Biology subjects

Minhas, V.

Publications and source records attributed to Minhas, V..

5 recordsLinked to original sources

bioRxiv2026

Genome-wide interrogation of genetic requirements for root colonization by CRISPRi-seq in Bacillus subtilis

Bacillus subtilis is a known plant growth promoting rhizobacterium, yet, the genetic basis of effective root colonization remains understudied. Here, we applied genome-wide CRISPR interference sequencing (CRISPRi-seq) to systematically identify genes required for early Arabidopsis thaliana root colonization by the B. subtilis strain 3610 . Our genome-wide inducible CRISPRi library was used to screen for bacterial fitness during hydroponic root colonization. A complementary RNA-seq in the same conditions further revealed a distinct transcriptional state of root-associated cells. We identified 249 genes impacting root colonization. We reveal a critical role of cell envelope remodeling during early root association, specifically wall teichoic acid synthesis and D-alanylation. Moreover, we observed a metabolic shift toward TCA-cycle-driven aerobic growth with uptake of fructose, active repression of sporulation, competence and prophage programs, as well as induction of secondary metabolite biosynthesis. Our work establishes CRISPRi-seq as a powerful tool for dissecting bacterial-plant interactions relevant to sustainable agriculture.

microbiology↗

bioRxiv2026

Epithelial innate immune sensing of pneumococci is inherently restricted to a small cellular minority across species and infection niches

Streptococcus pneumoniae colonises the nasopharynx asymptomatically yet causes life-threatening invasive disease. How it navigates early epithelial immune surveillance to cause disease remains unclear. Conventional innate immune models predict coordinated, population-wide epithelial responses to bacterial infection. Using single-cell RNA sequencing, RNA fluorescence in situ hybridization and in vivo mouse and zebrafish models, pneumococcal infection is instead shown to activate innate immune genes including chemokine, NF-{kappa}B regulatory, and prostaglandin pathway genes, in only 1-4% of lung epithelial cells. This restriction is seemingly pneumococcal-specific as Escherichia coli triggers responses in over 40% of the same cells. Strikingly, nasopharyngeal epithelial cells show complete immune silence to pneumococci while responding robustly to E. coli and Staphylococcus aureus, suggesting niche-specific immune evasion. Additionally, pharmacological inhibition of COX-2 significantly increased mortality in a zebrafish meningitis model, identifying prostaglandin signalling as a protective host response during invasive disease. Competence-associated surface remodelling contributes modestly and incrementally to immune restriction, while the predominant dampening is competence-independent. These findings challenge canonical epithelial immunity models against bacterial infection and provide a cellular framework for understanding pneumococcal commensalism and pathogenesis. Significance statementClassical innate immune models predict that bacterial infection triggers coordinated, population-wide transcriptional responses across the epithelium. Using single-cell RNA sequencing, RNA fluorescence in situ hybridization, and in vivo zebrafish and mouse models, we show that Streptococcus pneumoniae, responsible for over one million deaths annually, activates innate immune genes in only 1-4% of lung epithelial cells. Escherichia coli triggers responses in over 40% of the same cells, demonstrating this restriction is pneumococcal-specific. Nasopharyngeal epithelial cells, the bacteriums primary colonization niche, show complete immune silence to pneumococci, suggesting niche-specific evolutionary adaptation. The prostaglandin pathway is identified as a protective host response during invasive disease. These findings challenge canonical models of epithelial immunity and provide a cellular framework for pneumococcal commensalism and pathogenesis.

microbiology↗

bioRxiv2025

MAIT cells protect in severe pneumococcal pneumonia by regulating neutrophil/macrophage antimicrobial activities

Mucosal-Associated Invariant T (MAIT) cells populate the lung tissue where they contribute to defense against respiratory infections. While MAIT cells have been implicated in host resistance to infections caused by Gram-negative bacteria, their contribution in immunity against Gram-positive bacteria-driven pneumonia is still enigmatic. Here, we demonstrate that both mouse and human MAIT cells are activated during severe infection caused by Streptococcus pneumoniae, the major cause of community-acquired bacterial pneumonia. Upon infection, lung MAIT cells undergo a transcriptional reprogramming associated with acquisition of potent antimicrobial properties. MAIT cell-deficient mice are more susceptible to pneumococcal pneumonia, including higher mortality, uncontrolled bacterial growth and dissemination, and impaired neutrophil and interstitial macrophage activity. Moreover, prophylactic stimulation of MAIT cells using cognate antigen protects from pneumococcus-induced lethal pneumonia. These findings demonstrate that MAIT cells are key cellular actors during Gram-positive bacterial infections.

immunology↗

bioRxiv2022

Competence remodels the pneumococcal cell wall providing resistance to fratricide and surface exposing key virulence factors

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation and virulence. Competent bacteria produce so called fratricins such as CbpD, that kill non-competent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall- and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

microbiology↗

bioRxiv2019

In Vivo Dual RNA-Seq Analysis Reveals the Basis for Differential Tissue Tropism of Clinical Isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is a genetically diverse human-adapted pathogen commonly carried asymptomatically in the nasopharynx. We have recently shown that a single nucleotide polymorphism (SNP) in the raffinose pathway regulatory gene rafR accounts for a significant difference in the capacity of clonally-related strains to cause localised versus systemic infection. Here we have used dual RNA-seq to show that this SNP extensively impacts both bacterial and host transcriptomes in infected lungs. It affects expression of bacterial genes encoding multiple sugar transporters, and fine-tunes carbohydrate metabolism, along with extensive rewiring of host transcriptional responses to infection, particularly expression of genes encoding cytokine and chemokine ligands and receptors. The dual RNA-seq data predicted a crucial role for differential neutrophil recruitment in the distinct virulence profiles of the infecting strains and single cell analysis revealed that while reduced expression of the RafR regulon driven by a single rafR SNP provides a clear advantage for pneumococci to colonize the ear, in the lung it leads to massive recruitment of neutrophils and bacterial clearance. Importantly, the observed disease outcomes were confirmed by in vivo neutrophil depletion showing that early detection of bacteria by the host in the lung environment is crucial for effective clearance. Thus, dual RNA-seq provides a powerful tool for understanding complex host-pathogen interactions and revealed how a single bacterial SNP can drive differential disease outcomes.

microbiology↗