Garrett Westmacott

Identification of differentially expressed proteins in the cervical mucosa of HIV-1-resistant sex workers.

Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance. Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was used to examine cervical mucosa of HIV-1 resistant women ( n = 10) for biomarkers of HIV-1 resistance. Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control groups ( n = 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume differences. Validation of these results on a larger sample of individuals will provide further evidence these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective microbicides against HIV-1.

Vaginal bacteria modify HIV tenofovir microbicide efficacy in African women

Analysis of HIV clinical trials shows that the vaginal microbiome underlies varied responses to microbicide-based preexposure prophylaxis. Vaginal microbiome influences HIV acquisition Tenofovir is a preexposure drug used to prevent HIV infection. In clinical trials, tenofovir was effective for men, but not women. Klatt et al. now show that tenofovir efficacy in women depends on the composition of the vaginal microbiome (see the Perspective by Tuddenham and Ghanem). In a clinical trial of 688 women, tenofovir was three times as effective among those with a Lactobacillus-dominant vaginal microbiome as it was among other women. Gardnerella vaginalis tended to predominate in the women for whom tenofovir was less effective, and the authors found that the organism could rapidly metabolize and thereby inactivate the drug. Science, this issue p. 938; see also p. 907 Antiretroviral-based strategies for HIV prevention have shown inconsistent results in women. We investigated whether vaginal microbiota modulated tenofovir gel microbicide efficacy in the CAPRISA (Centre for the AIDS Program of Research in South Africa) 004 trial. Two major vaginal bacterial community types—one dominated by Lactobacillus (59.2%) and the other where Gardnerella vaginalis predominated with other anaerobic bacteria (40.8%)—were identified in 688 women profiled. Tenofovir reduced HIV incidence by 61% (P = 0.013) in Lactobacillus-dominant women but only 18% (P = 0.644) in women with non-Lactobacillus bacteria, a threefold difference in efficacy. Detectible mucosal tenofovir was lower in non-Lactobacillus women, negatively correlating with G. vaginalis and other anaerobic bacteria, which depleted tenofovir by metabolism more rapidly than target cells convert to pharmacologically active drug. This study provides evidence linking vaginal bacteria to microbicide efficacy through tenofovir depletion via bacterial metabolism.

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

Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

Evaluation of MALDI-TOF mass spectroscopy methods for determination of Escherichia coli pathotypes

It is rapidly becoming apparent that many E. coli pathotypes cause a considerable burden of human disease. Surveillance of these organisms is difficult because there are few or no simple, rapid methods for detecting and differentiating the different pathotypes. MALDI-TOF mass spectroscopy has recently been rapidly and enthusiastically adopted by many clinical laboratories as a diagnostic method because of its high throughput, relatively low cost, and adaptability to the laboratory workflow. To determine whether the method could be adapted for E. coli pathotype differentiation the Bruker Biotyper methodology and a second methodology adapted from the scientific literature were tested on isolates representing eight distinct pathotypes and two other groups of E. coli. A total of 136 isolates was used for this study. Results confirmed that the Bruker Biotyper methodology that included extraction of proteins from bacterial cells was capable of identifying E. coli isolates from all pathotypes to the species level and, furthermore, that the Bruker extraction and MALDI-TOF MS with the evaluation criteria developed in this work was effective for differentiating most pathotypes.

Proteomic Analysis of a NAP1 Clostridium difficile Clinical Isolate Resistant to Metronidazole

Background Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates. Methodology/Principal Findings NAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R. Conclusions/Significance This study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair.

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.

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

ABSTRACT 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.

MicroRNA abundance is altered in synaptoneurosomes during prion disease

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimers disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

MALDI-TOF MS detection of carbapenemase activity in clinical isolates of Enterobacteriaceae spp., Pseudomonas aeruginosa, and Acinetobacter baumannii compared against the Carba-NP assay

MALDI-TOF MS detection of carbapenemase-activity in Gram-negative bacteria was compared against the Carba-NP assay. MALDI-TOF MS detected activity from 99% of the strains, from all types of carbapenemase (200/202), while Carba-NP assays detected activity from 85% (45/53) of the tested isolates and could not consistently identify OXA- or GES carbapenemase activity.

Epstein-Barr virus in oral shedding of children with multiple sclerosis

Objective: To investigate Epstein-Barr virus (EBV) oral shedding frequency and EBV genetic diversity in pediatric patients with multiple sclerosis (MS). Methods: This was a prospective case-control study. We used PCR-based assays to detect viral DNA in the monthly mouth swabs of 22 pediatric patients with MS and 77 age- and sex-matched healthy controls. EBV-positive samples were further analyzed for sequence variation in the EBV BCRF1 (ebvIL-10) gene using direct DNA sequencing methods, and in the EBV LMP1 gene by mass spectrometry. Results: Nineteen of the 22 (86.4%) children with MS were seropositive for remote EBV infection compared to 35 out of 77 (45.5%) healthy controls (p = 0.008). Baseline analysis of mouth swabs revealed a higher proportion of EBV-positive samples from EBV-seropositive patients with MS compared to EBV-seropositive healthy controls (52.6% vs 20%, p = 0.007). Longitudinal analysis of monthly swabs revealed average EBV detection rates of 50.6% in patients with MS and 20.4% in controls (p = 0.01). The oral shedding frequencies of Herpesviruses herpes simplex virus–1, cytomegalovirus, human herpesvirus (HHV)-6, and HHV-7 did not differ between groups. Changes in the predominant EBV genetic variants were detected more frequently in patients with MS; however, no specific EBV genetic variant was preferentially associated with MS. Conclusion: Children with MS demonstrate abnormally increased rates of EBV viral reactivation and a broader range of genetic variants, suggesting a selective impairment in their immunologic control of EBV.