Biology subjects

Anil, A.

Publications and source records attributed to Anil, A..

7 recordsLinked to original sources

bioRxiv2026

Gram-negative-dominated polymicrobial microbiome of necrotizing soft tissue infections from North India: an integrated culture and 16S rRNA metagenomics prospective cohort study

Necrotizing soft tissue infections (NSTIs) carry 10 to 30% mortality. Current empirical antimicrobial guidance derives almost entirely from Western cohorts dominated by Streptococcus pyogenes and aerobic-anaerobic consortia, yet whether this microbial paradigm applies to tropical, high-antimicrobial-pressure settings has not been tested with culture-independent methods. We did a prospective cohort study of 169 patients with intraoperatively confirmed NSTI at a North Indian tertiary center (2021 to 2024). Wound tissue underwent aerobic and anaerobic culture, QIIME2-based 16S rRNA gene amplicon sequencing (V3-V4), and targeted SYBR Green quantitative PCR (qPCR) for Acinetobacter baumannii and S. pyogenes. The wound microbiota was overwhelmingly Gram-negative and polymicrobial, anchored by A. baumannii (culture, 33.7%; metagenomics, 49.1%; qPCR, 37.9%), Escherichia coli (32.0%), and Klebsiella pneumoniae (20.7%); S. pyogenes contributed only 4.7% of culture-positive cases. Polymicrobial wounds had higher Shannon diversity (2.59 versus 2.33; P = 0.048) and discrete community composition (PERMANOVA R2 = 0.511; P = 0.010). Culture-metagenomics agreement ranged from almost perfect for Escherichia ({kappa} = 0.849) to slight for Streptococcus ({kappa} = 0.131). North Indian NSTIs present a microbial picture distinct from the Western paradigm, with implications for empirical therapy. IMPORTANCENecrotizing soft tissue infections kill rapidly, and physicians must choose antibiotics before laboratory results are available. Globally, treatment guidelines assume the dominant pathogen is Streptococcus pyogenes, a Gram-positive organism, because nearly all molecular evidence comes from Western countries. We show that in a large Indian cohort, the infection is instead dominated by Gram-negative bacteria, especially Acinetobacter baumannii, with S. pyogenes rarely present. Polymicrobial status, not wound location or injury mechanism, most strongly determines community composition. These findings challenge the one-size-fits-all antibiotic approach and argue for region-specific empirical regimens prioritizing Gram-negative coverage in tropical, high-antimicrobial-pressure settings.

microbiology↗

bioRxiv2026

Fibrin barriers limit vancomycin penetration into staphylococcal abscess communities and maintain S. aureus in an unstressed, transcriptionally-responsive state

During Staphylococcus aureus infection, bacteria are frequently organized into staphylococcal abscess communities (SACs), multicellular bacterial aggregates encased within a host-derived fibrin barrier. Fibrin barriers are thought to insulate SAC-resident bacteria from host immune cells, as strains unable to form these barriers exhibit marked attenuation of virulence in animal infection models. However, the clinical consequences of these structures on antibiotic penetration, and whether the structures also impact bacterial physiology and alter antibiotic susceptibility, remains understudied. Here, we built upon prior work to develop a three-dimensional (3D) collagen gel matrix-based model, utilizing human coagulation components, that supports high throughput in vitro staphylococcal community (SC) formation and is compatible with timelapse microscopy and bulk transcriptional profiling. Using this system, we show that the fibrin barrier protects SCs from vancomycin by restricting drug penetration. Furthermore, we show that the fibrin barrier impacts transcriptional responses, leading to both shared and distinct transcriptional profiles between WT and fibrin barrier-deficient SCs. Together, these findings expand our understanding of SC fibrin barriers beyond immune evasion to include modulation of antibiotic interactions and bacterial physiological state.

microbiology↗

bioRxiv2025

Proteome profiling reveals HES1-driven mitotic catastrophe in ovarian serous carcinoma

Ovarian high-grade serous cancer (HGSC) is an aggressive subtype of epithelial ovarian cancer. Here, we identify BX-912, a phosphoinositide-dependent kinase 1 (PDPK1) inhibitor, as a promising therapeutic agent for HGSC. BX-912 suppressed HGSC growth as a single agent and synergized with olaparib independently of BRCA status. Unexpectedly, BX-912 treatment induced multinucleation, a phenotype not observed with other PDPK1 inhibitors. Proteome Integral Solubility Alteration (PISA) profiling revealed the transcription factor HES1 as a functional target of BX-912. Structural modeling showed that BX-912 binds the Orange domain of HES1, while its WRPW motif mediates interactions with protein partners, including the AP2 endocytic protein complex, coordinating their nuclear accumulation that leads to a mitotic catastrophe. Furthermore, cell cycle analyses showed that BX-912 combined with olaparib synergistically enhanced DNA damage and G2-M arrest. Our study demonstrates the value of proteomics for revealing hidden drug activities. It also identifies potential inhibition strategies for HES1, which is commonly overexpressed in HGSC. Additionally, this study proposes a novel strategy of targeting consecutive cell cycle phases to enhance treatment efficacy in HGSC.

cancer biology↗

bioRxiv2025

Staphylococcus aureus exhibits spatiotemporal heterogeneity in Sae activity during kidney abscess development

Virulence factors are required for bacterial pathogens to establish infection, however, their expression can be energetically costly, and must be tightly controlled to avoid fitness costs. Expression can be controlled at specific stages during infection (temporal regulation) or expressed by small subsets of the bacterial population (spatial regulation). There has been a great deal of interest in developing virulence factor-targeting strategies to combat Staphylococcus aureus infection, but the spatiotemporal regulation of the virulence factor master regulatory systems (Agr, Sae) has not been explored during kidney abscess formation. This information is critical for the design of therapeutics targeting these pathways. Here, we utilized a fluorescent transcriptional reporter approach to visualize dynamics in Agr and Sae activity during abscess formation in the mouse kidney. We categorized kidney abscess formation into four stages, then defined spatiotemporal gene expression. Agr signalling appeared inactive in the kidney; consistent with this, agr mutant abscesses fully developed. In contrast, we observed heterogeneous (ON/OFF) activity of Sae at early stages where bacteria were found intracellularly within neutrophils. Sae activity increased as abscesses developed, and heterogeneity in spatial patterning was observed, but patterns varied between abscesses suggesting distinct microenvironments within individual abscesses. Consistent with a requirement for Sae activity during abscess development, the sae mutant did not develop abscesses past early stages. These results have implications for the genes regulated by Agr and Sae, and suggest a requirement for Sae activity during kidney abscess development. ImportanceInfections with Staphylococcus aureus pose a serious public health threat due to high levels of antibiotic resistance and limited efficacy of alternative therapeutics. There has been a great deal of interest in developing novel therapeutics that target virulence factors essential during infection. However, it remains largely unknown if these factors are required at specific stages of the infection, and whether all bacterial cells or a limited subset express them. Here we sought to examine virulence factor expression using fluorescent reporter strains that would indicate activity of two master regulators of virulence in S. aureus, Agr and Sae. While Agr appeared inactive during kidney abscess development, the Sae system exhibited heterogeneity, increased expression at later stages, and was required for abscess progression. These results provide critical information for the development of virulence factor-targeting strategies for kidney abscess treatment.

microbiology↗

bioRxiv2021

Pyruvate oxidase as a key determinant of pneumococcal viability during transcytosis across the blood-brain barrier endothelium

Streptococcus pneumoniae (SPN / pneumococcus), invades myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defences governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs) formed following SPN internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which has been previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar metabolizing enzyme that catalyses oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of SpxB catalysed reaction, was also found to improve pneumococcal intracellular survival, by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. Expectedly, a {Delta}spxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting in its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. AUTHOR SUMMARYEukaryotic cells which constitute host barriers have innate immune defences to restrict microbial passage into sterile compartments. This necessitates need for pathogens to devise strategies to evade these, for successful establishment of disease. In this study, by focussing on the blood-brain barrier endothelium, we investigate the mechanisms which enable the opportunistic pathogen Streptococcus pneumoniae to traverse host barriers. Pyruvate oxidase, a pneumococcal sugar metabolizing enzyme was found to play a critical role in this key event, owing to production of acetyl phosphate and hydrogen peroxide via its enzymatic activity. On one hand, acetyl phosphate, by contributing to activation of acid tolerance stress response, enabled pneumococci to maintain viability in the lethal acidic pH of the lysosome-like vacuoles inside brain endothelium. On the other, hydrogen peroxide, was found to oxidise and inactivate a subset of degradative lysosomal enzymes. This two-pronged approach, aided by pyruvate oxidase, enabled pneumococci to evade intracellular degradation for successful transcytosis across the endothelium. Thus, pyruvate oxidase is a key determinant of pneumococcal virulence and hence can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

microbiology↗

bioRxiv2020

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-{pi} interaction, governing formation of the transmembrane {beta}-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with loss of pore forming ability of Ply facilitating a benign association of SPN in an alternative, intracellular host niche. AUTHOR SUMMARYStreptococcus pneumoniae, the main causative agent of pneumonia, triggers inflammation and tissue damage by expressing a pore-forming toxin, pneumolysin (Ply). Ply-induced inflammation drives pneumococcal transmission from nasopharynx (its primary reservoir), but also contributes to host mortality, limiting its occupiable habitats. Here, we uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways. This commensal-like lifestyle, resembling that of members of the mitis group of Streptococci, might have evolved within ST306 by loss of function ply mutations, compensating for limited nasopharyngeal carriage capacity by facilitating adaptation to an alternate niche.

microbiology↗

bioRxiv2018

High-resolution regulatory maps connect cardiovascular risk variants to disease related pathways

Genetic variant landscape of cardiovascular disease (CVD) is dominated by non-coding variants among which many occur within putative enhancers regulating the expression levels of relevant genes. It is crucial to assign the genetic variants to their correct gene both to gain insights into perturbed functions and better assess the risk of disease. In this study, we generated high-resolution genomic interaction maps (~750 bases) in aortic endothelial, smooth muscle and THP-1 macrophages using Hi-C coupled with sequence capture targeting 25,429 features including variants associated with CVD. We detected interactions for 761 CVD risk variants obtained by genome-wide association studies (GWAS) and identified novel as well as established functions associated with CVD. We were able to fine-map 331 GWAS variants using interaction networks, thereby identifying additional genes and functions. We also discovered a subset of risk variants interacting with multiple promoters and the expression levels of such genes were correlated. The presented resource enables functional studies of cardiovascular disease providing novel approaches for its diagnosis and treatment.

genomics↗