Biology subjects

Hussain, F.

Publications and source records attributed to Hussain, F..

9 recordsLinked to original sources

bioRxiv2026

Functional decoding reveals a hidden regulatory layer of the Salmonella transcriptome during infection

Bacterial transcriptomes contain extensive, largely unexplored regulatory information beyond annotated genes, including small regulatory RNAs (sRNAs), yet distinguishing functionally active transcripts from the broader non-coding transcriptome remains a fundamental challenge, particularly in the context of host-pathogen interactions. Here, we develop an unbiased highthroughput functional screening strategy to decode the regulatory potential of the Salmonella enterica transcriptome during macrophage infection. A pooled expression library comprising 875 RNA fragments derived from infection-relevant conditions was screened for effects on bacterial invasion and intracellular survival, revealing distinct and stage-specific regulatory activities. Functional characterization uncovered non-canonical sRNAs originating from 5' untranslated regions that differentially modulate virulence-associated programs spanning the SPI-1, SPI-4 and SPI-2 pathogenicity islands. One attenuates the SPI-1 and SPI-4 secretion systems, limiting bacterial adhesion and invasion, whereas the other promotes invasion-associated programs while repressing pathways supporting intracellular survival. Rather than acting solely on individual virulence determinants, these regulatory activities reveal a broader layer of posttranscriptional control that coordinates bacterial adaptation across the dynamic host environment. Our study establishes a scalable strategy for extracting functional regulatory information from condition-specific bacterial transcriptomes and provides a framework for uncovering context-dependent RNA-mediated control of complex host-associated phenotypes.

microbiology↗

bioRxiv2025

VIRGO2: Unveiling the Functional and Ecological Complexity of the Vaginal Microbiome with an Enhanced Non-Redundant Gene Catalog

Despite the importance of the cervicovaginal microbiome, the mechanisms that govern its composition and drive its impact on host physiology remain poorly understood. This study expands our understanding of the function and ecology of the vaginal microbiome using VIRGO2, an enhanced non-redundant gene catalog comprising over 1.7 million well-annotated genes from body-site specific microbes and viruses. Analyses using VIRGO2 revealed novel insights, including the identification of previously uncharacterized vaginal bacteria, features of the vaginal mycobiome and phageome, and differential expression of bacterial carbohydrate catabolic genes. Constructed from over 2,500 metagenomes and 4,000 bacterial genomes, VIRGO2 broadens geographic representation and microbial diversity compared to its predecessor. This updated catalog enables more precise profiling of taxonomic and functional composition from metagenomic and metatranscriptomic datasets. VIRGO2 is a critical resource for integrative analyses of vaginal microbial communities and their interactions with host tissues, thereby enhancing our mechanistic understanding of vaginal health and disease.

microbiology↗

bioRxiv2024

An unprecedented small RNA-riboswitch interaction controls expression of a bifunctional pump that is essential for Staphylococcus aureus infection

Maintaining manganese and iron homeostasis is critical for the human pathogen Staphylococcus aureus. To counteract metal-based host defense strategies (e.g., nutritional immunity, metal poisoning), S. aureus uses a combination of metal-sensing transcription factors and regulatory RNAs to maintain metal homeostasis. In this study, we uncovered an unprecedented interaction between a cis- and a trans-acting regulatory RNA controlling a conditionally essential gene, mntY, encoding a Mn efflux pump. This broadly conserved RNA-RNA interaction between a Fe-responsive sRNA and a Mn-sensing riboswitch allows the integration of Feand Mn-related stresses, notably encountered at the infection site, to fine-tune mntY expression. Remarkably, deletion of the mntY gene is strongly pleomorphic, causing growth defects, altering virulence factor expression, immune evasion, and survival during infection. We demonstrated that MntY is critical for the adaptation of S. aureus to both low and high Mn environments, due to its dual role in metalation of Mn-dependent exoenzymes and Mn detoxification. These findings point to MntY as a promising new therapeutic target to combat multidrug-resistant staphylococcal infections.

microbiology↗

bioRxiv2024

Interpretable machine learning reveals a diverse arsenal of anti-defenses in bacterial viruses

Antagonistic interactions with viruses are an important driver of the ecology and evolution of bacteria, and associating genetic signatures to these interactions is of fundamental importance to predict viral infection success. Recent studies have highlighted that bacteria possess a large, rapidly changing arsenal of defense genes and that viruses can neutralize at least some of these genes with matching anti-defenses. However, a broadly applicable approach for discovering the genetic underpinnings of such interactions is missing since typically used methods such as comparative genomics are limited by the rampant horizontal gene transfer and poor annotation of viral and bacterial genes. Here we show that genes that allow the viruses to overcome bacterial defenses can be systematically identified using an interpretable machine-learning approach even when using diverse bacteria-virus infection data. To verify the predictions, we experimentally characterized eight previously unknown anti-defense proteins in viruses specific for Vibrio bacteria and showed that they counteract a wide range of bacterial immune systems, including AbiH, AbiU, Septu, DRT, CBASS, and Retron. The power of our computational approach is highlighted by the identification of anti-defense proteins that inhibit non-homologous defense systems, which we verify for Retron and AbiH. We suggest that the computational prediction based on experimental interactions offers a promising avenue to unravel the genetic mechanisms of co-evolution between bacteria and their viruses.

microbiology↗

bioRxiv2023

Balanced cell division is secured by two different regulatory sites in OxyS RNA

By interfering with cell division, the Escherichia coli oxidative stress-induced OxyS small RNA brings about cell cycle arrest thus allowing DNA damage repair. Cell division and cell elongation are opposing functions to the extent that inhibition of cell division requires a parallel inhibition of cell elongation for the cells to survive. In this study, we report that in addition to cell division OxyS inhibits mepS encoding an essential peptidoglycan endopeptidase responsible for cell elongation. Furthermore, a phylogenetic evolutionary target conservation analysis of OxyS homologs to identify OxyS most common function revealed that the majority of OxyS targets belong to the category of "cell cycle" followed by "peptidoglycan metabolism", especially cell elongation. mepS is the most frequent target of this category, predicted to be regulated by OxyS in 64 % of the 146 organisms studied. We suggest that cell cycle arrest and balancing between cell division and cell elongation are important and conserved functions of the oxidative stress induced sRNA OxyS.

microbiology↗

bioRxiv2021

Genetic determinism of phage-bacteria coevolution in natural populations

Coevolution between bacteriophage (or phage) and their bacterial host is thought to be key for the coexistence of these antagonists. Recent studies have revealed the major role of mobile genetic elements in the emergence of phage resistant hosts but how phage escape these defenses in the wild remained to be explored. Here we show a striking parallel in phage evolving counter defenses to host defenses in natural population. We established a large collection of phages and their bacterial hosts and we explored the genetic structure of their interaction. We find that clearly delineated genomic clusters of phage are specific for distinct clades within a bacterial species, Vibrio crassostreae, yet while all phages can adsorb, only a subset of hosts are killed due to intracellular defense mechanisms. Host genomes contain multiple mobile defense genes and susceptibility to phage is negatively correlated with genome size. Phages also display extensive gene content variation, but their genome size remains conserved. We show that this gene content variation in hosts and phage is due to rapid turnover of genes involved in defense and escape, and that by exchanging anti-defense genes, phages irreversibly switch host. This could be indicative of co-evolution following the matching-allele-model of specificity and the spatial and temporal variability of phage infectivity further suggests that negative-frequency dependent selection drives phage-vibrio coevolutionary dynamics. We propose a "pan-escape system" that can be shared among phages by homologous recombination within a population that infects a bacterial host.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa biofilms display carbohydrate ligands for CD206 and CD209 that interfere with their receptor function

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of carbohydrate polymers, nucleic acids and proteins. Carbohydrates within P. aeruginosa biofilms include neutral and mannose-rich Psl, and cationic Pel composed of N-acetyl-galactosamine and N-acetyl-glucosamine. Here we show that P. aeruginosa biofilms display ligands for the C-type lectin receptors mannose receptor (MR, CD206) and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209). Binding of MR and DC-SIGN to P. aeruginosa biofilms is carbohydrate-and calcium-dependent and extends to biofilms formed by clinical isolates. Confocal analysis of P. aeruginosa biofilms shows abundant DC-SIGN ligands among bacteria aggregates while MR ligands concentrate into discrete clusters. DC-SIGN ligands are also detected in planktonic P. aeruginosa cultures and depend on the presence of the common polysaccharide antigen. Carbohydrates purified from P. aeruginosa biofilms are recognised by DC-SIGN and MR; both receptors preferentially bind the high molecular weight fraction (HMW; >132,000Da) with KDs in the nM range. HMW preparations contain 74.9-80.9% mannose, display -mannan segments and alter the morphology of human dendritic cells without causing obvious changes in cytokine responses. Finally, HMW interferes with the endocytic activity of cell-associated MR and DC-SIGN. This work identifies MR and DC-SIGN as receptors for bacterial biofilms and highlights the potential for biofilm-associated carbohydrates as immunomodulators through engagement of C-type lectin receptors. Author SummarySelective engagement of pattern recognition receptors during infection guides the decision-making process during induction of immune responses. This work identifies mannose-rich carbohydrates within bacterial biofilms as novel molecular patterns associated with bacterial infections. P. aeruginosa biofilms and biofilm-derived carbohydrates bind two important lectin receptors, MR (CD206) and DC-SIGN (CD209), involved in recognition of self and immune evasion. Abundance of MR and DC-SIGN ligands in the context of P. aeruginosa biofilms could impact immune responses and promote chronic infection.

microbiology↗

bioRxiv2018

Receptor Mediated Delivery of Cas9-Nanobody Induces Cisplatin Synthetic Dose Sensitivity

The CRISPR/Cas9 system has shown great potential for precisely editing genomic DNA sequences by introducing site-specific DNA cuts that are subsequently repaired by the cell. However, delivery of the CRISPR ribonucleoprotein remains an understudied area and hinders realizing the full potential of the system. We prepared Cas9 ribonucleoprotein complexes chemically conjugated to the 7D12 nanobody and demonstrate receptor-mediated transfection of Cas9 into A549 non-small-cell lung cancer cells via binding to the epithelial growth factor receptor for subsequent cell internalization. We further show that transfection with a Cas9 ribonucleoprotein targeting the BRCA2 gene results in an enhanced sensitivity to the chemotherapeutic drug Cisplatin, and thereby induces a synthetic dose lethality in A549 cells.

biochemistry↗

bioRxiv2018

Efficient Homology Directed Repair by Cas9:DNA Localization and Cationic Polymeric Transfection in Mammalian Cells

Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.\n\nSummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

biochemistry↗