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Salmonella enterica serovar Typhimurium can cross the epithelial barrier using either the invasion-associated type III secretion system (T3SS-1) or a T3SS-1-independent mechanism that remains poorly characterized. Here we show that flagellum-mediated motility supported a T3SS-1-independent pathway for entering ileal Peyer’s patches in the mouse model. Flagellum-dependent invasion of Peyer’s patches required energy taxis toward nitrate, which was mediated by the methyl-accepting chemotaxis protein (MCP) Tsr. Generation of nitrate in the intestinal lumen required inducible nitric oxide synthase (iNOS), which was synthesized constitutively in the mucosa of the terminal ileum but not in the jejunum, duodenum, or cecum. Tsr-mediated invasion of ileal Peyer’s patches was abrogated in mice deficient for Nos2 , the gene encoding iNOS. We conclude that Tsr-mediated energy taxis enables S . Typhimurium to migrate toward the intestinal epithelium by sensing host-derived nitrate, thereby contributing to invasion of Peyer’s patches. IMPORTANCE Nontyphoidal Salmonella serovars, such as S. enterica serovar Typhimurium, are a common cause of gastroenteritis in immunocompetent individuals but can also cause bacteremia in immunocompromised individuals. While the invasion-associated type III secretion system (T3SS-1) is important for entry, S . Typhimurium strains lacking a functional T3SS-1 can still cross the intestinal epithelium and cause a disseminated lethal infection in mice. Here we observed that flagellum-mediated motility and chemotaxis contributed to a T3SS-1-independent pathway for invasion and systemic dissemination to the spleen. This pathway required the methyl-accepting chemotaxis protein (MCP) Tsr and energy taxis toward host-derived nitrate, which we found to be generated by inducible nitric oxide synthase (iNOS) in the ileal mucosa prior to infection. Collectively, our data suggest that S . Typhimurium enhances invasion by actively migrating toward the intestinal epithelium along a gradient of host-derived nitrate emanating from the mucosal surface of the ileum. Nontyphoidal Salmonella serovars, such as S. enterica serovar Typhimurium, are a common cause of gastroenteritis in immunocompetent individuals but can also cause bacteremia in immunocompromised individuals. While the invasion-associated type III secretion system (T3SS-1) is important for entry, S . Typhimurium strains lacking a functional T3SS-1 can still cross the intestinal epithelium and cause a disseminated lethal infection in mice. Here we observed that flagellum-mediated motility and chemotaxis contributed to a T3SS-1-independent pathway for invasion and systemic dissemination to the spleen. This pathway required the methyl-accepting chemotaxis protein (MCP) Tsr and energy taxis toward host-derived nitrate, which we found to be generated by inducible nitric oxide synthase (iNOS) in the ileal mucosa prior to infection. Collectively, our data suggest that S . Typhimurium enhances invasion by actively migrating toward the intestinal epithelium along a gradient of host-derived nitrate emanating from the mucosal surface of the ileum.

A novel link between the unfolded protein response and NOD1/2 innate immune signalling, showing that NOD1/2 are required for ER-stress-induced IL-6 production in response to infection with Brucella abortus . NOD1and NOD2 link ER stress to inflammatory disease Inflammation due to endoplasmic reticulum (ER) stress is seen in a number of inflammatory diseases, including Crohn's disease and type 2 diabetes and ulcerative colitis. These authors show that activation of ER stress during infection with Brucella abortus is a pathogen-induced process that is sensed by the NOD1 and NOD2 proteins, two pathogen recognition receptors that induce pro-inflammatory responses mediated by activation of NF-κB. Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes 1 , 2 . ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α 3 . Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway 4 . Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus , which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells 5 , is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonie crypt hyperplasia in mice, thereby gaining an edge during its competition with the gut microbiota through an unknown mechanism. Here, we show that by triggering colonie crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygénation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the -secretase inhibitor dibenzazepine to diminish Notch-driven colonie crypt hyperplasia curtailed the fitness advantage conferred by aerobic respiration during C. rodentium infection. We conclude that C. rodentium uses its T3SS to induce histopathological lesions that generate an intestinal microenvironment in which growth of the pathogen is fueled by aerobic respiration.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.

Drug treatment against liver cancer has limited efficacy due to heterogeneous response among liver cancer subtypes. In addition, the functional biophysical phenotypes which arise from this heterogeneity and contribute to aggressive invasive behavior remain poorly understood. This study interrogated how heterogeneity in liver cancer subtypes contributes to differences in invasive phenotypes and drug response. Utilizing histological analysis, quantitative 2D invasion metrics, reconstituted 3D hydrogels, and bioinformatics, our study linked cytoskeletal dynamics to differential invasion profiles and drug resistance in liver cancer subtypes. We investigated cytoskeletal regulation in 2D and 3D culture environments using two liver cancer cell lines, SNU-475 and HepG2, chosen for their distinct cytoskeletal features and invasion profiles. For SNU-475 cells, a model for aggressive liver cancer, many cytoskeletal inhibitors abrogated 2D migration but only some suppressed 3D migration. For HepG2 cells, cytoskeletal inhibition did not significantly affect 3D migration but did affect proliferative capabilities and spheroid core growth. This study highlights cytoskeleton driven phenotypic variation, their consequences and coexistence within the same tumor, as well as efficacy of targeting biophysical phenotypes that may be masked in traditional screens against tumor growth. Treatment for liver cancer is complicated by its various subtypes, which show different responses to anticancer drugs. This study demonstrates the effectiveness of targeting biophysical phenotypes related to cytoskeleton properties that are usually masked in traditional drug screens.

The spread of multidrug-resistant zoonotic pathogens, such as Salmonella , within livestock is of concern for food safety. The spread of Salmonella on the farm is escalated by superspreaders, which shed the pathogen at high numbers with their feces. The spread of multidrug-resistant zoonotic pathogens, such as Salmonella , within livestock is of concern for food safety. The spread of Salmonella on the farm is escalated by superspreaders, which shed the pathogen at high numbers with their feces. However, there are currently no biomarkers to identify potential superspreaders. Kempf and coworkers determined that a potent early inflammatory response to Salmonella infection and changes in the microbiota composition are associated with the superspreader phenotype in pigs (F. Kempf, G. Cordoni, A.M. Chaussé, R. Drumo, et al., mSystems , in press, https://doi.org/10.1128/msystems.00852-22 ). Since these biomarkers only develop during Salmonella infection, additional work is needed to predict animals that have the potential to become superspreaders.

Background Foodborne pathogens, including Salmonella enterica serovar Typhimurium ( S . Typhimurium), pose a significant threat to both human health and livestock productivity. The pandemic S. Typhimurium ST34 clone acquired a genomic island (SGI-4) conferring high copper resistance, an adaptation relevant in the context of the widespread use of copper sulphate at therapeutic levels in pig farming. We investigated how high dietary copper influences the piglet gut microbiota and Salmonella -microbiota interactions that may explain the global spread of S. Typhimurium ST34. Results An on-farm study combined with faecal shotgun metagenomics revealed that several potential Salmonella competitor species, including Bifidobacterium , Escherichia , and Lactobacillus , were less abundant in piglets on high-copper diets. Anaerobic and aerobic culturing alongside whole genome sequencing of 131 species and copper sulphate susceptibility testing identified copper resistance gene acquisition in selected microbes, particularly within Escherichia . Niche competition assays demonstrated that copper resistance is critical for inter-species competition under high-copper conditions, with Salmonella ’s Type VI Secretion System providing a distinct advantage over Escherichia in the copper-modified niche. Conclusions Our findings suggest that copper supplementation alters the piglet gut environment, impacting competitive dynamics between pathogenic and commensal bacteria, likely to influence the zoonotic transmission of pathogens. 7HGfSp5tFGWTF9yHxnok5s Video Abstract

Wolfram syndrome is a rare multisystem disease characterized by childhood-onset diabetes mellitus and progressive neurodegeneration. Most cases are attributed to pathogenic variants in a single gene, Wolfram syndrome 1 (WFS1). There currently is no diseasemodifying treatment for Wolfram syndrome, as the molecular consequences of the loss of WFS1 remain elusive. Because diabetes mellitus is the first diagnosed symptom of Wolfram syndrome, we aimed to further examine the functions of WFS1 in pancreatic β cells in the context of hyperglycemia. Knockout (KO) of WFS1 in rat insulinoma (INS1) cells impaired calcium homeostasis and protein kinase B/Akt signaling and, subsequently, decreased cell viability and glucose-stimulated insulin secretion. Targeting calcium homeostasis with reexpression of WFS1, overexpression of WFS1’s interacting partner neuronal calcium sensor-1 (NCS1), or treatment with calpain inhibitor and ibudilast reversed deficits observed in WFS1-KO cells. Collectively, our findings provide insight into the disease mechanism of Wolfram syndrome and highlight new targets and drug candidates to facilitate the development of a treatment for this disorder and similar diseases.

Montane ecosystems occur throughout the world, and harbor many endemic species. They also provide key ecological services, including the catchment of water resources and the storage of organic carbon. These ecosystems are vulnerable to global climate change and increasing human pressures, including forestry and their conversion to arable land. In the extensive and biodiverse Mexican montane regions, ongoing deforestation and conversion to arable lands has led to diminished ecosystem health and services. Here, we undertook a comprehensive evaluation of carbon stocks within Mexican montane habitats in the Flora and Fauna Conservation Area of Nevado de Toluca. This aimed to integrate these habitats into Mexican and global census of forest carbon, the first step needed to convert on carbon credit markets to incentivise conservation of this region by local communities. Our study evaluated both, living biomass and belowground soil organic carbon in sites within forests, alpine grasslands and converted arable land. We addressed the following questions: (1) What are the organic carbon stocks, including the soil component, of our studied montane habitats? (2) What are the avoided CO2 emissions from maintaining natural forests and preventing conversion to arable land? And (3) Within our study area, are organic carbon stocks in the soil correlated to carbon stocks in aboveground living biomass? We found whole ecosystem organic carbon stocks ranged from 68 Mg OC ha-1 in unburnt alpine grasslands to 668 Mg OC ha-1 in Abies religiosa forests. By avoiding conversion of the Abies religiosa forests to arable lands, we show that emissions of 1,122 to 1,671 Mg CO2 ha-1 are avoided. Notably, the belowground soil organic carbon stock comprised ≥ 40% of the total ecosystem organic carbon stock. We recommend soil organic carbon stocks should be included within Mexican and global forestry carbon stock inventories, and should be considered within voluntary carbon-credit markets used to incentivize the conservation of Mexican montane habitats.

Listeria monocytogenes is a Gram-positive bacterial pathogen that has been used for decades to study bacterial infection and immunity. Murine infection models demonstrate that sublethal infections with L. monocytogenes cause only a mild disease but lead to the development of long-lived cell-mediated immunity (CMI), mediated by antigen-specific cytotoxic CD8+ T-cells. During infection L. monocytogenes is phagocytosed by antigen presenting cells but escapes the phagosome and accesses the host cell cytosol by secreting the virulence factor LLO, encoded by the gene hly. Importantly, robust immunization requires live bacteria that can enter the cytosol of host cells. In contrast, Δhly mutants remain in the phagosome (do not enter the cytosol) and are poor inducers of CMI. Furthermore, during co-administration of both cytosolic and Δhly strains simultaneously into mice, the Δhly mutant blocked CMI, indicating that the Δhly mutant suppresses immunity normally elicited by the cytosolic strain. Additionally, Δhly mutants induce the expression of the immunosuppressive cytokine IL-10, that inhibits the expression of inflammatory cytokines and the development of CMI. In chapter two, we identify the genetic and molecular determinants of IL-10 induction and immunosuppression by phagosome confined bacteria by screening 6000 Δhly L. monocytogenes transposon mutants for either enhanced or diminished capacity to induce IL-10 in macrophages. The transposon screen identified two classes of bacterial mutants. The first class led to diminished IL-10 induction and contained a transposon insertion in the gene that encodes the enzyme Lgt that is required for the formation of mature bacterial lipoproteins. We found that macrophages induced elevated IL-10 secretion in response to phagosome confined bacteria by detecting bacterial lipoproteins through the host receptor TLR2. The second class of mutants led to greater IL-10 induction and contained transposon insertions in the genes encoding two known cell-wall modifying enzymes, pgdA and oatA. The ΔhlyΔpgdAΔoatA triple mutant was known to be susceptible to lysozyme-dependent killing within the macrophage and induced over 200% more IL-10 than the Δhly mutant. This suggested that killed phagosomal bacteria were being degraded and thus releasing their nucleic acids within the phagosome that were then being detected by nucleic acid sensing phagosomal TLRs, leading to the robust induction of IL-10. Macrophages lacking both phagosomal TLRs and TLR2, no longer secreted IL-10 during infection with the ΔhlyΔpgdAΔoatA mutant. Although both TLR2 and endosomal TLR pathways led to IL-10 induction in macrophages, the detection of nucleic acids was the main immunosuppressive pathway since mice defective for phagosomal TLR localization were no longer suppressed for the development of CMI by the Δhly mutant. In conclusion, the work in chapter two identified both the bacterial and host molecular determinants that lead to immunosuppression during vaccinations with phagosome confined L. monocytogenes Δhly mutants.The work presented in chapter three sought to enhance L. monocytogenes-based cancer vaccines. A current effort in the field of tumor therapy involves converting tumors that respond poorly to cancer immunotherapy, termed immunologically “cold” tumors, into “hot” tumors by injecting antitumoral agents directly into the tumor microenvironment. However, the mechanisms that lead to effective intratumoral therapy are not fully understood and it is not clear whether it is beneficial to target the tumor cells or the immune cells during intratumoral therapy. The work in chapter three demonstrates that intratumoral injections with cytosolic L. monocytogenes strains are therapeutic in mice, whereas phagosome confined mutants are poorly therapeutic. In vitro infections demonstrated that L. monocytogenes infected macrophages approximately 100 times more efficiently than tumor cells. Nevertheless, in vitro infection of tumor cells suggested that L. monocytogenes could kill tumor cells directly by overgrowing within the tumor cell cytosol. We, therefore, hypothesized that enhancing L. monocytogenes’ ability to invade tumor cells would also enhance intratumoral therapy by enhancing the cold to hot conversion of tumors. A selection was performed for spontaneous L. monocytogenes mutants that were enhanced for invasion of tumor cells in vitro, with the goal of identifying the major bacterial determinants that contribute to tumor cell invasion, and to determine if invasion of tumor cells contributes to I.T. therapy.The selection identified two gain of function mutations in the master virulence regulator PrfA, that lock PrfA into a constitutively active form, termed PrfA*. Additionally, a novel mutation in the virulence gene ActA (ActAE144L) was identified that also conferred enhanced invasion into mouse tumor cells, indicating that ActA could be used to test if enhanced invasion contributes to intratumoral therapy. However, ActA did not play a beneficial role during intratumoral therapy in mice. Analysis of L. monocytogenes mutants identified that the majority of PrfA*-dependent-enhanced invasion was due to the virulence factor InlB, an internalization factor known to facilitate entry into non-phagocytic hepatocytes that express the hepatocyte growth factor receptor c-Met. Finally, InlB contributed significantly to intratumoral therapy in two commonly studied types of mouse tumors, CT26 and B16.F10. In conclusion, this work showed that in vitro genetic selections with attenuated bacterial pathogens can identify the relevant genes and pathways that affect tumor cell invasion. These results revealed that bacterial internalization factors that affect the invasive capacity of L. monocytogenes into non-phagocytic cells contributed significantly to intratumoral therapy.