genes, respectively. Collectively, this study demonstrates that FlhF may directly regulate the transcription of flagellar genes by binding to their promoters as a transcriptional regulator, which will be helpful in understanding the mechanism of FlhF in flagellar biosynthetic and bacterial flagellation in general

Antiviral activity of a novel mixture of natural antimicrobials, in vitro, and in a chicken infection model in vivo.

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2