Biology subjects

Dougan, G.

Publications and source records attributed to Dougan, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond connectivity: dispersal mortality and Allee effects prevent bobcat recolonisation despite habitat availability

Wildlife crossing infrastructure is promoted to restore connectivity for fragmented populations, but its effectiveness at enabling natural recolonisation remains untested. We tested this using a spatially explicit agent-based model parameterised with GPS telemetry data from bobcats (Lynx rufus) in New Jersey, USA. By integrating movement behaviour, stochastic demography, habitat suitability, and traffic-dependent mortality risk, we simulated 50-year recolonisation dynamics across a highly urbanised landscape. Despite extensive unoccupied suitable habitat, natural recolonisation completely failed across all scenarios, with vehicle-induced mortality during dispersal acting as the primary limiting factor and turning the matrix into a demographic sink. Even an idealised mitigation scenario in which mortality at high-mortality crossings was reduced to zero failed to produce a self-sustaining population. Although dispersal increased, individuals at the recolonisation front remained too sparse to overcome the mate-finding Allee effect. Sensitivity analysis confirmed that the recolonisation-failure result is robust to {+/-}50% variation in per-crossing mortality and {+/-}25% variation in disperser survival. Restoring structural connectivity is not, in itself, a sufficient intervention for recovering low-density carnivore populations facing a high-mortality matrix. Instead disperser survival and local density at the recolonisation front are the rate-limiting determinants. In such systems translocation rather than crossing-structure investment is more likely to result in recolonisation success.

ecology↗

bioRxiv2023

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were found across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

microbiology↗

bioRxiv2022

Genomic analysis unveils the role of genome degradation events and gene flux in the emergence and persistence of S. Paratyphi A lineages

Paratyphoid fever caused by S. Paratyphi A is endemic in parts of Asia and Sub-Saharan Africa. The proportion of enteric fever cases caused by S. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory of S. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study "Surveillance of Enteric Fever in India (SEFI)." In the study period (2017-2020), S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis documented that the genome degradation events and gene acquisitions or losses play a major role in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 pseudogene-forming mutations possibly associated with the emergence of lineages were identified. Pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We also identified that the six characteristic missense mutations associated with the lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A confer only a low structural impact and would therefore have minimal impact on vaccine effectiveness. Since S. Paratyphi A is human restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and intervention by means of genomic surveillance would be continually needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today.

genomics↗

bioRxiv2022

Obesity associated with attenuated tissue immune cell responses in COVID-19

Obesity is common and associated with more severe COVID-19, proposed to be in part related to an adipokine-driven pro-inflammatory state. Here we analysed single cell transcriptomes from bronchiolar lavage in three adult cohorts, comparing obese (Ob, body mass index (BMI) >30m2) and non-obese (N-Ob, BMI <30m2). Surprisingly, we found that Ob subjects had attenuated lung immune/inflammatory responses in SARS-CoV-2 infection, with decreased expression of interferon (IFN), IFN{gamma} and tumour necrosis factor (TNF) alpha response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Analysis of peripheral blood immune cells in an independent adult cohort showed a similar, but less marked, reduction in type I IFN and IFN{gamma} response genes, as well as decreased serum IFN, in Ob patients with SARS-CoV-2. Nasal immune cells from Ob children with COVID-19 also showed reduced enrichment of IFN and IFN{gamma} response genes. Altogether, these findings show blunted tissue immune responses in Ob COVID-19 patients, with clinical implications.

immunology↗

bioRxiv2021

Dissemination and pathogenesis of toxigenic Clostridium perfringens strains linked to neonatal intensive care units and Necrotising Enterocolitis

BackgroundClostridium perfringens is an anaerobic toxin-producing bacterium that has long been associated with intestinal diseases, particularly in neonatal humans and animals. More recently, infant gut microbiome studies have suggested an important link between C. perfringens and the devastating preterm-associated disease Necrotising Enterocolitis (NEC), but in-depth studies on this pathogen (genomics and mechanistic) are lacking. Methods/MaterialsWe isolated and whole-genome sequenced 274 infant-associated C. perfringens isolates from 5 hospitals across the UK between 2011-2016 (including longitudinal samples from 31 individuals). We performed in-depth genomic analyses, phenotypically characterised pathogenic traits of 10 strains (including 4 C. perfringens from NEC patients) and established a novel oral-challenge C57BL/6 mouse infection model for microbe-host studies. ResultsPore-forming toxin encoding genes pfoA and cpb2 were enriched within hypervirulent lineages that exclusively consisted of C. perfringens-associated NEC (CPA-NEC) strains, in addition to overabundance of colonisation factors. Importantly, we identified a circulating C. perfringens variant, eventually linked to a fatal CPA-NEC case. The variant was detected consistently within 6 individuals in two sister hospitals across a 40-day window, demonstrating for the first time the intra- and inter-hospital dissemination of C. perfringens. CPA-NEC isolates were determined phenotypically to be more virulent (linked with overabundance of gene pfoA) than isolates obtained from non-NEC preterm babies. In addition, two pfoA-positive CPA-NEC C. perfringens strains were confirmed to induce clinical inflammatory tissue lesions in vivo. ConclusionsHypervirulent lineages are linked to CPA-NEC, potentially due to the production of pore-forming toxins, coupled with higher metabolic, transmission, and pathogenic capacities. These studies indicate C. perfringens is an important bacterial pathogen in preterm infants and highlights the requirement for further investigation into development of intervention and therapeutic strategies.

microbiology↗

bioRxiv2021

FXR inhibition reduces ACE2 expression, SARS-CoV-2 infection and may improve COVID-19 outcome

Prevention of SARS-CoV-2 entry in cells through the modulation of viral host receptors, such as ACE2, could represent a new therapeutic approach complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive. Here, we identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19-affected tissues, including the gastrointestinal and respiratory systems. We demonstrate that FXR antagonists, including the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulate ACE2 levels, and reduce susceptibility to SARS-CoV-2 infection in lung, cholangiocyte and gut organoids. We then show that therapeutic levels of UDCA downregulate ACE2 in human organs perfused ex situ and reduce SARS-CoV-2 infection ex vivo. Finally, we perform a retrospective study using registry data and identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death. In conclusion, we identify a novel function of FXR in controlling ACE2 expression and provide evidence that this approach could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials.

microbiology↗

bioRxiv2021

Inhibitory concentrations of ciprofloxacin induce an adaptive response promoting the intracellular survival of Salmonella Typhimurium

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fuelled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of S. Typhimurium to differing concentrations of ciprofloxacin, dependent on their minimum inhibitory concentrations (MIC), and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium are able to recover from inhibitory concentrations of ciprofloxacin, and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium have increased capacity for intracellular replication in comparison to untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. ImportanceAntimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin, and for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.

microbiology↗

bioRxiv2021

Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya

Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control. In a 3 year case-control study, we investigated typhoid among children aged <16 years (4,670 febrile cases and 8,549 age matched controls) living in an informal settlement, Nairobi, Kenya. 148 S. Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1%. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype58), with the majority of each (76% and 88%) being multidrug-resistant strains in 3 sublineages of H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled. The high rate of multidrug-resistant H58 S. Typhi, and the close phylogenetic relationships between carriers and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting.

physiology↗

bioRxiv2021

Novel gut pathobionts confound results in a widely used mouse model of human inflammatory disease

The mammalian gut microbiota consists of hundreds of anaerobic bacterial species that shape intestinal homeostasis and influence host immune responses. Although the causal roles of specific human gut bacterial species in health and disease are emerging, the role of indigenous gut bacteria in driving immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 laboratory mice designed to identify and validate the causes of disease variability in the widely used dextran sulphate sodium (DSS) mouse model of inflammatory bowel disease. Using microbiome analysis, coupled with machine learning and targeted anaerobic culturing, we identified and isolated the novel gut pathobiont species Duncaniella muricolitica and Alistipes okayasuensis and fulfilled Kochs postulates in mice to show that each pathobiont exerts dominant effects in the DSS model leading to variable treatment responses. We show these pathobiont species are common, but not ubiquitous, in mouse facilities around the world, raising experimental design opportunities for improved mouse models of human intestinal diseases.

microbiology↗

bioRxiv2021

High-content imaging to phenotype antimicrobial effects on individual bacteria at scale

High-content imaging (HCI) is technique for screening multiple cells in high resolution to detect subtle morphological and phenotypic variation. The method has been commonly deployed on model eukaryotic cellular systems, often for screening new drugs and targets. HCI is not commonly utilised for studying bacterial populations but may be powerful tool in understanding and combatting antimicrobial resistance. Consequently, we developed a high-throughput method for phenotyping bacteria under antimicrobial exposure at the scale of individual bacterial cells. Imaging conditions were optimised on an Opera Phenix confocal microscope (Perkin-Elmer) and novel analysis pipelines were established for both Gram-negative bacilli and Gram-positive cocci. The potential of this approach was illustrated using isolates of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. HCI enabled the detection and assessment of subtle morphological characteristics, undetectable through conventional phenotypical methods, that could reproducibly distinguish between bacteria exposed to different classes of antimicrobials with distinct modes of action (MOA). In addition, distinctive responses were observed between susceptible and resistant isolates. By phenotyping single bacterial cells, we observed intra-population differences, which may be critical in identifying persistence or emerging resistance during antimicrobial treatment. The work presented here outlines a comprehensive method for investigating morphological changes at scale in bacterial populations under specific perturbation. ImportanceHigh-content imaging (HCI) is a microscopy technique that permits the screening of multiple cells simultaneously in high resolution to detect subtle morphological and phenotypic variation. The power of this methodology is that is can generate large datasets comprised of multiple parameters taken from individual cells subjected to range of different conditions. We aimed to develop novel methods for using HCI to study bacterial cells exposed to a range of different antibiotic classes. Using an Opera Phenix confocal microscope (Perkin-Elmer) and novel analysis pipelines we created a method to study the morphological characteristics of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus when exposed to antibacterial drugs with differing modes of action. By imaging individual bacterial cells at high resolution and scale, we observed intra-population differences associated with different antibiotics. The outlined methods are highly relevant for how we begin to better understand and combat antimicrobial resistance.

microbiology↗

bioRxiv2020

Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time

BackgroundEnterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 fatalities per year, with the highest rates of burden, ca 75 million cases per year, amongst children under five years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. ResultsWe used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. The plasmids carrying ETEC virulence genes were compared to other available long-read sequenced ETEC strains using blastn. The ETEC reference strains harbour between two and five plasmids, including virulence, antibiotic resistance and phage-plasmids. The virulence plasmids carrying the colonisation factors are highly conserved as shown by comparison with plasmids with other ETEC strains and confirm that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. ConclusionWe confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

genomics↗

bioRxiv2020

A global resource for genomic predictions of antimicrobial resistance and surveillance of Salmonella Typhi at Pathogenwatch

BackgroundMicrobial whole-genome sequencing (WGS) is now increasingly used to inform public health investigations of infectious disease. This approach has transformed our understanding of the global population structure of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever. WGS has been particularly informative for understanding the global spread of multi-drug resistant (MDR) typhoid. As WGS capacity becomes more decentralised, there is a growing opportunity for collaboration and sharing of surveillance data within and between countries to inform disease control policies. This requires freely available, community driven tools that reduce the barriers to access genomic data for public health surveillance and that deliver genomic data on a global scale. MethodsHere we present the Pathogenwatch (https://pathogen.watch/styphi) scheme for S. Typhi, a web application enabling the rapid identification of genomic markers of antimicrobial resistance (AMR) and contextualization with public genomic data to identify high-risk clones at a population level. Data are delivered in single genome reports or in collections of genomes combined with geographic and other data using trees, maps and tables. ResultsWe show that the clustering of S. Typhi genomes in Pathogenwatch is comparable to established bioinformatics methods, and that genomic predictions of AMR are largely concordant with phenotypic drug susceptibility data. We demonstrate the public health utility of Pathogenwatch with examples selected from over 4,300 public genomes available in the application. ConclusionsPathogenwatch democratises genomic epidemiology of S. Typhi by providing an intuitive entry point for the analysis of WGS and linked epidemiological data, enabling international public health monitoring of the emergence and spread of high risk clones.

genomics↗

bioRxiv2020

The genetic signatures of Salmonella Typhi carriage in the human gallbladder

Despite recent gains in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains an enigma. Aiming to understand if S. Typhi in the gallbladder are vital for transmission and/or adapted for long-term colonisation we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneous sequences from organisms isolated from the blood of acute patients.S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes but reflective of the general population. However, gallbladder isolates had a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p=2.8x10-9), respectively. This variation was associated with a higher prevalence of nonsense mutations in the gallbladder isolates in the predominant genotype. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Particularly, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to a 134Kb deletion. Long-term gallbladder carriage of S. Typhi results in atypically long branch lengths that can distinguish between carriage and acute infection. Our data strongly suggests typhoid carriers are unlikely to play a principal role in disease transmission in endemic settings, but that the hostile environment of the human gallbladder may generate new antigenic variants through immune selection.

microbiology↗

bioRxiv2020

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

microbiology↗

bioRxiv2020

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage {Phi}HKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here we report the comprehensive molecular characterization of {Phi}HKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for optimal growth in human blood, confers resistance to neutrophil killing, and degrades neutrophil extracellular traps (NETs). Investigating single, double and triple isogenic knockout mutants of the {Phi}HKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the etiology and pathogenesis of scarlet fever-causing GAS mediated by phage {Phi}HKU.vir exotoxins.

microbiology↗

bioRxiv2020

A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities

The COVID-19 pandemic is expanding at an unprecedented rate. As a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. Academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. Here, we describe the process of developing a SARS-Cov2 diagnostic workflow in a conventional academic Containment Level 2 (CL2) laboratory. Our outline includes simple SARS-Cov2 deactivation upon contact, the methods for a quantitative real-time reverse transcriptase PCR (qRT-PCR) detecting SARS-Cov2, a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. This was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. Within 14 days, we created a validated COVID-19 diagnostics service for healthcare workers in our local hospital. The described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame.

molecular biology↗

bioRxiv2019

Discovery of a Pseudomonas aeruginosa Type VI secretion system toxin targeting bacterial protein synthesis using a global genomics approach

The Type VI secretion system (T6SS) is a bacterial weapon which delivers toxic effectors to kill competitors or subvert some of their key functions. Here we use transposon directed insertion-site sequencing (TraDIS) to identify T6SS toxins associated with the H1-T6SS, one of the three T6SS machines found in Pseudomonas aeruginosa. This approach identified several putative toxin-immunity pairs, including Tse8-Tsi8. Full characterization of this protein pair demonstrated that Tse8 is delivered by the VgrG1a spike complex into prey cells where it targets the transamidosome, a multiprotein complex involved in protein synthesis in bacteria lacking either one or both of the asparagine or glutamine tRNA synthases. Our data suggests that Tse8 combines as a non-cognate component of the transamidosome complex, reducing fitness by limiting the ability of the cell to synthesize proteins. This is the first demonstration of a T6SS toxin affecting protein synthesis, expanding the range of cellular components targeted by this bacterial weapon. The success of the current study validates the use of our TraDIS approach as a tool to drastically expand the repertoire of T6SS toxins in any T6SS-encoding bacterium.

microbiology↗

bioRxiv2019

Rora regulates activated T helper cells during inflammation

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

immunology↗