Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells.

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

Germicidal efficacy of continuous and pulsed ultraviolet-C radiation on pathogen models and SARS-CoV-2.

Ultraviolet radiation's germicidal efficacy depends on several parameters, including wavelength, radiant exposure, microbial physiology, biological matrices, and surfaces. In this work, several ultraviolet radiation sources (a low-pressure mercury lamp, a KrCl excimer, and four UV LEDs) emitting continuous or pulsed irradiation were compared. The greatest log reductions in E. coli cells and B. subtilis endospores were 4.1 ± 0.2 (18 mJ cm-2) and 4.5 ± 0.1 (42 mJ cm-2) with continuous 222 nm, respectively. The highest MS2 log reduction observed was 2.7 ± 0.1 (277 nm at 3809 mJ cm-2). Log reductions of SARS-CoV-2 with continuous 222 nm and 277 nm were ≥ 3.4 ± 0.7, with 13.3 mJ cm-2 and 60 mJ cm-2, respectively. There was no statistical difference between continuous and pulsed irradiation (0.83-16.7% [222 nm and 277 nm] or 0.83-20% [280 nm] duty rates) on E. coli inactivation. Pulsed 260 nm radiation (0.5% duty rate) at 260 nm yielded significantly greater log reduction for both bacteria than continuous 260 nm radiation. There was no statistical difference in SARS-CoV-2 inactivation between continuous and pulsed 222 nm UV-C radiation and pulsed 277 nm radiation demonstrated greater germicidal efficacy than continuous 277 nm radiation. Greater radiant exposure for all radiation sources was required to inactivate MS2 bacteriophage. Findings demonstrate that pulsed irradiation could be more useful than continuous UV radiation in human-occupied spaces, but threshold limit values should be respected. Pathogen-specific sensitivities, experimental setup, and quantification methods for determining germicidal efficacy remain important factors when optimizing ultraviolet radiation for surface decontamination or other applications.

Dysregulation of B lymphocyte development in the SKG mouse model of rheumatoid arthritis.

Rheumatoid arthritis is a chronic and systemic inflammatory disease that affects approximately 1% of the world's population and is characterised by joint inflammation, the destruction of articular cartilage and bone, and many potentially life-threatening extraarticular manifestations. B lymphocytes play a central role in the pathology of rheumatoid arthritis as the precursors of autoantibody secreting plasma cells, as highly potent antigen-presenting cells, and as a source of various inflammatory cytokines, however, the effects of rheumatoid arthritis on B lymphocyte development remain poorly understood. Here, we analyse B lymphocyte development in murine models of rheumatoid arthritis, quantifying all the subsets of B cell precursors in the bone marrow and splenic B cells using flow cytometry. We demonstrate a severe reduction in pre-B cells and immature B cells in the bone marrow of mice with active disease, despite no major effects on the mature naïve B cell numbers. The loss of B cell precursors in the bone marrow of the affected mice was associated with a highly significant reduction in the proportion of Ki67+ cells, indicating impaired cell proliferation, while the viability of the B cell precursors was not significantly affected. We also observed some mobilisation of the B cell precursor cells into the mouse spleen, demonstrated with flow cytometry and pre-B colony forming units assays. In summary, the current work demonstrates a severe dysregulation in B lymphocyte development in murine rheumatoid arthritis, with possible implications for B cell repertoire formation, tolerance induction, and disease mechanisms.

Salmonella enterica subsp. enterica virulence potential can be linked to higher survival within a dynamic in vitro human gastrointestinal model.

Salmonella enterica subsp. enterica is one of the leading causes of human foodborne infections and several outbreaks are now associated with the consumption of fresh fruit and vegetables. This study aims at evaluating whether Salmonella virulence can be linked to an enhanced ability to survive successive digestive environments. Thirteen S. enterica strains were selected according to high and low virulence phenotypes. Lettuce inoculated separately with each S. enterica strain was used as food matrix in the TNO gastrointestinal model (TIM-1) of the human upper gastrointestinal tract. During the passage in the stomach, counts determined using PMA-qPCR were 2-5 logs higher than the cultivable counts for all strains indicating the presence of viable but non-cultivable cells. Bacterial growth was observed in the duodenum compartment after 180 min for all but one strain and growth continued into the ileal compartment. After passage through the simulated gastrointestinal tract, both virulent and avirulent S. enterica strains survived but high virulence strains had a significantly (p = 0.004) better average survival rate (1003 %-3753 %) than low virulence strains (from 25 % to 3730%). The survival rates of S. enterica strains could be linked to the presence of genes associated with acid and bile resistance and their predicted products. The presence of single nucleotide polymorphisms may also impact the function of virulence associated genes and play a role in the resulting phenotype. These data provide an understanding of the relationship between measured virulence potential and survival of S. enterica during dynamic simulated gastrointestinal transit.

A Loss-of-Function Mutation in the Integrin Alpha L (Itgal) Gene Contributes to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Collaborative Cross Strain CC042.

Salmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8+ T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4+, CD8+) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 (Stsl6 and Stsl7) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L (Itgal) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene.

USP18 lack in microglia causes destructive interferonopathy of the mouse brain.

Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called "microgliopathies". However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. Here, we identified the ubiquitin-specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence. We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon-induced genes, thereby terminating IFN signaling. The Usp18-mediated control was independent from its catalytic activity but instead required the interaction with Ifnar2. Additionally, the absence of Ifnar1 restored microglial activation, indicating a tonic IFN signal which needs to be negatively controlled by Usp18 under non-diseased conditions. These results identify Usp18 as a critical negative regulator of microglia activation and demonstrate a protective role of Usp18 for microglia function by regulating the Ifnar pathway. The findings establish Usp18 as a new molecule preventing destructive microgliopathy.

Identification of new loci involved in the host susceptibility to Salmonella Typhimurium in collaborative cross mice.

BACKGROUND: Salmonella is a Gram-negative bacterium causing a wide range of clinical syndromes ranging from typhoid fever to diarrheic disease. Non-typhoidal Salmonella (NTS) serovars infect humans and animals, causing important health burden in the world. Susceptibility to salmonellosis varies between individuals under the control of host genes, as demonstrated by the identification of over 20 genetic loci in various mouse crosses. We have investigated the host response to S. Typhimurium infection in 35 Collaborative Cross (CC) strains, a genetic population which involves wild-derived strains that had not been previously assessed. RESULTS: One hundred and forty-eight mice from 35 CC strains were challenged intravenously with 1000 colony-forming units (CFUs) of S. Typhimurium. Bacterial load was measured in spleen and liver at day 4 post-infection. CC strains differed significantly (P < 0.0001) in spleen and liver bacterial loads, while sex and age had no effect. Two significant quantitative trait loci (QTLs) on chromosomes 8 and 10 and one suggestive QTL on chromosome 1 were found for spleen bacterial load, while two suggestive QTLs on chromosomes 6 and 17 were found for liver bacterial load. These QTLs are caused by distinct allelic patterns, principally involving alleles originating from the wild-derived founders. Using sequence variations between the eight CC founder strains combined with database mining for expression in target organs and known immune phenotypes, we were able to refine the QTLs intervals and establish a list of the most promising candidate genes. Furthermore, we identified one strain, CC042/GeniUnc (CC042), as highly susceptible to S. Typhimurium infection. CONCLUSIONS: By exploring a broader genetic variation, the Collaborative Cross population has revealed novel loci of resistance to Salmonella Typhimurium. It also led to the identification of CC042 as an extremely susceptible strain.

Enterobacteria and host resistance to infection.

Enterobacteriaceae are a large family of Gram-negative, non-spore-forming bacteria. Although many species exist as part of the natural flora of animals including humans, some members are associated with both intestinal and extraintestinal diseases. In this review, we focus on members of this family that have important roles in human disease: Salmonella, Escherichia, Shigella, and Yersinia, providing a brief overview of the disease caused by these bacteria, highlighting the contribution of animal models to our understanding of their pathogenesis and of host genetic determinants involved in susceptibility or resistance to infection.

Complex genetics architecture contributes to Salmonella resistance in AcB60 mice.

Human infection with Salmonella is of global public health concern. In low- and middle-income countries, Salmonella infection is a major source of disease in terms of both mortality and morbidity, while in high-income nations, the pathogen is an ongoing threat to food security. The outcome of infection with Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) in mouse models is dependent upon a coordinated and complex immune response. A panel of recombinant congenic strains (RCS) derived from the reciprocal double backcross of A/J and C57BL/6J mice has been screened for their susceptibility to Salmonella infection, and the RCS AcB60 was identified to be the most resistant strain to Salmonella infection, more resistant than the parental strain A/J. These mice are known to carry resistant alleles at three well-defined Salmonella susceptibility loci, Slc11a1 Ity (solute carrier family 11 member 1; Immunity to Typhimurium locus), Pklr Ity4 (pyruvate kinase liver and red blood cell; Ity4 locus), and Ity5. In the current study, we used interval mapping to validate a locus on Chr 15, named Ity8, linked to Salmonella resistance in AcB60 mice. Global gene expression analysis during infection identified AcB60-specific expression of genes involved in Ccr7 signaling, including downstream effector Mapk11 (mitogen-activated protein kinase 11), located within the Ity8 interval, and representing a potential positional candidate gene. An additional region on Chr 18 of C57BL/6J descent was shown to be associated with increase resistance in AcB60. These observations provide an opportunity to achieve new insight into the complex genetics of resistance to Salmonella infection in the context of mouse models of human infection with Salmonella Typhimurium.

Altered IFN-γ-mediated immunity and transcriptional expression patterns in N-Ethyl-N-nitrosourea-induced STAT4 mutants confer susceptibility to acute typhoid-like disease.

Salmonella enterica is a ubiquitous Gram-negative intracellular bacterium that continues to pose a global challenge to human health. The etiology of Salmonella pathogenesis is complex and controlled by pathogen, environmental, and host genetic factors. In fact, patients immunodeficient in genes in the IL-12, IL-23/IFN-γ pathway are predisposed to invasive nontyphoidal Salmonella infection. Using a forward genomics approach by N-ethyl-N-nitrosourea (ENU) germline mutagenesis in mice, we identified the Ity14 (Immunity to Typhimurium locus 14) pedigree exhibiting increased susceptibility following in vivo Salmonella challenge. A DNA-binding domain mutation (p.G418_E445) in Stat4 (Signal Transducer and Activator of Transcription Factor 4) was the causative mutation. STAT4 signals downstream of IL-12 to mediate transcriptional regulation of inflammatory immune responses. In mutant Ity14 mice, the increased splenic and hepatic bacterial load resulted from an intrinsic defect in innate cell function, IFN-γ-mediated immunity, and disorganized granuloma formation. We further show that NK and NKT cells play an important role in mediating control of Salmonella in Stat4(Ity14/Ity14) mice. Stat4(Ity14/Ity14) mice had increased expression of genes involved in cell-cell interactions and communication, as well as increased CD11b expression on a subset of splenic myeloid dendritic cells, resulting in compromised recruitment of inflammatory cells to the spleen during Salmonella infection. Stat4(Ity14/Ity14) presented upregulated compensatory mechanisms, although inefficient and ultimately Stat4(Ity14/Ity14) mice develop fatal bacteremia. The following study further elucidates the pathophysiological impact of STAT4 during Salmonella infection.

Functional validation of the genetic architecture of Salmonella Enteritidis persistence in 129S6 mice.

The Gram-negative bacteria, Salmonella, cause a broad spectrum of clinical diseases in humans, ranging from asymptomatic carriage to life-threatening sepsis. We have designed an experimental model to study the contribution of genetic factors to the persistence of Salmonella Enteritidis during the late phase of infection in 129S6/SvEvTac and C57BL/6J mice. C57BL/6J mice cleared the bacteria from their reticuloendothelial system within a period of 42 days, whereas the 129S6 mice still presented a high bacterial load. Using this model, we have identified ten Salmonella Enteritidis susceptibility loci (Ses1, Ses1.1, and Ses3-Ses10) associated with bacterial persistence in target organs of 129S6/SvEvTac mice using a two-locus epistasis QTL linkage mapping approach. Significant statistical interactions were detected between Ses1 on chromosome 1 and Ses5 on chromosome 7 and between Ses1 and Ses4 on chromosome X. In this study, we functionally validated the genetic architecture of Salmonella persistence in 129S6 mice using single- (129S6.B6-Ses1.2 that combines Ses1 and Ses1.1 loci, 129S6.B6-Ses4, and 129S6.B6-Ses5) and double-congenic mice (129S6.B6-Ses1.2/Ses4 and 129S6.B6-Ses1.2/Ses5). These experiments demonstrate functional interactions between Ses1.2 and Ses4 or Ses5 that improve Salmonella Enteritidis clearance, validating the critical role played by gene-gene interactions in the contribution to bacterial clearance heritability. Improved bacterial clearance in double-congenic mice could be explained by the impact of Ses4 and Ses5 in combination with Ses1.2 on TH polarization since a TH2 bias (decreased Ifng and increased Il4 mRNA levels and reduced IgG2a immunoglobulins in the serum) was observed in 129S6.B6-Ses1.2/Ses5 mice and a TH17 (high Il17 expression) bias in 129S6.B6-Ses1.2/Ses4.

Elevated mitochondrial reactive oxygen species generation affects the immune response via hypoxia-inducible factor-1alpha in long-lived Mclk1+/- mouse mutants.

Mitochondrial reactive oxygen species (ROS) are believed to stabilize hypoxia-inducible factor (HIF)-1alpha, a transcriptional regulator of the immune response. Mclk1 encodes a mitochondrial protein that is necessary for ubiquinone biosynthesis. Heterozygote Mclk1(+/-) mutant mice are long-lived despite increased mitochondrial ROS and decreased energy metabolism. In this study, Mclk1(+/-) mutant mice in the C57BL/6J background displayed increased basal and induced expression of HIF-1alpha in liver and macrophages in association with elevated expression of inflammatory cytokines, in particular TNF-alpha. Mutant macrophages showed increased classical and decreased alternative activation, and mutant mice were hypersensitive to LPS. Consistent with these observations in vivo, knock-down of Mclk1 in murine RAW264.7 macrophage-like cells induced increased mitochondrial ROS as well as elevated expression of HIF-1alpha and secretion of TNF-alpha. We used an antioxidant peptide targeted to mitochondria to show that altered ROS metabolism is necessary for the enhanced expression of HIF-1alpha, which, in turn, is necessary for increased TNF-alpha secretion. These findings provide in vivo evidence for the action of mitochondrial ROS on HIF-1alpha activity and demonstrate that changes in mitochondrial function within physiologically tolerable limits modulate the immune response. Our results further suggest that altered immune function through a limited increase in HIF-1alpha expression can positively impact animal longevity.

N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αß signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium.

To deepen our knowledge of the natural host response to pathogens, our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3 Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (ubiquitin-specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-gamma production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-gamma production during infection. The reduced IFN-gamma levels, along with the increased inflammation, rationalize the S. Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death.

A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloid leukemia-like syndrome in BXH-2 mice.

BXH-2 mice develop a fatal myeloid leukemia by a two-step mutagenic process. First, a BXH-2-specific recessive mutation causes a myeloproliferative syndrome. Second, retroviral insertions alter oncogenes or tumor suppressors, resulting in clonal expansion of leukemic cells. We have identified a recessive locus on chromosome 8 (Myls) that is responsible for myeloproliferation in BXH-2. This Myls interval has been narrowed down to 2 Mb and found to contain several positional candidates, including the interferon consensus sequence-binding protein 1 gene (Icsbp, also known as interferon regulatory factor 8 [IRF8]). We show that BXH-2 mice carry a mutation (915 C to T) resulting in an arginine-to-cysteine substitution at position 294 within the predicted IRF association domain of the protein. Although expression of Icsbp1 mRNA transcripts is normal in BXH-2 splenocytes, these cells are unable to produce interleukin 12 and interferon-gamma in response to activating stimuli, confirming that R294C behaves as a loss-of-function mutation. Myeloproliferation in BXH-2 mice is concomitant to increased susceptibility to Mycobacterium bovis (BCG) despite the presence of resistance alleles at the Nramp1 locus. These results suggest a two-step model for chronic myeloid leukemia in BXH-2, in which inactivation of Icsbp1 predisposes to myeloproliferation and immunodeficiency. This event is required for retroviral replication, and subsequent insertional mutagenesis that causes leukemia in BXH-2 mice.

The C3H/HeJ inbred mouse is a model of vesico-ureteric reflux with a susceptibility locus on chromosome 12.

Vesico-ureteric reflux is the most common congenital anomaly of the urinary tract, characterized by a defective uretero-vesical junction with retrograde urine flow from the bladder toward the kidneys. Because there is strong evidence for a genetic basis for some cases of vesico-ureteric reflux, we screened 11 inbred mouse strains for reflux and kidney size and identified one strain, C3H/HeJ, that has a 100 percent incidence of vesico-ureteric reflux with otherwise normal kidneys at birth. These mice are predisposed to reflux as a result of a defective uretero-vesical junction characterized by a short intravesical ureter. This defect results from a delay in urinary tract development initially manifested by a ureteric bud arising from a more caudal location along the mesonephric duct. In contrast, C57BL/6J mice (resistant to reflux at birth) have long intravesical ureters, normally positioned ureteric buds, and no delay in urinary tract development. Genome-wide and additional fine mapping of backcross mice, derived from C3H/HeJ and C57BL/6J crosses, identified a significant reflux susceptibility locus, Vurm1, on chromosome 12 (peak logarithm of the odds=7.39). The C3H/HeJ mouse is a model of vesico-ureteric reflux without renal malformation, and further characterization of this model will allow for the identification of a pathway important for urinary tract development, a finding that will serve as a model for the human disorder.

Combining Whole-Genome Sequencing and Multimodel Phenotyping To Identify Genetic Predictors of Salmonella Virulence.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.

The Salmonella enterica Plasmidome as a Reservoir of Antibiotic Resistance.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infection.

Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defences through strategically targeting host proteins implicated in restricting infection. Therefore, to gain insight into the host-pathogen interactions governing Salmonella infection, we performed an in vivo genome-wide mutagenesis screen to uncover key host defence proteins. This revealed an uncharacterized role of CYRI (FAM49B) in conferring host resistance to Salmonella infection. We show that CYRI binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerization. However, unlike CYFIP proteins, CYRI negatively regulates RAC1 signalling, thereby attenuating processes such as macropinocytosis, phagocytosis and cell migration. This enables CYRI to counteract Salmonella at various stages of infection, including bacterial entry into non-phagocytic and phagocytic cells as well as phagocyte-mediated bacterial dissemination. Intriguingly, to dampen its effects, the bacterial effector SopE, a RAC1 activator, selectively targets CYRI following infection. Together, this outlines an intricate host-pathogen signalling interplay that is crucial for determining bacterial fate. Notably, our study also outlines a role for CYRI in restricting infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes. This provides evidence implicating CYRI cellular functions in host defence beyond Salmonella infection.

Molecular genetic analysis of two loci (Ity2 and Ity3) involved in the host response to infection with Salmonella typhimurium using congenic mice and expression profiling.

Numerous genes have been identified to date that contribute to the host response to systemic Salmonella Typhimurium infection in mice. We have previously identified two loci, Ity2 and Ity3, that control survival to Salmonella infection in the wild-derived inbred MOLF/Ei mouse using a (C57BL/6J x MOLF/Ei)F(2)cross. We validated the existence of these two loci by creating congenic mice carrying each quantitative trait locus (QTL) in isolation. Subcongenic mice generated for each locus allowed us to define the critical intervals underlying Ity2 and Ity3. Furthermore, expression profiling was carried out with the aim of identifying differentially expressed genes within the critical intervals as potential candidate genes. Genomewide expression arrays were used to interrogate expression differences in the Ity2 congenics, leading to the identification of a new candidate gene (Havcr2, hepatitis A virus cellular receptor 2). Interval-specific oligonucleotide arrays were created for Ity3, identifying one potential candidate gene (Chi3l1, chitinase 3-like 1) to be pursued further. The combination of the use of congenics in QTL confirmation and fine mapping and in the identification of candidate genes by expression profiling has been successful and represents a step toward quantitative gene(s) identification.

TLR4 gene dosage contributes to endotoxin-induced acute respiratory inflammation.

Toll-like receptor (TLR)4 is critical for endotoxin recognition and cellular responses. Using Tlr4 transgenic mice, we investigated the influence of Tlr4 gene dosage on acute respiratory response to endotoxin. Transgenic mice expressing three, six, or 30 copies of Tlr4, control, and Tlr4-deficient mice received intranasal administration of lipopolysaccharide (LPS; 10 ug), and the airway response was analyzed by plethysmography, lung histology, cell recruitment, cytokine and chemokine secretion and protein leakage into the bronchoalveolar space. We demonstrate that overexpression of Tlr4 augmented a LPS-induced bronchoconstrictive effect, as well as tumor necrosis factor and CXC chemokine ligand 1 (keratinocyte-derived chemokine) production. Neutrophil recruitment, microvascular and alveolar epithelial injury with protein leak in the airways, and damage of the lung microarchitecture were Tlr4 gene dose-dependently increased. Therefore, the TLR4 expression level determines the extent of acute pulmonary response to inhaled endotoxin, and TLR4 may thus be a valuable target for immunointervention in acute lung inflammation as a result of endotoxins.