Vaginal microbiome-hormonal contraceptive interactions associate with the mucosal proteome and HIV acquisition.

Alterations to the mucosal environment of the female genital tract, such as genital inflammation, have been associated with increased HIV acquisition in women. As the microbiome and hormonal contraceptives can affect vaginal mucosal immunity, we hypothesized these components may interact in the context of HIV susceptibility. Using previously published microbiome data from 685 women in the CAPRISA-004 trial, we compared relative risk of HIV acquisition in this cohort who were using injectable depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (NET-EN), and combined oral contraceptives (COC). In women who were Lactobacillus-dominant, HIV acquisition was 3-fold higher in women using DMPA relative to women using NET-EN or COC (OR: 3.27; 95% CI: 1.24-11.24, P = 0.0305). This was not observed in non-Lactobacillus-dominant women (OR: 0.95, 95% CI: 0.44-2.15, P = 0.895) (interaction P = 0.0686). Higher serum MPA levels associated with increased molecular pathways of inflammation in the vaginal mucosal fluid of Lactobacillus-dominant women, but no differences were seen in non-Lactobacillus dominant women. This study provides data suggesting an interaction between the microbiome, hormonal contraceptives, and HIV susceptibility.

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.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.

Molecular Signatures of Immune Activation and Epithelial Barrier Remodeling Are Enhanced during the Luteal Phase of the Menstrual Cycle: Implications for HIV Susceptibility.

UNLABELLED: The variable infectivity and transmissibility of HIV/SHIV has been recently associated with the menstrual cycle, with particular susceptibility observed during the luteal phase in nonhuman primate models and ex vivo human explant cultures, but the mechanism is poorly understood. Here, we performed an unbiased, mass spectrometry-based proteomic analysis to better understand the mucosal immunological processes underpinning this observed susceptibility to HIV infection. Cervicovaginal lavage samples (n = 19) were collected, characterized as follicular or luteal phase using days since last menstrual period, and analyzed by tandem mass spectrometry. Biological insights from these data were gained using a spectrum of computational methods, including hierarchical clustering, pathway analysis, gene set enrichment analysis, and partial least-squares discriminant analysis with LASSO feature selection. Of the 384 proteins identified, 43 were differentially abundant between phases (P < 0.05, ≥2-fold change). Cell-cell adhesion proteins and antiproteases were reduced, and leukocyte recruitment (interleukin-8 pathway, P = 1.41E-5) and extravasation proteins (P = 5.62E-4) were elevated during the luteal phase. LASSO/PLSDA identified a minimal profile of 18 proteins that best distinguished the luteal phase. This profile included cytoskeletal elements and proteases known to be involved in cellular movement. Gene set enrichment analysis associated CD4(+) T cell and neutrophil gene set signatures with the luteal phase (P < 0.05). Taken together, our findings indicate a strong association between proteins involved in tissue remodeling and leukocyte infiltration with the luteal phase, which may represent potential hormone-associated mechanisms of increased susceptibility to HIV. IMPORTANCE: Recent studies have discovered an enhanced susceptibility to HIV infection during the progesterone-dominant luteal phase of the menstrual cycle. However, the mechanism responsible for this enhanced susceptibility has not yet been determined. Understanding the source of this vulnerability will be important for designing efficacious HIV prevention technologies for women. Furthermore, these findings may also be extrapolated to better understand the impact of exogenous hormone application, such as the use of hormonal contraceptives, on HIV acquisition risk. Hormonal contraceptives are the most widely used contraceptive method in sub-Saharan Africa, the most HIV-burdened area of the world. For this reason, research conducted to better understand how hormones impact host immunity and susceptibility factors important for HIV infection is a global health priority.

Mass Spectrometry-Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping.

BACKGROUND: Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS: On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS: Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 µg or 1.465 × 10(7) cells) level and close correlation (r(2) > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS: MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

Highly Pathogenic H5N1 and Novel H7N9 Influenza A Viruses Induce More Profound Proteomic Host Responses than Seasonal and Pandemic H1N1 Strains.

Influenza A viruses (IAV) are important human and animal pathogens with potential for causing pandemics. IAVs exhibit a wide spectrum of clinical illness in humans, from relatively mild infections by seasonal strains to acute respiratory distress syndrome during infections with some highly pathogenic avian influenza (HPAI) viruses. In the present study, we infected A549 human cells with seasonal H1N1 (sH1N1), 2009 pandemic H1N1 (pdmH1N1), or novel H7N9 and HPAI H5N1 strains. We used multiplexed isobaric tags for relative and absolute quantification to measure proteomic host responses to these different strains at 1, 3, and 6 h post-infection. Our analyses revealed that both H7N9 and H5N1 strains induced more profound changes to the A549 global proteome compared to those with low-pathogenicity H1N1 virus infection, which correlates with the higher pathogenicity these strains exhibit at the organismal level. Bioinformatics analysis revealed important modulation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response in infection. Cellular fractionation and Western blotting suggested that the phosphorylated form of NRF2 is not imported to the nucleus in H5N1 and H7N9 virus infections. Fibronectin was also strongly inhibited in infection with H5N1 and H7N9 strains. This is the first known comparative proteomic study of the host response to H7N9, H5N1, and H1N1 viruses and the first time NRF2 is shown to be implicated in infection with highly pathogenic strains of influenza.

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization-Time of Flight-Based Peptide Mass Fingerprinting.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

Sequence-level and dual-phase identification of Salmonella flagellum antigens by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Forty-three reference strains involving the 24 most common serovars of Salmonella enterica were examined by using a mass spectrometry-based H antigen typing platform (MS-H). The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonella flagella at the molecular level without antiserum and phase inversion.

A systems biology examination of the human female genital tract shows compartmentalization of immune factor expression.

Many mucosal factors in the female genital tract (FGT) have been associated with HIV susceptibility, but little is known about their anatomical distribution in the FGT compartments. This study comprehensively characterized global immune factor expression in different tissue sites of the lower and upper FGT by using a systems biology approach. Tissue sections from the ectocervix, endocervix, and endometrium from seven women who underwent hysterectomy were analyzed by a combination of quantitative mass spectrometry and immunohistochemical staining. Of the >1,000 proteins identified, 281 were found to be differentially abundant in different tissue sites. Hierarchical clustering identified four major functional pathways distinguishing compartments, including innate immune pathways (acute-phase response, LXR/RXR) and development (RhoA signaling, gluconeogenesis), which were enriched in the ectocervix/endocervix and endometrium, respectively. Immune factors important for HIV susceptibility, including antiproteases, immunoglobulins, complement components, and antimicrobial factors, were most abundant in the ectocervix/endocervix, while the endometrium had a greater abundance of certain factors that promote HIV replication. Immune factor abundance is heterogeneous throughout the FGT and shows unique immune microenvironments for HIV based on the exposure site. This may have important implications for early events in HIV transmission and site-specific susceptibility to HIV in the FGT.

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.

High pH reversed-phase chromatography as a superior fractionation scheme compared to off-gel isoelectric focusing for complex proteome analysis.

MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off-gel IEF (OG-IEF) and high pH RP (Hp-RP) column chromatography have both been successfully utilized as a first-dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12-fraction replicate analysis, Hp-RP resulted in more peptides and proteins identified than OG-IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp-RP. This leads to a more uniform distribution of total and unique peptides for Hp-RP across all fractions collected. These results suggest that fractionation by Hp-RP over OG-IEF is the better choice for typical complex proteome analysis.

Increased genital mucosal cytokines in Canadian women associate with higher antigen-presenting cells, inflammatory metabolites, epithelial barrier disruption, and the depletion of L. crispatus.

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.

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.

Pregnancy associates with alterations to the host and microbial proteome in vaginal mucosa.

PROBLEM: Pregnant women are at increased risk of HIV acquisition, but the biological mechanisms contributing to this observation are not well understood. METHOD OF STUDY: Here, we assessed host immune and microbiome differences in the vaginal mucosa of healthy pregnant and non-pregnant women using a metaproteomics approach. Cervicovaginal lavage (CVL) samples were collected from 23 pregnant and 25 non-pregnant women. RESULTS: Mass spectrometry analysis of CVL identified 550 human proteins and 376 bacterial proteins from 11 genera. Host proteome analysis indicated 56 human proteins (10%) were differentially abundant (P < .05) between pregnant and non-pregnant women, including proteins involved in angiogenesis (P = 3.36E-3), cell movement of phagocytes (P = 1.34E-6), and permeability of blood vessels (P = 1.27E-4). The major bacterial genera identified were Lactobacillus, Gardnerella, Prevotella, Megasphaera, and Atopobium. Pregnant women had higher levels of Lactobacillus species (P = .017) compared with non-pregnant women. Functional pathway analysis indicated that pregnancy associated with changes to bacterial metabolic pathway involved in energy metabolism, which were increased in pregnant women (P = .035). CONCLUSION: Overall, pregnant women showed differences in the cervicovaginal proteome and microbiome that may be important for HIV infection risk.

Detection of Antimicrobial Resistance Using Proteomics and the Comprehensive Antibiotic Resistance Database: A Case Study.

PURPOSE: Antimicrobial resistance (AMR), especially multidrug resistance, is one of the most serious global threats facing public health. The authors proof-of-concept study assessing the suitability of shotgun proteomics as an additional approach to whole-genome sequencing (WGS) for detecting AMR determinants. EXPERIMENTAL DESIGN: Previously published shotgun proteomics and WGS data on four isolates of Campylobacter jejuni are used to perform AMR detection by searching the Comprehensive Antibiotic Resistance Database, and their detection ability relative to genomics screening and traditional phenotypic testing measured by minimum inhibitory concentration is assessed. RESULTS: Both genomic and proteomic approaches identify the wild-type and variant molecular determinants responsible for resistance to tetracycline and ciprofloxacin, in agreement with phenotypic testing. In contrast, the genomic method identifies the presence of the ß-lactamase gene, blaOXA-61 , in three isolates. However, its corresponding protein product is detected in only a single isolate, consistent with results obtained from phenotypic testing.

Intestinal proteomic analysis of a novel non-human primate model of experimental colitis reveals signatures of mitochondrial and metabolic dysfunction.

Animal models recapitulating features of chronic colitis, such as ulcerative colitis, Crohn's disease, or HIV infection, are critical to study disease pathogenesis and test novel therapeutics. In this study, we used a proteomics approach to explore the molecular intestinal response in two rhesus macaque (RM) animal models of experimentally induced colitis using dextran sulfate sodium (DSS) and simian immunodeficiency virus (SIV) infection. Proteomic analysis detected more than 2500 proteins in colonic tissue collected from 30 RMs. Differential protein expression analysis revealed a protein expression pattern in DSS-treated RMs resembling the proteome of human ulcerative colitis. In a group of 12 DSS-treated RMs compared to 6 with no treatment, decrease in expression of proteins related to mitochondrial energy metabolism, including fatty acid metabolism was noted, while innate immune activation pathways, including complement and coagulation proteins were upregulated. SIV infection of RMs resulted in increased innate immune responses related to viral defense. Proteomic signatures of barrier damage were apparent in both DSS treatment or SIV infection. These results demonstrate that DSS treatment in a non-human primate model resembles features of human ulcerative colitis, making this a promising tool to study important immunological mechanisms in inflammatory bowel disease.

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.

Comparison of genomes and proteomes of four whole genome-sequenced Campylobacter jejuni from different phylogenetic backgrounds.

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 C. jejuni isolates. We previously characterized the presence and expression of CJIE4 prophage proteins in four C. jejuni 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.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.IMPORTANCE The Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.