Biology subjects

Hachani, A.

Publications and source records attributed to Hachani, A..

10 recordsLinked to original sources

bioRxiv2026

Characterisation of novel Campylobacter jejuni Type VI secretion system (T6SS) effectors and exploration of the roles of the C. jejuni T6SS in bacterial antagonism and human host cell interaction

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

microbiology↗

bioRxiv2025

A Practical Resource for Multi-Omics Data Integration in Microbial Systems

The increasing availability of microbial multi-omics datasets has created new opportunities to explore complex biological systems. However, exploration remains limited by the lack of accessible, reproducible workflows that integrate multiple omics layers and deliver easily interpretable visualisations of functional and pathway-level insights. Here, we present an R-based workflow for integrated analysis and network-based pathway visualisation of microbial multi-omic data. The workflow enables microbiologists to analyse transcriptomic, proteomic, and metabolomic datasets either individually or in combination, apply univariate and multivariate approaches for biomarker discovery, and generate easily interpretable visualisations of functional and pathway-level signatures. Implemented as multi-step R Markdown, it leverages widely-used open-source tools, including mixOmics for biomarker identification and omics integration and clusterProfiler for pathway and functional enrichment analyses, with a new network-based integration and visualisation. Its flexible design supports a range of experimental structures and facilitates comparisons across strains, omics layers, and conditions, making it suitable for researchers with limited computational expertise. We demonstrate its utility using a publicly available Streptococcus pyogenes dataset, revealing both shared and strain-specific functional responses to human serum. This workflow provides a comprehensive and adaptable framework for systematic multi-omics analysis, improving accessibility and reproducibility and facilitating functional interpretation of microbial responses to diverse environments. Data summaryThe code for this workflow is available on GitHub (https://github.com/warasinee/Multiomics_Case_Study). Datasets from our previously published study (1) were used to showcase the functionality and practical utility of the workflow. The multi-omics Streptococcus pyogenes dataset used in this study is available in the following public repositories: Gene Expression Omnibus (GSE152821; GSE152822; GSE152823; GSE152824, GSE152826), Proteomics Identifications Database (PXD020863), and MetaobLights (MTBLS2324) (1). Impact StatementHigh-throughput omics technologies are transforming our understanding of how microbes adapt to diverse environments and cause disease. The integration of diverse omics layers at a systems level, combining transcriptomics, proteomics, and metabolomics data to identify signature molecules, pathways, and their interactions, remains challenging. Here, we present an R-based bioinformatic workflow designed for microbiology research, which connects existing tools and customised functions to streamline data integration and interpretation. The workflow links biomarkers to functional pathways, visualises results in an interactive network context, and is designed for flexibility and reproducibility. This practical resource lowers technical barriers to microbial multi-omics analysis, providing user-friendly access for dataset exploration and integration and supporting interpretation of system-level microbial adaptation in environmental, clinical, and industrial contexts.

systems biology↗

bioRxiv2025

Integrated multi-omics reveals coordinated Staphylococcus aureus metabolic, iron transport and stress responses to human serum

Bloodstream infections caused by Staphylococcus aureus remain a leading cause of mortality worldwide. Our understanding of S. aureus survival and persistence in human serum, a cell-free fraction of blood hostile for bacteria, is still limited. Here, we applied multivariate data integration methods and network analysis to a multi-omic dataset generated from five clinically prevalent S. aureus genotypes exposed to human serum. We observed and then confirmed using isogenic mutants, the significant roles of gapdhB, sucA, sirA, sstD, and perR in bacterial survival in serum. These data show that metabolic versatility in carbon source usage, iron transport and resistance to oxidative stress are interlinked and central to S. aureus fitness in serum, representing potential S. aureus vulnerabilities that could be exploited therapeutically. IMPORTANCEBloodstream infections caused by Staphylococcus aureus are associated with mortality rates of up to 30%. However, the molecular mechanisms that enable this pathogen to survive in human serum, a nutrient-limited and immunologically hostile environment remain poorly understood. By integrating multi-omic data from five clinically relevant S. aureus genotypes and validating key signatures using mutants, we identified conserved genetic determinants critical for bacterial survival in serum. Our findings highlight the interconnected roles of carbohydrate metabolic flexibility, iron acquisition, and oxidative stress resistance in shaping S. aureus adaptation to serum. This work advances our understanding of microbial strategies to survive in the bloodstream and demonstrates the potential of multi-omic integration to uncover therapeutic vulnerabilities in bacterial pathogens.

microbiology↗

bioRxiv2025

Simple sequence repeats power Staphylococcus aureus adaptation

Understanding Staphylococcus aureus adaptation is foundational for addressing the challenges posed by this pathogen. Using evolutionary convergence analysis on 7099 S. aureus genomes we uncovered frequent, reversible loss-of-function (LoF) mutations caused by simple sequence repeats (SSRs). Functional assays showed SSRs are potent, length-dependent switches that activate and deactivate genes, a mechanism exemplified by frequent LoF mutations in mutL (DNA mismatch repair) that causes a hypermutator phenotype. Analysis of over 600 episodes of human S. aureus colonisation and infection confirmed laboratory findings and showed that mutL SSR hypermutability behaves like a phase-variable evolutionary capacitor that unlocks subsequent adaptive SSR variations. We also experimentally demonstrate that SSRs facilitate foreign DNA uptake (hsdR restriction endonuclease) and lead to gentamicin-resistant small colony variants that are known to cause persistent infections (menF menadione biosynthesis). Furthermore, the phenomenon is widespread, with 27 of 29 diverse human microbiome samples harbouring S. aureus with distinct SSR subpopulations, indicative of niche-specific adaptations. Thus, SSRs are potent reservoirs of previously unrecognized S. aureus genetic heterogeneity that confer rapid adaptive capacity, including in antigens targeted by past vaccine trials. A deeper understanding of SSR evolutionary trajectories has the potential to improve treatments and to anticipate S. aureus responses to new therapies.

microbiology↗

bioRxiv2025

PI3Kδ Bridges Microbial Surveillance with Antigen Presentation to Reinforce Intestinal Immunity

Phosphoinositide 3-kinase delta (PI3K{delta}) is essential for immune cell functions, preventing immunodeficiency and inflammation; however, its role in dendritic cell (DC)-mediated immune regulation remains unknown. Here, we report a key role for DC-intrinsic PI3K{delta} in linking microbial recognition with antigen presentation for effective T-cell priming. Using genetic and functional assays, we demonstrate that PI3K{delta} deficiency in DCs leads to broad dysregulation of intestinal CD4 T-cell immunity, characterized by impaired regulatory T-cell expansion and increased susceptibility to colitis. DC-based studies show that PI3K{delta} links pattern-recognition-receptor signaling to MHC class I- and II-restricted presentation of phagosome-associated antigens by facilitating NOX2-dependent oxidative burst through RAC2, while mitigating inflammasome activation. In contrast, PI3K{delta} deficiency disrupts phagosomal pH balance, leading to accelerated acidification and proteolysis of antigens, impairing T-cell activation. Our study identifies PI3K{delta} as a key coordinator of phagosome dynamics, important for DC adaptive programming that shapes T-cell responses and supports intestinal immune homeostasis.

immunology↗

bioRxiv2025

Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a {Delta}fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

microbiology↗

bioRxiv2024

Quorum sensing and DNA methylation play active roles in clinical Burkholderia phase variation

Phenotypic diversity in bacteria often results from adaptation to changing environmental conditions and is exemplified by variable colony morphotypes. Discrete genomic changes and modulation in gene expression occur in Burkholderia pseudomallei undergoing adaptation. Alternatively, adapted colony morphotype variants of species belonging to the Burkholderia cepacia complex (Bcc) lose a complete replicon (the pC3 virulence megaplasmid), which affects their production of virulence factors. We report that variants arising in Burkholderia ambifaria clinical isolates - with affected phenotypes - have retained their pC3, suggesting that another phase variation mechanism can take place in this Bcc species. Proteomic and phenotypic characterisation showed that morphotype variants of B. ambifaria strains CEP0996 (pC3-null) and HSJ1 (pC3-positive) share similarities in phenotypes controlled by the Cep quorum sensing system. Thus, we determined the role of quorum sensing in B. ambifaria HSJ1 phase variation and confirmed that the main quorum sensing system Cep is important for the emergence of variants. As DNA methylation is one of the main epigenetic factors involved in bacterial phase variation that regulates some virulence factors of the Bcc species Burkholderia cenocepacia, we hypothesized that B. ambifaria HSJ1 phase variation could also be regulated by adenosine DNA methylation. By deleting the three putative adenosine DNA methyltransferases, we found that an orphan type II DNA methyltransferase prevents the emergence of phase variants. This is the first study to report quorum sensing and adenosine DNA methylation as two antagonistic systems independently controlling phase variation. ImportanceSome Burkholderia species are pathogenic to plants, animals, or humans. In immunocompromised individuals, such as those with cystic fibrosis, infection with Burkholderia cepacia complex (Bcc) bacteria can lead to "cepacia syndrome." In the Australian Aboriginal population, melioidosis caused by B. pseudomallei is prevalent, particularly among those with diabetes or alcoholism. Burkholderias phenotypic plasticity, including colony morphotype variation (CMV), enables rapid adaptation to diverse environments, enhancing survival and pathogenicity. This study reveals phase variation as a new CMV mechanism within the Bcc group. We found that quorum sensing and DNA methylation are involved in phase variation. Understanding the underlying CMV mechanisms could lead to the development of targeted therapies against these highly antibiotic-resistant bacteria.

microbiology↗

bioRxiv2023

The WalKR two-component regulator coordinates cell wall homeostasis with DNA replication in Staphylococcus aureus

Among the 16 two-component systems (TCSs) in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodelling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including ChIP-seq, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS); translation (rplK); DNA compaction (hup); initiation of DNA replication (dnaA, hup); and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by co-ordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Collectively, our findings address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics.

microbiology↗

bioRxiv2022

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (Intracellular Toxicity of S. aureus) to quantify intracellula cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

microbiology↗

bioRxiv2022

Niche-specific genome degradation and convergent evolution shaping Staphylococcus aureus adaptation during severe infections

During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr-mediated adaptation we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.

genomics↗