The CJIE1 prophage of Campylobacter jejuni affects protein expression in growth media with and without bile salts

BACKGROUND: The presence of Campylobacter jejuni temperate bacteriophages has increasingly been associated with specific biological effects. It has recently been demonstrated that the presence of the prophage CJIE1 is associated with increased adherence and invasion of C. jejuni isolates in cell culture assays. RESULTS: Quantitative comparative proteomics experiments were undertaken using three closely related isolates with CJIE1 and one isolate without CJIE1 to determine whether there was a corresponding difference in protein expression levels. Initial experiments indicated that about 2% of the total proteins characterized were expressed at different levels in isolates with or without the prophage. Some of these proteins regulated by the presence of CJIE1 were associated with virulence or regulatory functions. Additional experiments were conducted using C. jejuni isolates with and without CJIE1 grown on four different media: Mueller Hinton (MH) media containing blood; MH media containing 0.1% sodium deoxycholate, which is thought to result in increased expression of virulence proteins; MH media containing 2.5% Oxgall; and MHwithout additives. These experiments provided further evidence that CJIE1 affected protein expression, including virulence-associated proteins. They also demonstrated a general bile response involving a majority of the proteome and clearly showed the induction of almost all proteins known to be involved with iron acquisition. The data have been deposited to the ProteomeXchange with identifiers PXD000798, PXD000799, PXD000800, and PXD000801. CONCLUSION: The presence of the CJIE1 prophage was associated with differences in protein expression levels under different conditions. Further work is required to determine what genes are involved in causing this phenomenon

Mx2 expression is associated with reduced susceptibility to HIV infection in highly exposed HIV seronegative Kenyan sex workers.

CAPRISA, 2015.Abstract available in pdf

Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells.

CAPRISA, 2016.Abstract available in pdf

DNA sequence heterogeneity of Campylobacter jejuni CJIE4 prophages and expression of prophage genes.

Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not been well characterized. In this study we describe investigations into the DNA sequence variability and protein expression in a second prophage, CJIE4. CJIE4 sequences were obtained de novo from DNA sequencing of five C. jejuni isolates, as well as from whole genome sequences submitted to GenBank by other research groups. These CJIE4 DNA sequences were heterogenous, with several different insertions/deletions (indels) in different parts of the prophage genome. Two variants of a 3-4 kb region inserted within CJIE4 had different gene content that distinguished two major conserved CJIE4 prophage families. Additional indels were detected throughout the prophage. Detection of proteins in the five isolates characterized in our laboratory in isobaric Tags for Relative and Absolute Quantitation (iTRAQ) experiments indicated that prophage proteins within each of the two large indel variants were expressed during growth of the bacteria on Mueller Hinton agar plates. These proteins included the extracellular DNase associated with resistance to DNA transformation and prophage repressor proteins. Other proteins associated with known or suspected roles in prophage biology were also expressed from CJIE4, including capsid protein, the phage integrase, and MazF, a type II toxin-antitoxin system protein. Together with the results previously obtained for the CJIE1 prophage these results demonstrate that sequence variability and expression of moron genes are both general properties of temperate bacteriophages in C. jejuni

A Functional Phenylacetic Acid Catabolic Pathway Is Required for Full Pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans Host Model▿

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in paaE, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of B. cenocepacia K56-2 in relation to its pathogenicity in the Caenorhabditis elegans model of infection. We demonstrated that targeted-insertion mutagenesis of paaA and paaE, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system, paaZ, coding for a putative ring opening enzyme, and paaF, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon source. paaA and paaE insertional mutants were attenuated for virulence, and expression of paaE in trans restored pathogenicity of the paaE mutant to wild-type levels. Interruption of paaZ and paaF slightly increased virulence. Using gene interference by ingested double-stranded RNA, we showed that the attenuated phenotype of the paaA and paaE mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in C. elegans. Taken together, our results demonstrate that B. cenocepacia possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in C. elegans

Detection of proteins associated with the CJIE1 prophage after growth on Mueller-Hinton agar in iTRAQ labelling experiments.

<p>Detection of proteins associated with the CJIE1 prophage after growth on Mueller-Hinton agar in iTRAQ labelling experiments.</p

Schematic diagram demonstrating differences in Group 1 CJIE4 prophage DNA sequences.

<p>Strains carried the large indel containing genes encoding CJE1439-Cj1442, plus strain 414. The diagram includes prophages investigated in this work (see Materials and Methods) plus those obtained from NCBI databases. Sizes of genes are approximate, and any non-coding sequence between genes has not been shown to conserve space.</p

Comparison of sequence annotation for five CJIE4 prophages.

<p>Comparison of sequence annotation for five CJIE4 prophages.</p

Summary data for the 4 complete sequenced isolates and genomes.

<p>Summary data for the 4 complete sequenced isolates and genomes.</p

Comparison of genomes and proteomes of four whole genome-sequenced <i>Campylobacter jejuni</i> from different phylogenetic backgrounds

<div><p>Whole genome sequencing (WGS) has been used to assess the phylogenetic relationships, virulence and metabolic differences, and the relationship between gene carriage and host or niche differentiation among populations of <i>C</i>. <i>jejuni</i> isolates. We previously characterized the presence and expression of CJIE4 prophage proteins in four <i>C</i>. <i>jejuni</i> isolates using WGS and comparative proteomics analysis, but the isolates were not assessed further. In this study we compare the closed, finished genome sequences of these isolates to the total proteome. Genomes of the four isolates differ in phage content and location, plasmid content, capsular polysaccharide biosynthesis loci, a type VI secretion system, orientation of the ~92 kb invertible element, and allelic differences. Proteins with 99% sequence identity can be differentiated using isobaric tags for relative and absolute quantification (iTRAQ) comparative proteomic methods. GO enrichment analysis and the type of artefacts produced in comparative proteomic analysis depend on whether proteins are encoded in only one isolate or common to all isolates, whether different isolates have different alleles of the proteins analyzed, whether conserved and variable regions are both present in the protein group analyzed, and on how the analysis is done. Several proteins encoded by genes with very high levels of sequence identity in all four isolates exhibited preferentially higher protein expression in only one of the four isolates, suggesting differential regulation among the isolates. It is possible to analyze comparative protein expression in more distantly related isolates in the context of WGS data, though the results are more complex to interpret than when isolates are clonal or very closely related. Comparative proteomic analysis produced log₂ fold expression data suggestive of regulatory differences among isolates, indicating that it may be useful as a hypothesis generation exercise to identify regulated proteins and regulatory pathways for more detailed analysis.</p></div