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

Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity

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

Human Papillomavirus 16 E6 and E7 Oncoproteins Alter the Abundance of Proteins Associated with DNA Damage Response, Immune Signaling and Epidermal Differentiation

The high-risk human papillomaviruses are oncogenic viruses associated with almost all cases of cervical carcinomas, and increasing numbers of anal, and oral cancers. Two oncogenic HPV proteins, E6 and E7, are capable of immortalizing keratinocytes and are required for HPV associated cell transformation. Currently, the influence of these oncoproteins on the global regulation of the host proteome is not well defined. Liquid chromatography coupled with quantitative tandem mass spectrometry using isobaric-tagged peptides was used to investigate the effects of the HPV16 oncoproteins E6 and E7 on protein levels in human neonatal keratinocytes (HEKn). Pathway and gene ontology enrichment analyses revealed that the cells expressing the HPV oncoproteins have elevated levels of proteins related to interferon response, inflammation and DNA damage response, while the proteins related to cell organization and epithelial development are downregulated. This study identifies dysregulated pathways and potential biomarkers associated with HPV oncoproteins in primary keratinocytes which may have therapeutic implications. Most notably, DNA damage response pathways, DNA replication, and interferon signaling pathways were affected in cells transduced with HPV16 E6 and E7 lentiviruses. Moreover, proteins associated with cell organization and differentiation were significantly downregulated in keratinocytes expressing HPV16 E6 + E7. High-risk HPV E6 and E7 oncoproteins are necessary for the HPV-associated transformation of keratinocytes. However their influence on the global dysregulation of keratinocyte proteome is not well documented. Here shotgun proteomics using TMT-labeling detected over 2500 significantly dysregulated proteins associated with E6 and E7 expression. Networks of proteins related to interferon response, inflammation and DNA damage repair pathways were altered

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

Basic abundance variation of S100 calcium-dependent family members collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.

<p>Basic abundance variation of S100 calcium-dependent family members collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.</p

Unbiased Proteomics Analysis Demonstrates Significant Variability in Mucosal Immune Factor Expression Depending on the Site and Method of Collection

<div><p>Female genital tract secretions are commonly sampled by lavage of the ectocervix and vaginal vault or via a sponge inserted into the endocervix for evaluating inflammation status and immune factors critical for HIV microbicide and vaccine studies. This study uses a proteomics approach to comprehensively compare the efficacy of these methods, which sample from different compartments of the female genital tract, for the collection of immune factors. Matching sponge and lavage samples were collected from 10 healthy women and were analyzed by tandem mass spectrometry. Data was analyzed by a combination of differential protein expression analysis, hierarchical clustering and pathway analysis. Of the 385 proteins identified, endocervical sponge samples collected nearly twice as many unique proteins as cervicovaginal lavage (111 vs. 61) with 55% of proteins common to both (213). Each method/site identified 73 unique proteins that have roles in host immunity according to their gene ontology. Sponge samples enriched for specific inflammation pathways including acute phase response proteins (p = 3.37×10⁻²⁴) and LXR/RXR immune activation pathways (p = 8.82×10⁻²²) while the role IL-17A in psoriasis pathway (p = 5.98×10⁻⁴) and the complement system pathway (p = 3.91×10⁻³) were enriched in lavage samples. Many host defense factors were differentially enriched (p<0.05) between sites including known/potential antimicrobial factors (n = 21), S100 proteins (n = 9), and immune regulatory factors such as serpins (n = 7). Immunoglobulins (n = 6) were collected at comparable levels in abundance in each site although 25% of those identified were unique to sponge samples. This study demonstrates significant differences in types and quantities of immune factors and inflammation pathways collected by each sampling technique. Therefore, clinical studies that measure mucosal immune activation or factors assessing HIV transmission should utilize both collection methods to obtain the greatest representation of immune factors secreted into the female genital tract.</p></div

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