Mapping the vaginal microbiota variations in women from a community clinic in Mumbai, India.

PURPOSE: The vaginal microbiome contributes significantly to women's reproductive health and fluctuates due to various physiological and pathological factors. The study's objective is to map the vaginal microbiome of non-pregnant women and evaluate variations based on various potential factors influencing vaginal milieu. METHODS: Fifty-two sexually active, non-pregnant women between 18 and 45 years were recruited from a community clinic and clinical history was recorded. Vaginal swabs were collected to assess the vaginal microbiome by sequencing the V3-V4 region of the 16S rRNA using the Illumina HiSeq platform, followed by data analysis with QIIME 2. Vaginal milieu was assessed by Nugent score and profiling cytokines in the cervico-vaginal lavage. RESULTS: Lactobacillus iners (34.3%) were the most abundant species in all women. Significant changes in abundance of genera (Lactobacillus, Prevotella and Anaerococcus), expression of pro-inflammatory cytokine IFN-γ and changes in alpha and beta diversity was observed in women having asymptomatic bacterial vaginosis (BV). Differences in beta diversity were seen between healthy women and women exhibiting presence of Candida spp. Variations in the abundance of genera (Lactobacillus, Bifidobacterium, Porphyromonas) were observed in women who had delivery less than twelve months back, probably as more of these women (50%, 53.7%) had higher abnormal Nugent score. CONCLUSION: Lactobacillus iners was the most prevalent vaginal species in women from a Mumbai community clinic. Maximum variations in the vaginal microbiome characterized by a perturbation of the Lactobacillus predominant vaginal microbiota are seen in those women who have asymptomatic BV and childbirth within last twelve months.

Membrane vesicles of Lactobacillus gasseri ATCC 19992 disrupt biofilms of vaginal pathogens.

Membrane vesicles (MVs) are bioactive, nano-sized entities produced by all organisms. MVs of L. gasseri ATCC 19992 were isolated and their effect on the biofilms of vaginal pathogens, G. vaginalis and S. aureus was studied. The L. gasseri MVs resulted in significant disruption of biofilms of the vaginal pathogens.

A novel gut microbiome-immune axis influencing pathology in HCMV infected infants with neonatal cholestasis.

The interplay of active HCMV infection with gut dysbiosis in the immunopathology of cholestasis in neonates and infants remains unexplored. In this study, we evaluated gut microbiome profiles and immune dysfunction in a cohort of HCMV infected cholestatic infants (IgM positive, N = 21; IgM negative, N = 25) compared to healthy infants, N = 10. HCMV infected IgM positive individuals exhibited increased clinical severity in terms of liver dysfunction, altered CD4+: CD8+ ratio, and elevated Granzyme B levels in cellular immune subsets. Gut microbiome analysis revealed distinct and differential diversity and composition within infected groups aligned with clinical severity reflected through the increased abundance of Gammaproteobacteria, reduced Bifidobacteria, and a unique signature mapping to the HCMV infected IgM negative group. Correlation analyses revealed associations between Bifidobacterium breve, Gammaproteobacteria, Firmicutes, Clostridia, Finegoldia magna, Veillonella dispar, and Granzyme B expressing immune cell subsets. Our study describes a novel gut microbiome-immune axis that may influence disease severity in cholestatic infants with active HCMV infection.

Cytomegalovirus reactivation in a SARS-CoV-2 infected woman experiencing fetal demise in the first trimester with fetal trisomy 21: A case report.

Cytomegalovirus (CMV) is the most common cause of congenital viral infections. Women seropositive for CMV prior to pregnancy can develop a non-primary CMV infection. Here, we present a case of first trimester pregnancy loss during active SARS-CoV-2 infection. There was no evidence of SARS-CoV-2 RNA in placenta and fetal tissue, but there was presence of congenital cytomegalovirus infection by nested PCR. To the best of our knowledge, this is the first report demonstrating association of early congenital CMV infection due to reactivation and fetal demise in a SARS-CoV-2 positive woman with fetal trisomy 21.

A literature review on correlation between HPV coinfection with C. trachomatis and cervical neoplasia - coinfection mediated cellular transformation.

Cervical cancer is the fourth most common cause of mortality worldwide. Persistent infection with high-risk human papillomaviruses (hrHPV) is a known significant risk factor in cervical neoplasia development (CN). Though HPV contributes to carcinogenesis, other factors provide an ideal niche for persistence of HPV, especially, coinfection with Chlamydia trachomatis (CT) which has been linked to CN development. CT infection is associated with inflammation, cell proliferation, EMT transition and anti-apoptotic processes. To better understand the correlation between HPV-CT coinfection and CN development, a literature review was conducted on the prevalence of HPV-CT coinfection focusing on the role of infection-induced inflammation as HPV-CT coinfection creates an environment for cellular transformation, activates an innate immune response and triggers EMT transition. Moreover, inflammation plays a crucial role in developing neoplasia as there is a decrease in effector cells and a change in the levels of players like ROS and miRs. CT infection induces chronic inflammation followed by cervical epithelial cell damage and increases susceptibility to HPV infection which may lead to cellular transformation. The literature search was performed based on a comprehensive investigation of publications in the PubMed journal database and Scopus, on the development of CN. We have reviewed the prevalence of HPV-CT infection and the factors increasing the risk of developing CN.

Opposing roles for sMAdCAM and IL-15 in COVID-19 associated cellular immune pathology.

Immune cell dysregulation and lymphopenia characterize COVID-19 pathology in moderate to severe disease. While underlying inflammatory factors have been extensively studied, homeostatic and mucosal migratory signatures remain largely unexplored as causative factors. In this study, we evaluated the association of circulating IL-6, soluble mucosal addressin cell adhesion molecule (sMAdCAM), and IL-15 with cellular dysfunction characterizing mild and hypoxemic stages of COVID-19. A cohort of SARS-CoV-2 infected individuals (n = 130) at various stages of disease progression together with healthy controls (n = 16) were recruited from COVID Care Centres (CCCs) across Mumbai, India. Multiparametric flow cytometry was used to perform in-depth immune subset characterization and to measure plasma IL-6 levels. sMAdCAM, IL-15 levels were quantified using ELISA. Distinct depletion profiles, with relative sparing of CD8 effector memory and CD4+ regulatory T cells, were observed in hypoxemic disease within the lymphocyte compartment. An apparent increase in the frequency of intermediate monocytes characterized both mild as well as hypoxemic disease. IL-6 levels inversely correlated with those of sMAdCAM and both markers showed converse associations with observed lympho-depletion suggesting opposing roles in pathogenesis. Interestingly, IL-15, a key cytokine involved in lymphocyte activation and homeostasis, was detected in symptomatic individuals but not in healthy controls or asymptomatic cases. Further, plasma IL-15 levels negatively correlated with T, B, and NK count suggesting a compensatory production of this cytokine in response to the profound lymphopenia. Finally, higher levels of plasma IL-15 and IL-6, but not sMAdCAM, were associated with a longer duration of hospitalization.

pH Stress Mediated Alteration in Protein Composition and Reduction in Cytotoxic Potential of Gardnerella vaginalis Membrane Vesicles.

The vagina of healthy women is predominantly colonized by lactobacilli but it also harbors a limited proportion of certain anaerobes such as Gardnerella vaginalis. An increase in G. vaginalis along with other anaerobes on account of perturbation in the vaginal microbiota is associated with bacterial vaginosis (BV). Although strategies adopted by G. vaginalis for survival and pathogenesis in a conducive environment (i.e., high vaginal pH, characteristic of BV) have been previously studied, the approaches potentially employed for adaptation to the low pH of the healthy vagina are unknown. In the present study, we investigated the effect of acidic stress on the modulation of the production and function of membrane vesicles (MVs) of G. vaginalis. pH stress led to a distortion of the bacterial cell morphology as well as an altered biogenesis of MVs, as revealed by transmission electron microscopy (TEM). Both qualitative and quantitative differences in protein content of MVs produced in response to pH stress were observed by flow cytometry. A significant change in the protein composition characterized by presence of chaperones despite a reduction in number of proteins was also noted in the stress induced MVs. Further, these changes were also reflected in the reduced cytotoxic potential toward vaginal epithelial cells. Although, these findings need to be validated in the in vivo settings, the modulation of G. vaginalis MV biogenesis, composition and function appears to reflect the exposure to acidic conditions prevailing in the host vaginal mileu in the absence of vaginal infection.

sMAdCAM: IL-6 Ratio Influences Disease Progression and Anti-Viral Responses in SARS-CoV-2 Infection.

The role of sMAdCAM, an important gut immune migratory marker, remains unexplored in COVID-19 pathogenesis considering recent studies positing the gut as a sanctuary site for SARS-CoV-2 persistence. Thus, assimilating profiles of systemic inflammatory mediators with sMAdCAM levels may provide insights into the progression of COVID-19 disease. Also, the role of these markers in governing virus specific immunity following infection remains largely unexplored. A cohort (n = 84) of SARS-C0V-2 infected individuals included a group of in-patients (n = 60) at various stages of disease progression together with convalescent individuals (n = 24) recruited between April and June 2020 from Mumbai, India. Follow-up of 35 in-patients at day 7 post diagnosis was carried out. Th1/Th2/Th17 cytokines along with soluble MAdCAM (sMAdCAM) levels in plasma were measured. Also, anti-viral humoral response as measured by rapid antibody test (IgG, IgM), Chemiluminescent Immunoassay (IgG), and antibodies binding to SARS-CoV-2 proteins were measured by Surface Plasmon Resonance (SPR) from plasma. IL-6 and sMAdCAM levels among in-patients inversely correlated with one another. When expressed as a novel integrated marker-sMIL index (sMAdCAM/IL-6 ratio)-these levels were incrementally and significantly higher in various disease states with convalescents exhibiting the highest values. Importantly, sMAdCAM levels as well as sMIL index (fold change) correlated with peak association response units of receptor binding domain and fold change in binding to spike respectively as measured by SPR. Our results highlight key systemic and gut homing parameters that need to be monitored and investigated further to optimally guide therapeutic and prophylactic interventions for COVID-19.

A novel ORF1a-based SARS-CoV-2 RT-PCR assay to resolve inconclusive samples.

BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.

Frequency of Effector Memory Cells Expressing Integrin α4ß7 Is Associated With TGF-ß1 Levels in Therapy Naïve HIV Infected Women With Low CD4+ T Cell Count.

Integrin α4ß7 expressing CD4+ T cells are preferred targets for HIV infection and are thought to be predictors of disease progression. Concurrent analysis of integrin α4ß7 expressing innate and adaptive immune cells was carried out in antiretroviral (ART) therapy naïve HIV infected women in order to determine its contribution to HIV induced immune dysfunction. Our results demonstrate a HIV infection associated decrease in the frequency of integrin α4ß7 expressing endocervical T cells along with an increase in the frequency of integrin α4ß7 expressing peripheral monocytes and central memory CD4+ T cells, which are considered to be viral reservoirs. We report for the first time an increase in levels of soluble MAdCAM-1 (sMAdCAM-1) in HIV infected individuals as well as an increased frequency and count of integrin ß7Hi CD8+ memory T cells. Correlation analysis indicates that the frequency of effector memory CD8+ T cells expressing integrin α4ß7 is associated with levels of both sMAdCAM-1 and TGF-ß1. The results of this study also suggest HIV induced alterations in T cell homeostasis to be on account of disparate actions of sMAdCAM-1 and TGF-ß1 on integrin α4ß7 expressing T cells. The immune correlates identified in this study warrant further investigation to determine their utility in monitoring disease progression.

Association of cervicovaginal dysbiosis mediated HPV infection with cervical intraepithelial neoplasia.

Cellular transformation to malignancy is a multifactorial process strongly linked with microbiome dysbiosis. The female reproductive tract (FRT) is inhabited by specific Lactobacillus spp which play a significant role in maintaining a homeostatic balance and providing resistance to perturbation. Any imbalance in the resident microbiota of the FRT results in cervicovaginal dysbiosis and increased predisposition to viral and bacterial infections. In the present review, we discuss the critical role played by the cervicovaginal microbiome in maintaining cervicovaginal homeostasis. Loss of the mutualistic relationship between cervicovaginal microbiota and the host leads to increased susceptibility to Human papilloma virus (HPV) infection. HPV in coinfection with Chlamydia trachomatis has been linked with increased risk for cellular transformation. The progression to cervical neoplasia is a multistep process regulated by cellular and epigenetic changes mediated by oncogenes and miRNA. Exosomes derived from the infected cells play an important role in the pathological development and progression to cervical neoplasia as they harbor the regulatory molecules like miRNA, proteins and prooncogenic factors which may facilitate cellular transformation.

The Role of Integrin α4ß7 Signaling in Human Immunodeficiency Virus-1 Pathogenesis and Viral Entry in Primary CD4+ T Cells As Revealed by Comparative Phosphoproteomic Signatures.

Integrin α4ß7, a CD4 independent receptor of human immunodeficiency virus-1 (HIV-1) gp120, defines a subset of CD4+T cells preferentially targeted by HIV. It is also considered as a promising therapeutic target for HIV-1 infection. Despite its role in HIV acquisition and disease progression, HIV-1-mediated integrin α4ß7 signaling has not been elucidated so far. In view of this, we determined phosphoproteomic signatures of HIV-1 gp120 signaling as well as signaling mediated by the integrin α4ß7 ligand, mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1), in primary CD4+ T cells. This is the first comprehensive report on MAdCAM-1 signaling, which is believed to enhance HIV-1 replication. Importantly, we identified proteins associated with both classical and nonclassical integrin functions. We observed that HIV-1 gp120 signaling is associated with proteins that have previously not been associated with HIV-1 pathogenesis and thus, need to be explored further. There was a significant overlap in proteins identified by both MAdCAM-1 and HIV-1 gp120 signaling, which most likely represents cellular processes triggered upon interaction of HIV-1 gp120 with integrin α4ß7. Pathway analysis revealed enrichment of processes that could facilitate viral replication as well as viral entry through endocytosis. Although these results warrant independent replication and further validation, they suggest the presence of additional potential therapeutic targets. These results also suggest that combinatorial approaches for targeting both HIV-1 gp120 and MAdCAM-1 signaling may be necessary for efficient control of HIV-1 infection as well as novel innovation strategies in HIV therapeutics.

Characterization of Gardnerella vaginalis membrane vesicles reveals a role in inducing cytotoxicity in vaginal epithelial cells.

Bacterial vaginosis (BV) is a common polymicrobial infection affecting women in the reproductive age and is associated with adverse obstetric and gynaecological outcomes. Gardnerella vaginalis is the most virulent anaerobic bacterial species predominantly associated with BV. However, a clear understanding of the mechanisms by which it contributes to the pathogenesis and persistence of BV is lacking. In this report, we demonstrate for the first time, the isolation of membrane vesicles (MVs) from G. vaginalis ATCC 14019. These MVs are approximately 120-260 nm in diameter. Proteomic characterization of the MVs by LC-MS/MS led to the identification of 417 proteins, including proteins involved in cellular metabolism as well as molecular chaperones and certain virulence factors. Immunoblot analysis of the MVs confirmed the presence of vaginolysin, the most well-characterized virulence factor of G. vaginalis. The exposure of the vaginal epithelial cells, VK2/E6E7 to the G. vaginalis MVs resulted in the internalization of the MVs. The MVs induced cytotoxicity and an increase in the levels of the pro-inflammatory cytokine, IL-8 in VK2 cells as well lysis of erythrocytes. The results of the study indicate that G. vaginalis MVs may be involved in the delivery of cytotoxic proteins and other virulence factors to the host cells and could thereby contribute towards enhancing the cellular damage associated with pathogenesis of BV.

Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

Broad substrate stereospecificity of the Mycobacterium tuberculosis 7-keto-8-aminopelargonic acid synthase: Spectroscopic and kinetic studies.

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the mycobacterium tuberculosis KAPA synthase with both L- AND D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 m(-1) s(-1)) is approximately 5 times slower than that with L-alanine (k = 399.4 m(-1) s(-1)). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed d-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 10(4) m(-1) s(-1)). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, d-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (Ki = 114.83 microm) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 microm), the next enzyme of the pathway.

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis.

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1s-1. The Km values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 microM and 308.28 microM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 micromoles/min/ml and 0.003 s-1, respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.

Polymyxin B: an ode to an old antidote for endotoxic shock.

Endotoxic shock, a syndrome characterized by deranged hemodynamics, coagulation abnormalities, and multiple system organ failure is caused by the release into the circulation of lipopolysaccharide (LPS), the structurally diverse component of Gram-negative bacterial outer membranes, and is responsible for 60% mortality in humans. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, neutralizes endotoxin but induces severe side effects in the process. The potent endotoxin neutralizing ability of PMB, however, offers possibilities for designing non-toxic therapeutic agents for combating endotoxicosis. Amongst the numerous approaches for combating endotoxic shock, peptide mediated neutralization of LPS seems to be the most attractive one. The precise mode of binding of PMB to LPS and the structural features involved therein have been elucidated only recently using a variety of biophysical approaches. These suggest that efficient neutralization of endotoxin by PMB is not achieved by mere binding to LPS but requires its sequestration from the membrane. Incorporation of this feature into the design of endotoxin neutralizing peptides should lead to the development of effective antidotes for endotoxic shock.