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Intracellular pathogens commonly reside within macrophages to find shelter from humoral defenses, but host cell death can expose them to the extracellular milieu. We find intracellular pathogens solve this dilemma by using virulence factors to generate a complement-dependent find-me signal that initiates uptake by a new phagocyte through efferocytosis. During macrophage death, Salmonella uses a type III secretion system to perforate the membrane of the pathogen-containing vacuole (PCV), thereby triggering complement deposition on bacteria entrapped in pore-induced intracellular traps (PITs). In turn, complement activation signals neutrophil efferocytosis, a process that shelters intracellular bacteria from the respiratory burst. Similarly, Brucella employs its type IV secretion system to perforate the PCV membrane, which induces complement deposition on bacteria entrapped in PITs. Collectively, this work identifies virulence factor-induced perforation of the PCV as a strategy of intracellular pathogens to generate a find-me signal for efferocytosis.

Summary The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis.

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.

The mammalian intestine is host to a microbial community that prevents pathogen expansion through unknown mechanisms, while antibiotic treatment can increase susceptibility to enteric pathogens. Here we show that streptomycin treatment depleted commensal, butyrate-producing Clostridia from the mouse intestinal lumen, leading to decreased butyrate levels, increased epithelial oxygenation, and aerobic expansion of Salmonella enterica serovar Typhimurium. Epithelial hypoxia and Salmonella restriction could be restored by tributyrin treatment. Clostridia depletion and aerobic Salmonella expansion were also observed in the absence of streptomycin treatment in genetically resistant mice but proceeded with slower kinetics and required the presence of functional Salmonella type III secretion systems. The Salmonella cytochrome bd-II oxidase synergized with nitrate reductases to drive luminal expansion, and both were required for fecal-oral transmission. We conclude that Salmonella virulence factors and antibiotic treatment promote pathogen expansion through the same mechanism: depletion of butyrate-producing Clostridia to elevate epithelial oxygenation, allowing aerobic Salmonella growth.

OBJECTIVES/SPECIFIC AIMS: Our long-term goal is to elucidate the molecular mechanisms and virulence factors that control the differential presentation of infection with Salmonella typhimurium and Salmonella typhi. The objectives of this study are to study the mechanisms that enable S. typhi to trigger a decreased inflammatory response in comparison with S. typhimurium and evade detection by the immune system, leading to the development of asymptomatic chronic carriage of S. typhi. METHODS/STUDY POPULATION: A loss of function eptB mutant S. typhimurium strain was generated. Lipopolysaccharide (LPS) was isolated from wild-type and eptB mutant S. typhimurium and wild-type S. typhi. Binding of LPS to recombinant intelectin was tested by dot blot and enzyme-linked immunosorbant assay (ELISA). C57BL/6 mice were infected with wild-type or eptB mutant S. typhimurium by oral gavage and inflammatory cytokines in the spleen, liver, and Peyer’s patches were measured by qPCR. RESULTS/ANTICIPATED RESULTS: LPS isolated from wild-type S. typhimurium is not bound by intelectin, a protein that has been proposed to function in innate immunity and that is known to be able to bind certain moieties within LPS. Conversely, LPS isolated from eptB mutant S. typhimurium and wild-type S. typhi, which lacks a functional eptB, is bound by intelectin. Mice infected with an eptB mutant S. typhimurium exhibit decreased expression of inflammatory cytokines in the spleen compared to mice infected with the wild type S. typhimurium, suggesting that loss of eptB function allows a nontyphoidal Salmonella serovar to mimic the stealth phenotype of typhoidal serovars. Together, these results suggest that loss of eptB function allows intelectin to bind to and detoxify Salmonella LPS, leading to decreased systemic inflammation during infection. DISCUSSION/SIGNIFICANCE OF IMPACT: These results have broad implications for how pathogens such as S. typhimurium induce systemic shock during infection and may also help to explain a mechanism for how S. typhi is able to evade immune detection and enhance dissemination to systemic sites, leading to development of the asymptomatic chronic carrier state. Further investigation of this novel virulence mechanism will mark a decisive step forward in understanding the mechanisms underlying the differential pathogenesis of S. typhimurium-induced gastroenteritis and S. typhi-induced typhoid fever. Additionally, these results contribute to our understanding of the interactions between host and pathogen in affecting disease presentation, which will have wide appeal among researchers interested in microbial pathogenesis and the contribution of host-pathogen interactions to health and disease.

Nuclear factor-κB (NF-κB) regulates cellular responses to inflammation and aging, and alterations in NF-κB signaling underlie the pathogenesis of multiple human diseases. Effective clinical therapeutics targeting this pathway remain unavailable. In primary human keratinocytes, we found that hypochlorite (HOCl) reversibly inhibited the expression of CCL2 and SOD2, two NF-κB–dependent genes. In cultured cells, HOCl inhibited the activity of inhibitor of NF-κB kinase (IKK), a key regulator of NF-κB activation, by oxidizing cysteine residues Cys114 and Cys115. In NF-κB reporter mice, topical HOCl reduced LPS-induced NF-κB signaling in skin. We further evaluated topical HOCl use in two mouse models of NF-κB–driven epidermal disease. For mice with acute radiation dermatitis, topical HOCl inhibited the expression of NF-κB–dependent genes, decreased disease severity, and prevented skin ulceration. In aged mice, topical HOCl attenuated age-dependent production of p16INK4a and expression of the DNA repair gene Rad50. Additionally, skin of aged HOCl-treated mice acquired enhanced epidermal thickness and proliferation, comparable to skin in juvenile animals. These data suggest that topical HOCl reduces NF-κB–mediated epidermal pathology in radiation dermatitis and skin aging through IKK modulation and motivate the exploration of HOCl use for clinical aims.

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.