Biology subjects

Wenner, N.

Publications and source records attributed to Wenner, N..

16 recordsLinked to original sources

bioRxiv2026

Competition against the intestinal microbiota selects for pathoadaptive traits in ESBL E. coli

The intestinal tract is a reservoir for Extended-Spectrum {beta}-Lactamase (ESBL)-producing Escherichia coli. Asymptomatic gut colonization by these pathobionts represents a major risk for extraintestinal infections. Despite clinical relevance, the genetic basis of gut colonization by ESBL E. coli remains poorly understood. Here, we determined how the microbiota shapes the fitness landscape of diverse ESBL E. coli strains, defining the functional requirements for intestinal colonization. In microbiota-depleted hosts, colonization relies mostly on metabolic functions. In contrast, in mice harbouring a microbiota, pathoadaptive functions associated with adhesion and biofilm formation are dominant determinants of E. coli fitness, together with accessory virulence functions. Consistent with these observations, experimental evolution in mice reveals convergent adaptation of ESBL E. coli to the presence of a complex microbiota through enhanced adhesion. These findings establish the microbiota as a major ecological driver of pathoadaptation in antibiotic-resistant pathobionts.

microbiology↗

bioRxiv2025

DMB labelling for detection and analysis of capsular polysaccharides

Bacterial capsules are major virulence factors enabling systemic infection by undermining innate and adaptive immunity. Capsular polysaccharides are also the antigen in some of the most successful antibacterial vaccines - including the pneumococcal, neisserial and Hib conjugate vaccines. However, it remains exceptionally challenging to study capsules, primarily due to their high chemical diversity and the limited methods available for their detection and analysis. We describe a robust biochemical method for detection and analysis of ABC transporter-dependent capsular polysaccharides, a major class of capsules that are associated with extraintestinal pathogenic Escherichia coli (ExPEC). The method involves release and fluorescent tagging of polysaccharides from the cell surface. Anion exchange chromatography of labelled samples reveals the presence, relative abundance, and length-distribution of these diverse polysaccharide antigens. The method provides a modern approach to detecting the capsule phenotype, bridging a critical gap left by the decline of serotyping assays. It will enhance our understanding of fundamental capsule biology and advance the development of capsule-targeting vaccines.

biochemistry↗

bioRxiv2024

Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence

AO_SCPLOWBSTRACTC_SCPLOWBacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognize and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognizes cognate motifs has not been fully understood. We have characterised BREX from pathogenic Salmonella and generated the first X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that larger BREX complexes are required for methyltransferase activity and defence. Through rational engineering of PglX, we broadened both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is the sole specificity factor for BREX activity, providing motif recognition for both phage defence and host methylation.

microbiology↗

bioRxiv2023

Phenotypic heterogeneity drives phage-bacteria coevolution in the intestinal tract

Phenotypic heterogeneity in bacteria can generate reversible resistance against various stressors, including predation by phages. This allows mixed populations of phenotypically resistant and sensitive bacteria to coexist with virulent phages. However, it remains unclear if these dynamics prevent the evolution of genetic resistance in bacteria and how they affect the evolution of phages. In this work, we focus on bistable alterations of the O-antigen (known as phase variation) in Salmonella Typhimurium (S.Tm) to study how heterogeneous phenotypic resistance affects phage-bacteria coevolution. Our findings reveal that phase variation allows a stable coexistence of S.Tm with a virulent T5-like phage in vitro. This coexistence is nevertheless short-lived when S.Tm and the phage interact within the intestinal tract of mice. In this context, the phage evolves to also infect phenotypically resistant S.Tm cells, incidentally altering infectivity on other Salmonella serovars. In return, the broader host range of the evolved phages drives the evolution of genetic resistance in S.Tm, which results in phage extinction. This work demonstrates that phenotypic heterogeneity profoundly influences the antagonistic coevolution of phages and bacteria, with outcomes intricately tied to the ecological context.

microbiology↗

bioRxiv2023

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi, and we performed an intensive comparative genomic analysis of 608 isolates obtained from fever surveillance at the Queen Elizabeth Hospital, Blantyre between 1996 and 2018. We discovered that following the upsurge of the well-characterised S. Typhimurium ST313 lineage 2 from 1999 onwards, two new multidrug-resistant sublineages designated 2.2 and 2.3, emerged in Malawi in 2006 and 2008, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineage 2.2 or 2.3. To identify factors that characterised the emergence of the prevalent ST313 sublineage 2.2, we performed genomic and functional analysis of two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). Comparative genomic analysis showed that the chromosome of ST313 lineage 2 and sublineage 2.2 were broadly similar, only differing by 29 SNPs and small indels and a 3kb deletion in the Gifsy-2 prophage region that spanned the sseI pseudogene. Lineage 2 and sublineage 2.2 have unique plasmid profiles that were verified by long read sequencing. The transcriptome was initially explored in 15 infection-relevant conditions and within macrophages. Differential gene expression was subsequently investigated in depth in the four most important in vitro growth conditions. We identified up-regulation of SPI2 genes in non-inducing conditions, and down-regulation of flagellar genes in D37712, compared to D23580. Following phenotypic confirmation of transcriptional differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed-growth in minimal media. We speculate that this competitive advantage is contributing to the continuing presence of sublineage 2.2 in Malawi.

bioinformatics↗

bioRxiv2022

Salmonella succinate utilisation is inhibited by multiple regulatory systems

Succinate is a potent immune signalling molecule that is present in the mammalian gut and within macrophages. Both of these niches are colonised by the pathogenic bacterium Salmonella enterica serovar Typhimurium during infection. Succinate is a C4-dicarboyxlate that can serve as a source of carbon for bacteria. When succinate is provided as the sole carbon source for in vitro cultivation, Salmonella and other enteric bacteria exhibit a slow growth rate and a long lag phase. This growth inhibition phenomenon was known to involve the sigma factor RpoS, but the genetic basis of the repression of bacterial succinate utilisation was poorly understood. Here, we used an experimental evolution approach to isolate fast-growing mutants during growth of S. Typhimurium on succinate containing minimal medium. Our approach reveals novel RpoS-independent systems that inhibit succinate utilisation. The CspC RNA binding protein restricts succinate utilisation, an inhibition that is antagonised by high levels of the small regulatory RNA (sRNA) OxyS. We discovered that the Fe-S cluster regulatory protein IscR inhibits succinate utilisation by repressing the C4-dicarboyxlate transporter DctA. The RNA chaperone Hfq, the exoribonuclease PNPase and their cognate sRNAs function together to repress succinate utilisation via RpoS induction. Furthermore, the ribose operon repressor RbsR is required for the complete RpoS-driven repression of succinate utilisation, suggesting a novel mechanism of RpoS regulation. Our discoveries shed light on redundant regulatory systems that tightly regulate the utilisation of succinate. We propose that the control of central carbon metabolism by multiple regulatory systems in Salmonella governs the infection niche-specific utilisation of succinate.

microbiology↗

bioRxiv2022

Genome-wide fitness analysis identifies genes required for in vitro growth and macrophage infection by African and Global Epidemic pathovariants of Salmonella Enteritidis

Salmonella Enteritidis is the second most common serovar associated with invasive non-typhoidal Salmonella (iNTS) disease in sub-Saharan Africa. Previously, genomic and phylogenetic characterisation of S. Enteritidis isolates from human bloodstream led to the discovery of the Central/Eastern African (CEAC) and West African clades, which were distinct from the gastroenteritis-associated Global Epidemic clade (GEC). The African S. Enteritidis clades have unique genetic signatures that include genomic degradation, novel prophage repertoires and multi-drug resistance, but the molecular basis for the enhanced propensity of African S. Enteritidis to cause bloodstream infection is poorly understood. We used transposon insertion sequencing (TIS) to identify the genetic determinants of the GEC representative strain P125109 and the CEAC representative strain D7795 for growth in three in vitro conditions (LB or minimal NonSPI2 and InSPI2 growth media), and for survival and replication in RAW 264.7 murine macrophages. We identified 207 in vitro-required genes that were common to both S. Enteritidis strains and also required by S. Typhimurium, S. Typhi and Escherichia coli, and 63 genes that were only required by individual S. Enteritidis strains. Similar types of genes were required by both P125109 and D7795 for optimal growth in particular media. Screening the transposon libraries during macrophage infection identified 177 P125109 and 201 D7795 genes that contribute to bacterial survival and replication in mammalian cells. The majority of these genes have proven roles in Salmonella virulence. Our analysis also revealed candidate strain-specific macrophage fitness genes, some of which represent potential novel Salmonella virulence factors. IMPACT STATEMENTInvasive non-typhoidal Salmonella (iNTS) disease is a systemic infection that has a high case fatality rate of 15% and is responsible for an estimated 66,500 deaths/year in sub-Saharan Africa. The main causative agents are pathovariants of Salmonella Typhimurium, known as S. Typhimurium ST313, and Salmonella Enteritidis (S. Enteritidis), known as Central/Eastern African (CEAC) and West African S. Enteritidis. Whilst the African S. Typhimurium pathovariant has been an active focus of research over the past decade, studies on African S. Enteritidis have been lacking. We used transposon insertion sequencing (TIS) to identify the genetic requirements of both African and Global Epidemic S. Enteritidis to grow in vitro and to infect murine macrophages. To our knowledge, this is the first genome-wide functional analysis of African S. Enteritidis under conditions relevant to infection of a mammalian host. We show that the gene sets required for growth under laboratory conditions and macrophage infection by African and Global Epidemic S. Enteritidis were broadly similar, and that the majority of the genes that contribute to survival and replication in macrophage already have proven roles in Salmonella virulence. Our analysis did identify candidate strain-specific macrophage fitness genes, some of which could be novel Salmonella virulence factors.

microbiology↗

bioRxiv2021

Biogenesis of a bacterial metabolosome for propanediol utilization

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both "Shell first" and "Cargo first" assembly pathways, unlike the {beta}-carboxysome structural analog which only involves the "Cargo first" strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

microbiology↗

bioRxiv2021

Opposing roles of Granzymes A and B in the immune response to intestinal infection

Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non- redundant roles. GzmB-/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA-/-mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/467595v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@2b2c9corg.highwire.dtl.DTLVardef@7de81eorg.highwire.dtl.DTLVardef@1209d2org.highwire.dtl.DTLVardef@42dffb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria. SIGNIFICANCEProteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.

microbiology↗

bioRxiv2021

Isolation and characterisation of bacteriophages with activity against invasive non-typhoidal Salmonella causing bloodstream infection in Malawi

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in sub-Saharan Africa.

microbiology↗

bioRxiv2020

Prophage-encoded phage defence proteins with cognate self-immunity

Temperate phages are pervasive in bacterial genomes, existing as vertically-inherited islands called prophages. Prophages are vulnerable to the predation of their host bacterium by exogenous phages. Here we identify BstA, a novel family of prophage-encoded phage defense proteins found in diverse Gram-negative bacteria. BstA drives potent suppression of phage epidemics through abortive infection. During lytic replication, the bstA-encoding prophage is not itself inhibited by BstA due to a self-immunity mechanism conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella and Escherichia prophages is functionally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, and cannot be exploited by heterologous BstA-encoding phages. BstA allows prophages to defend host cells against exogenous phage attack, without sacrificing their own lytic autonomy.

microbiology↗

bioRxiv2019

A window into lysogeny: Revealing temperate phage biology with transcriptomics

Integrated phage elements, known as prophages, are a pervasive feature of bacterial genomes. Prophages can enhance the fitness of their bacterial hosts by conferring beneficial functions, such as virulence, stress tolerance or phage resistance, which are encoded by accessory loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, novel prophage accessory loci are challenging to identify based on DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and often represent functionally-important accessory loci. Using this approach we identify a novel anti-sense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1 and immunity to closely-related phages. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.

microbiology↗

bioRxiv2019

The diversity, evolution and ecology of Salmonella in venomous snakes

BackgroundReptile-associated Salmonella are a major, but often neglected cause of both gastrointestinal and bloodstream infection globally. The diversity of Salmonella enterica has not yet been determined in venomous snakes, however other cold-blooded animals have been reported to carry a broad range of Salmonella bacteria. We investigated the prevalence and assortment of Salmonella in a collection of venomous snakes in comparison with non-venomous reptiles.\n\nMethodology/Principle FindingsWe used a combination of selective enrichment techniques and whole-genome sequencing. We established a unique dataset of reptilian isolates to study Salmonella enterica species-level evolution and ecology and investigated differences between phylogenetic groups. We observed that 91% of venomous snakes carried Salmonella, and found substantial diversity between the serovars (n=58) carried by reptiles. The Salmonella serovars belonged to four of the six Salmonella enterica subspecies: diarizonae, enterica, houtanae and salamae. Subspecies enterica isolates were distributed among two distinct phylogenetic clusters, previously described as clade A (52%) and clade B (48%). We identified metabolic differences between S. diarizonae, S. enterica clade A and clade B involving growth on lactose, tartaric acid, dulcitol, myo-inositol and allantoin.\n\nSignificanceWe present the first whole genome-based comparative study of the Salmonella bacteria that colonise venomous and non-venomous reptiles and shed new light on Salmonella evolution. The findings raise the possibility that venomous snakes are a reservoir for human Salmonellosis in Africa. The proximity of venomous snakes to human dwellings in rural Africa may result in contaminated faecal matter being shed on surfaces and in water sources used for human homes and to irrigate salad crops. Because most of the venomous snakes had been captured in Africa, we conclude that the high level of Salmonella diversity reflects the African environmental niches where the snakes have inhabited.\n\nAuthor SummarySalmonella enterica is a remarkable bacterial species that causes Neglected Tropical Diseases globally. The burden of disease is greatest in some of the most poverty-afflicted regions of Africa, where salmonellosis frequently causes bloodstream infection with fatal consequences. The bacteria have the ability to colonise the gastrointestinal tract of a wide range of animals including reptiles. Direct or indirect contact between reptiles and humans can cause Salmonellosis. In this study, we determined the prevalence and diversity of Salmonella in a collection of African venomous snakes for the first time. Using the power of genomics, we showed that the majority of venomous snakes (91%) carry Salmonella, two thirds of which belonged to a subspecies of S. enterica called enterica, which is associated with most cases of human salmonellosis. Within the S. enterica subspecies we identified two evolutionary groups which display distinct growth patterns on infection relevant carbon sources. Our findings could have particular significance in Africa where venomous snakes wander freely around human dwellings and potentially shed contaminated faecal matter in water sources and on surfaces in rural homes.

microbiology↗

bioRxiv2018

Adding function to the genome of African Salmonella ST313

Salmonella Typhimurium ST313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580, and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe, revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in sixteen infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium SalComD23580: bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by 3 independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolise this carbon source has been negatively selected during ST313 evolution. The data revealed a novel plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multi-condition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

microbiology↗

bioRxiv2017

The role of a single non-coding nucleotide in the evolution of an epidemic African clade of Salmonella

Introductory ParagraphSalmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely-related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by just 1000 single-nucleotide polymorphisms (SNPs). We hypothesised that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes.\n\nHere we identified 3,597 transcriptional start sites (TSS) of the ST313 strain D23580, and searched for a gene expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance and modulated virulence in the chicken infection model. The PgtE protease is known to mediate systemic infection in animal models. We propose that high levels of expression PgtE of by African S. Typhimurium ST313 promotes bacterial survival and bacterial dissemination during human infection.\n\nOur finding of a functional role for an extra-genic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on non-coding regions of the genome.

molecular biology↗