Infection-generated electric field in gut epithelium drives bidirectional migration of macrophages.

Many bacterial pathogens hijack macrophages to egress from the port of entry to the lymphatic drainage and/or bloodstream, causing dissemination of life-threatening infections. However, the underlying mechanisms are not well understood. Here, we report that Salmonella infection generates directional electric fields (EFs) in the follicle-associated epithelium of mouse cecum. In vitro application of an EF, mimicking the infection-generated electric field (IGEF), induces directional migration of primary mouse macrophages to the anode, which is reversed to the cathode upon Salmonella infection. This infection-dependent directional switch is independent of the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. The switch is accompanied by a reduction of sialic acids on glycosylated surface components during phagocytosis of bacteria, which is absent in macrophages challenged by microspheres. Moreover, enzymatic cleavage of terminally exposed sialic acids reduces macrophage surface negativity and severely impairs directional migration of macrophages in response to an EF. Based on these findings, we propose that macrophages are attracted to the site of infection by a combination of chemotaxis and galvanotaxis; after phagocytosis of bacteria, surface electrical properties of the macrophage change, and galvanotaxis directs the cells away from the site of infection.

Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization.

Young age is a risk factor for prolonged colonization by common pathogens residing in their upper respiratory tract (URT). Why children present with more persistent colonization is unknown and there is relatively little insight into the host-pathogen interactions that contribute to persistent colonization. To identify factors permissive for persistent colonization during infancy, we utilized an infant mouse model of Streptococcus pneumoniae colonization in which clearance from the mucosal surface of the URT requires many weeks to months. Loss of a single bacterial factor, the pore-forming toxin pneumolysin (Ply), and loss of a single host factor, IL-1α, led to more persistent colonization. Exogenous administration of Ply promoted IL-1 responses and clearance, and intranasal treatment with IL-1α was sufficient to reduce colonization density. Major factors known to affect the duration of natural colonization include host age and pneumococcal capsular serotype. qRT-PCR analysis of the uninfected URT mucosa showed reduced baseline expression of genes involved in IL-1 signaling in infant compared to adult mice. In line with this observation, IL-1 signaling was important in initiating clearance in adult mice but had no effect on early colonization of infant mice. In contrast to the effect of age, isogenic constructs of different capsular serotype showed differences in colonization persistence but induced similar IL-1 responses. Altogether, this work underscores the importance of toxin-induced IL-1α responses in determining the outcome of colonization, clearance versus persistence. Our findings about IL-1 signaling as a function of host age may provide an explanation for the increased susceptibility and more prolonged colonization during early childhood.

Malaria Parasite-Mediated Alteration of Macrophage Function and Increased Iron Availability Predispose to Disseminated Nontyphoidal Salmonella Infection.

Disseminated infections with nontyphoidal Salmonella (NTS) are a significant cause of child mortality in sub-Saharan Africa. NTS infection in children is clinically associated with malaria, suggesting that malaria compromises the control of disseminated NTS infection. To study the mechanistic basis for increased NTS susceptibility, we utilized a model of concurrent infection with Salmonella enterica serotype Typhimurium and Plasmodium yoelii nigeriensis (P. yoelii). Underlying malaria blunted monocyte expression of Ly6C, a marker for inflammatory activation, and impaired recruitment of inflammatory cells to the liver. Hepatic mononuclear phagocytes expressed lower levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor and showed increased levels of production of interleukin-10 and heme oxygenase-1, indicating that the underlying malaria modifies the activation state and inflammatory response of mononuclear phagocytes to NTS. P. yoelii infection also increased intracellular iron levels in liver mononuclear cells, as evidenced by elevated levels of ferritin and by the rescue of an S Typhimurium tonB feoB mutant defective for iron uptake. In addition, concurrent P. yoelii infection partially rescued the systemic colonization defect of an S Typhimurium spiB mutant defective for type III secretion system 2 (T3SS-2), indicating that the ability of phagocytic cells to limit the spread of S Typhimurium is impaired during concurrent P. yoelii infection. These results show that concurrent malaria increases susceptibility to disseminated NTS infection by blunting macrophage bactericidal mechanisms and providing an essential nutrient that enhances bacterial growth.

Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion.

Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator-activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward ß-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.

Disseminated infections with antibiotic-resistant non-typhoidal Salmonella strains: contributions of host and pathogen factors.

Non-typhoidal Salmonella enterica serovars (NTS) are generally associated with gastroenteritis; however, the very young and elderly, as well as individuals with compromised immunity, are at risk of developing disseminated infection that can manifest as bacteremia or focal infections at systemic sites. Disseminated NTS infections can be fatal and are responsible for over 600 000 deaths annually. Most of these deaths are in sub-Saharan Africa, where multidrug-resistant NTS clones are currently circulating in a population with a high proportion of individuals that are susceptible to disseminated disease. This review considers how genome degradation observed in African NTS isolates has resulted in phenotypic differences in traits related to environmental persistence and host-pathogen interactions. Further, it discusses host mechanisms promoting susceptibility to invasive infection with NTS in individuals with immunocompromising conditions. We conclude that mechanistic knowledge of how risk factors compromise immunity to disseminated NTS infection will be important for the design of interventions to protect against systemic disease.

Virulence factors enhance Citrobacter rodentium expansion through aerobic respiration.

Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonic 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 colonic crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygenation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the γ-secretase inhibitor dibenzazepine to diminish Notch-driven colonic 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.

Energy Taxis toward Host-Derived Nitrate Supports a Salmonella Pathogenicity Island 1-Independent Mechanism of Invasion.

UNLABELLED: 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.

Loss of Multicellular Behavior in Epidemic African Nontyphoidal Salmonella enterica Serovar Typhimurium ST313 Strain D23580.

UNLABELLED: Nontyphoidal Salmonella enterica serovar Typhimurium is a frequent cause of bloodstream infections in children and HIV-infected adults in sub-Saharan Africa. Most isolates from African patients with bacteremia belong to a single sequence type, ST313, which is genetically distinct from gastroenteritis-associated ST19 strains, such as 14028s and SL1344. Some studies suggest that the rapid spread of ST313 across sub-Saharan Africa has been facilitated by anthroponotic (person-to-person) transmission, eliminating the need for Salmonella survival outside the host. While these studies have not ruled out zoonotic or other means of transmission, the anthroponotic hypothesis is supported by evidence of extensive genomic decay, a hallmark of host adaptation, in the sequenced ST313 strain D23580. We have identified and demonstrated 2 loss-of-function mutations in D23580, not present in the ST19 strain 14028s, that impair multicellular stress resistance associated with survival outside the host. These mutations result in inactivation of the KatE stationary-phase catalase that protects high-density bacterial communities from oxidative stress and the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype. However, we found that like 14028s, D23580 is able to elicit an acute inflammatory response and cause enteritis in mice and rhesus macaque monkeys. Collectively, these observations suggest that African S. Typhimurium ST313 strain D23580 is becoming adapted to an anthroponotic mode of transmission while retaining the ability to infect and cause enteritis in multiple host species. IMPORTANCE: The last 3 decades have witnessed an epidemic of invasive nontyphoidal Salmonella infections in sub-Saharan Africa. Genomic analysis and clinical observations suggest that the Salmonella strains responsible for these infections are evolving to become more typhoid-like with regard to patterns of transmission and virulence. This study shows that a prototypical African nontyphoidal Salmonella strain has lost traits required for environmental stress resistance, consistent with an adaptation to a human-to-human mode of transmission. However, in contrast to predictions, the strain remains capable of causing acute inflammation in the mammalian intestine. This suggests that the systemic clinical presentation of invasive nontyphoidal Salmonella infections in Africa reflects the immune status of infected hosts rather than intrinsic differences in the virulence of African Salmonella strains. Our study provides important new insights into the evolution of host adaptation in bacterial pathogens.

Mast cells and histamine alter intestinal permeability during malaria parasite infection.

Co-infections with malaria and non-typhoidal Salmonella serotypes (NTS) can present as life-threatening bacteremia, in contrast to self-resolving NTS diarrhea in healthy individuals. In previous work with our mouse model of malaria/NTS co-infection, we showed increased gut mastocytosis and increased ileal and plasma histamine levels that were temporally associated with increased gut permeability and bacterial translocation. Here, we report that gut mastocytosis and elevated plasma histamine are also associated with malaria in an animal model of falciparum malaria, suggesting a broader host distribution of this biology. In support of mast cell function in this phenotype, malaria/NTS co-infection in mast cell-deficient mice was associated with a reduction in gut permeability and bacteremia. Further, antihistamine treatment reduced bacterial translocation and gut permeability in mice with malaria, suggesting a contribution of mast cell-derived histamine to GI pathology and enhanced risk of bacteremia during malaria/NTS co-infection.

Inflammation-associated alterations to the intestinal microbiota reduce colonization resistance against non-typhoidal Salmonella during concurrent malaria parasite infection.

Childhood malaria is a risk factor for disseminated infections with non-typhoidal Salmonella (NTS) in sub-Saharan Africa. While hemolytic anemia and an altered cytokine environment have been implicated in increased susceptibility to NTS, it is not known whether malaria affects resistance to intestinal colonization with NTS. To address this question, we utilized a murine model of co-infection. Infection of mice with Plasmodium yoelii elicited infiltration of inflammatory macrophages and T cells into the intestinal mucosa and increased expression of inflammatory cytokines. These mucosal responses were also observed in germ-free mice, showing that they are independent of the resident microbiota. Remarkably, P. yoelii infection reduced colonization resistance of mice against S. enterica serotype Typhimurium. Further, 16S rRNA sequence analysis of the intestinal microbiota revealed marked changes in the community structure. Shifts in the microbiota increased susceptibility to intestinal colonization by S. Typhimurium, as demonstrated by microbiota reconstitution of germ-free mice. These results show that P. yoelii infection, via alterations to the microbial community in the intestine, decreases resistance to intestinal colonization with NTS. Further they raise the possibility that decreased colonization resistance may synergize with effects of malaria on systemic immunity to increase susceptibility to disseminated NTS infections.

Transient Loss of Protection Afforded by a Live Attenuated Non-typhoidal Salmonella Vaccine in Mice Co-infected with Malaria.

In immunocompetent individuals, non-typhoidal Salmonella serovars (NTS) are associated with gastroenteritis, however, there is currently an epidemic of NTS bloodstream infections in sub-Saharan Africa. Plasmodium falciparum malaria is an important risk factor for invasive NTS bloodstream in African children. Here we investigated whether a live, attenuated Salmonella vaccine could be protective in mice, in the setting of concurrent malaria. Surprisingly, mice acutely infected with the nonlethal malaria parasite Plasmodium yoelii 17XNL exhibited a profound loss of protective immunity to NTS, but vaccine-mediated protection was restored after resolution of malaria. Absence of protective immunity during acute malaria correlated with maintenance of antibodies to NTS, but a marked reduction in effector capability of Salmonella-specific CD4 and CD8 T cells. Further, increased expression of the inhibitory molecule PD1 was identified on memory CD4 T cells induced by vaccination. Blockade of IL-10 restored protection against S. Typhimurium, without restoring CD4 T cell effector function. Simultaneous blockade of CTLA-4, LAG3, and PDL1 restored IFN-γ production by vaccine-induced memory CD4 T cells but was not sufficient to restore protection. Together, these data demonstrate that malaria parasite infection induces a temporary loss of an established adaptive immune response via multiple mechanisms, and suggest that in the setting of acute malaria, protection against NTS mediated by live vaccines may be interrupted.

Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function.

Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection.

Malaria-associated L-arginine deficiency induces mast cell-associated disruption to intestinal barrier defenses against nontyphoidal Salmonella bacteremia.

Coinfection with malaria and nontyphoidal Salmonella serotypes (NTS) can cause life-threatening bacteremia in humans. Coinfection with malaria is a recognized risk factor for invasive NTS, suggesting that malaria impairs intestinal barrier function. Here, we investigated mechanisms and strategies for prevention of coinfection pathology in a mouse model. Our findings reveal that malarial-parasite-infected mice, like humans, develop L-arginine deficiency, which is associated with intestinal mastocytosis, elevated levels of histamine, and enhanced intestinal permeability. Prevention or reversal of L-arginine deficiency blunts mastocytosis in ileal villi as well as bacterial translocation, measured as numbers of mesenteric lymph node CFU of noninvasive Escherichia coli Nissle and Salmonella enterica serotype Typhimurium, the latter of which is naturally invasive in mice. Dietary supplementation of malarial-parasite-infected mice with L-arginine or L-citrulline reduced levels of ileal transcripts encoding interleukin-4 (IL-4), a key mediator of intestinal mastocytosis and macromolecular permeability. Supplementation with L-citrulline also enhanced epithelial adherens and tight junctions in the ilea of coinfected mice. These data suggest that increasing L-arginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans.