Biology subjects

Hinton, J. C. D.

Publications and source records attributed to Hinton, J. C. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Bloodstream-associated Salmonella Typhimurium and Enteritidis iNTS pathovariants hyper-replicate in human macrophages

Invasive non-typhoidal Salmonella (iNTS) are a major cause of bloodstream infections in sub-Saharan Africa, yet the host-pathogen interaction mechanisms remain poorly understood. Here, we developed and optimised a human macrophage infection model based on PMA-differentiated THP-1 cells to investigate infection dynamics of clinically relevant Salmonella Typhimurium and Salmonella Enteritidis strains. We compared intracellular survival and replication of gastroenteritis-associated and bloodstream-associated pathovariants, including S. Typhimurium ST313 Lineage 2, the novel S. Typhimurium ST313 Lineage 3 and the understudied S. Enteritidis Central/Eastern African (CEAC) clades. Our results reveal that the CEAC S. Enteritidis and ST313 S. Typhimurium iNTS pathovariants hyper-replicate within host cells, compared to global epidemic isolates. The cellular model achieved robust pro-inflammatory polarisation of human macrophages, while revealing limitations in modelling macrophage plasticity. Overall, this work defines clear differences in intracellular behaviour of Salmonella pathovariants and establishes a robust experimental framework for future studies on invasive disease pathogenesis and therapeutic interventions that target iNTS bacteria.

microbiology↗

bioRxiv2025

Comparative phenotypic, genomic and transcriptomic characterisation of two Salmonella Typhimurium strains for a first-in-human challenge model

BackgroundInvasive non-typhoidal Salmonella (iNTS) disease remains a major public health challenge in sub-Saharan Africa. Salmonella Typhimurium is responsible for the majority of cases, with specific lineages being associated with increased risk of bloodstream infection. We have recently developed a Salmonella Typhimurium controlled human infection model (CHIM) to better understand disease pathogenesis and to provide a platform to test candidate vaccines. Selecting appropriate challenge strainsis a central design consideration in developing challenge model protocols. We describe the rationale, manufacture, and detailed characterisation of the two Salmonella Typhimurium strains used in the first-in-human NTS CHIM. MethodsTwo Salmonella Typhimurium strains, 4/74 (ST19) and D23580 (ST313), were selected from the UK Health Security Agency National Collection of Type Cultures and manufactured under Good Manufacturing Practice (GMP) conditions. Challenge agent stocks underwent microbial limits testing, viability and stability assessments, and phenotypic characterisation including growth kinetics, motility, acid sensitivity, and antibiotic susceptibility. Whole-genome sequencing was performed to confirm genetic stability post-manufacture. The effect of pre-challenge handling conditions was assessed in saline and sodium bicarbonate buffers, and bacterial survival was evaluated under simulated gastric and intestinal conditions using a modified Rossett-Rice model. Transcriptomic profiling was undertaken to determine whether sodium bicarbonate exposure altered expression of key virulence genes. ResultsBoth strains retained their expected phenotypic characteristics, including reduced motility and melibiose utilisation in D23580. GMP stocks remained pure, viable, and stable post-manufacture, with no unexpected genomic mutations detected. Both strains were susceptible to clinically relevant antibiotics used in the study. Survival was maintained in neutral and mildly acidic conditions, with significant reduction below pH 3.5. Stability was preserved for up to one hour in saline buffer and during simulated gastric transit in the Rossett-Rice model. Transcriptomic analysis showed no significant changes in Salmonella pathogenicity island-1 or 2, or flagellar gene expression following sodium bicarbonate exposure ConclusionsThese findings confirm that the Salmonella Typhimurium 4/74 and D23580 strains retained genomic and phenotypic integrity post-manufacture. The resulting challenge stocks provide the foundation for an ongoing NTS CHIM that aims to advance understanding of NTS and iNTS pathogenesis and support candidate vaccine testing.

microbiology↗

bioRxiv2025

Salmonella effector kinase SteC is activated by host-mediated phosphorylation

The pathogen Salmonella, which causes significant human morbidity and mortality, encodes an effector kinase, SteC, which mediates actin polymerisation and cell migration. Given the minimal nature of its kinase domain, it remains unclear how SteC is catalytically active and how this activity is regulated. Here, we show that SteC is activated following the phosphorylation of the highly conserved S379 residue by a host kinase. Phosphorylation of S379 dramatically increases nucleotide binding affinity of SteC, enabling substrate phosphorylation and promoting actin polymerisation. Further mutational analysis identified the functional role of HD and DGD motifs that likely mimic the HxD and DFG motifs of eukaryotic kinases. Meanwhile, the C-tail of SteC, encompassing amino acids 429-457, is essential for function following translocation from Salmonella, but dispensable for catalysis in vitro. Overall, our findings uncover two previously unappreciated mechanisms that mediate the activity of the only Salmonella effector kinase within the host. HIGHLIGHTSSteC is phosphorylated at S379 in mammalian cells SteC phosphorylation is critical for kinase activity and actin polymerisation Phosphorylation at S379 promotes nucleotide binding The C-tail of SteC is not required for catalysis but is essential for activity in infected cells

microbiology↗

bioRxiv2024

Colicin Receptor CirA Enhances Salmonella Typhimuriums Resistance to Colicin Ib in the Absence of the Cognate Immunity Protein

Intestinal microbiota play a central role in colonisation resistance providing a fundamental barrier to infection to enteric pathogens. An important mechanism of colonisation resistance involves the production of antimicrobial peptides, such as colicins. Pore-forming colicins, synthesised by Escherichia coli (E. coli) strains, target competing bacteria in their environmental niche, whilst the producing cells are safeguarded by specific immunity proteins. Notably, non-typhoidal Salmonella Typhimurium strains can produce a narrow-spectrum protein toxin colicin IB (ColIb) providing a competitive edge against susceptible Enterobacteriaceae strains. However, the multi-drug resistant and systemically invasive iNTS (invasive non-Typhoidal Salmonella) S. Typhimurium D23580 strain poses an interesting case. The strain lacks colicin Ib production and the corresponding immunity protein, but its potential vulnerability in a colicin-rich gastrointestinal milieu remains uninvestigated. In this study, S. Typhimurium D23580 exhibited resistance to colicin Ib under tested conditions, despite the absence of the immunity gene imm. Intriguingly, in colicin Ib-producing S. Typhimurium strains, the imm gene appeared functionally redundant in contrast to our current understanding. ColIb binds to the outer membrane protein CirA and is translocated to the inner membrane where it forms a pore in sensitive bacteria dissipating the electrochemical potential. Through a series of experimental approaches, including the use of Escherichia coli and S. Typhimurium cirA deletion mutants, promoter-swap techniques, and gene complementation, we identified that the colicin resistance phenotype in S. Typhimurium was partly attributable to the CirA receptor. This finding suggests a complex interplay in the microbial resistance to colicins, highlighting the intricacies of microbial interactions within the gastrointestinal environment.

microbiology↗

bioRxiv2024

Molecular basis of the biogenesis of a protein organelle for ethanolamine utilization

Many pathogenic bacteria use proteinaceous ethanolamine-utilization microcompartments (Eut BMCs) to facilitate the catabolism of ethanolamine, an abundant nutrient in the mammalian gut. The ability to metabolize ethanolamine gives pathogens a competitive edge over commensal microbiota which can drive virulence in the inflamed gut. Despite their critical functions, the molecular mechanisms underlying the synthesis of Eut BMCs in bacterial cells remain elusive. Here, we report a systematic study for dissecting the molecular basis underlying Eut BMC assembly in Salmonella. We determined the functions of individual building proteins in the structure and function of Eut BMCs and demonstrated that EutQ plays an essential role in both cargo encapsulation and Eut BMC formation through specific association with the shell and cargo enzymes. Furthermore, our data reveal that Eut proteins can self-assemble to form cargo and shell aggregates independently in vivo, and that the biogenesis of Eut BMCs follows a unique "Shell-first" pathway. Cargo enzymes exhibit dynamic liquid-like organization within the Eut BMC. These discoveries provide mechanistic insights into the structure and assembly of the Eut BMC, which serves as a paradigm for membrane-less organelles. It opens up new possibilities for therapeutic interventions for infectious diseases.

cell biology↗

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↗

bioRxiv2024

Global diversity and evolution of Salmonella Panama, an understudied serovar causing gastrointestinal and invasive disease worldwide: a genomic epidemiology study

BackgroundNontyphoidal Salmonella (NTS) is a globally important bacterial pathogen, typically associated with foodborne gastrointestinal infection. Some NTS serovars can also colonise normally sterile sites in humans to cause invasive NTS (iNTS) disease. One understudied Salmonella enterica serovar which is responsible for a significant number of cases of iNTS disease is Panama. Despite global dissemination, numerous outbreaks, and a reported association with iNTS disease, S. enterica serovar Panama (S. Panama) has not been investigated in detail. MethodsUsing combined epidemiological and whole genome sequencing data we analysed 836 S. Panama genomes derived from historical collections, national surveillance datasets, and publicly available data. The collection represents all inhabited continents and includes isolates collected between 1931 and 2019. Maximum likelihood and Bayesian phylodynamic approaches were used to determine population structure & evolutionary history, and to infer geo-temporal dissemination. A combination of different bioinformatic approaches utilising short-read and long-read data were used to characterise geographic and clade-specific trends in antimicrobial resistance (AMR), and genetic markers for invasiveness. FindingsWe identified the presence of multiple geographically linked S. Panama clades, and regional trends in antimicrobial resistance profiles. Most isolates were pan-susceptible to antibiotics and belonged to clades circulating in the United States of America, Latin America, and the Caribbean. Multidrug resistant (MDR) isolates in our collection belonged to two phylogenetic clades circulating in Europe and Asia/Oceania, which exhibited the highest invasiveness indices based on the conservation of 196 extra-intestinal predictor genes. InterpretationThis first large-scale phylogenetic analysis of S. Panama revealed important information about population structure, AMR, global ecology, and genetic markers of invasiveness of the identified genomic subtypes. Our findings provide an important baseline for understanding S. Panama infection in the future. The presence of MDR clades with an elevated invasiveness index should be monitored by ongoing surveillance as such clades may pose an increased public health risk. FundingThe project was supported by both a Global Challenges Research Fund (GCRF) data & resources grant BBS/OS/GC/000009D and the BBSRC Core Capability Grant to the Earlham Institute BB/CCG1720/1. JCDH received a Wellcome Trust Investigator award (grant number 222528/Z/21/Z). CVP was supported by the John Lennon Memorial Scholarship from the University of Liverpool and a Fee Bursary Award from the Institute of Integrative Biology at the University of Liverpool. FXW was awarded the Institut Pasteur; Sante publique France; the Fondation Le Roch-Les Mousquetaires; and the French governments Investissement dAvenir programme, Laboratoire dExcellence "Integrative Biology of Emerging Infectious Diseases" (grant number ANR-10-LABX-62-IBEID). MDS was supported by a BBSRC grant (KSB: BB/V009184/1). RJBengtsson was supported by an MRC grant (KSB: MR/R020787/1). RJBennett was supported by a BBSRC DTP (BB/M011186/1). DJI was supported by a National Health and Medical Research Council (NHMRC) Emerging Leadership Fellowship (GNT1195210). BPH was supported by NHMRC Leadership Fellowship (GNT1196103). KSB and EVR are affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Gastrointestinal Infections at University of Liverpool in partnership with the United Kingdom Health Security Agency, in collaboration with University of Warwick. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care or UKHSA. MAC is affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) (NIHR200892) in Genomics and Enabling Data at University of Warwick in partnership with the UK Health Security Agency (UKHSA), in collaboration with Universities of Cambridge and Oxford. MAC is based at UKHSA. The views expressed are those of the author(s) and not necessarily those of the NIHR, the Department of Health and Social Care or the UK Health Security Agency. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. No payment was received by any pharmaceutical company or other agency to write this article. The authors were not precluded from accessing data in the study, and accept responsibility to submit for publication. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSalmonella Panama has consistently been reported as a frequently isolated Salmonella serovar in national surveillance datasets, causing both sporadic cases and larger outbreaks. However, this picture has been masked due to most of the focus being placed on the top two serovars associated with nontyphoidal Salmonella (NTS) and invasive NTS disease: Typhimurium and Enteritidis. Previous works on S. Panama have determined transmission to include human faeces and breast milk, as well as non-human sources such as environmental reservoirs and animals (reptiles and pigs used for food). In contrast to most of the Salmonella serovars causing gastroenteritis, S. Panama has also been associated with a range of systemic infections including septicaemia, meningitis, and osteomyelitis, mostly affecting infants. The patterns of antimicrobial resistance of this pathogen were not well known, with studies reporting a mixture of different antimicrobial resistance profiles. Added value of this studyHere we conducted a large-scale study of 836 globally relevant S. Panama isolates to understand global population structure and disease ecology. The S. Panama genomes used in this study were sourced from a combination of historical collections (including the first ever isolated strain of this serovar dating from 1931), national surveillance datasets, and publicly available data. Some of these isolates had been linked to travel, allowing an inferred location for understanding population structure. Overall, this assembled S. Panama collection spanned a range of 88 years and covered 45 countries and regions of all inhabited continents. We applied bacterial phylodynamic approaches to determine population structure, evolutionary history, and clade-specific trends of invasiveness and antimicrobial resistance correlated with geo-temporal parameters. Implications of all the available evidenceThis study revealed the population structure and global ecology of S. Panama, indicating that multidrug resistant S. Panama circulates in European and Asian regions, and presents an increased number of genetic markers for extra-intestinal invasiveness. The transmission and expansion of antimicrobial-resistant S. Panama strains presented in this study highlight the significance of supporting control and monitoring efforts from an international perspective.

genomics↗

bioRxiv2023

Temporal profiling of Salmonella transcriptional dynamics during macrophage infection using a comprehensive reporter library

The transcriptome of Salmonella enterica serovar Typhimurium (S. Tm) dynamically responds to the rapid environmental shifts intrinsic to S. Tm lifestyle, exemplified by entry into the Salmonella-containing vacuole (SCV) within macrophages. Intracellular S. Tm must respond to the acidity of the SCV, accumulation of reactive oxygen/nitrogen species, and fluctuations in nutrient availability. Despite thorough RNA-seq-based investigations, the precise transcriptional timing of the expression of many secretion systems, metabolic pathways, and virulence effectors involved in infection has yet to be elucidated. Here, we construct a comprehensive library of GFP-reporter strains representing [~]3,000 computationally identified S. Tm promoter regions to study the dynamics of transcriptional regulation. We quantified promoter activity during in vitro growth in defined and complex media and throughout the timeline of intracellular infection of RAW 246.7 macrophages. Using bulk measurements and single-cell imaging, we uncovered condition-specific transcriptional regulation and population-level heterogeneity in the activity of virulence-related promoters, including SPI2 genes such as ssaR and ssaG. We discovered previously unidentified transcriptional activity from 234 genes, including ones with novel activity during infection that are associated with pathogenecity islands and are involved in metabolism and metal homeostasis. Our library and data sets should provide powerful resources for systems-level interrogation of Salmonella transcriptional dynamics.

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

A widespread family of WYL-domain transcriptional regulators co-localises with diverse phage defence systems and islands

AO_SCPLOWBSTRACTC_SCPLOWBacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into "defence islands", providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterise a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 [A], the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localised with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.

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↗

bioRxiv2021

Niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the cytosol, and the bacterial genes required for cytosolic colonization, remain unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes/sRNAs with cytosol-specific or vacuole-specific expression signatures. Using these genes/sRNAs as environmental biosensors, we defined that Salmonella is exposed to iron and manganese deprivation and oxidative stress in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS and sitA-mntH. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, had a cytosolic-specific expression profile. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. Therefore, this archetypical vacuole-adapted pathogen requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol. Author SummaryIntracellular pathogens reside either within a membrane-bound vacuole or are free-living in the cytosol and their virulence programs are tailored towards survival within a particular intracellular compartment. Some bacterial pathogens (such as Salmonella enterica) can successfully colonize both intracellular niches, but how they do so is unclear. Here we have exploited the parallel intracellular lifestyles of S. enterica in epithelial cells to identify the niche-specific bacterial expression profiles and environmental cues encountered by S. enterica. We have also discovered bacterial genes that are required for colonization of the cytosol, but not the vacuole. Our results advance our understanding of pathogen-adaptation to alternative replication niches and highlight an emerging concept in the field of bacteria-host cell interactions.

microbiology↗

bioRxiv2020

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

microbiology↗