ABSTRACTMembers of theRoseobacterlineage of marine bacteria are prolific surface colonizers in marine coastal environments, and antimicrobial secondary metabolite production has been hypothesized to provide a competitive advantage to colonizing roseobacters. Here, we report that the roseobacterPhaeobactersp. strain Y4I produces the blue pigment indigoidine via a nonribosomal peptide synthase (NRPS)-based biosynthetic pathway encoded by a novel series of genetically linked genes:igiBCDFE. A Tn5-based random mutagenesis library of Y4I showed a perfect correlation between indigoidine production by thePhaeobacterstrain and inhibition ofVibrio fischerion agar plates, revealing a previously unrecognized bioactivity of this molecule. In addition, igiD null mutants (igiD encoding the indigoidine NRPS) were more resistant to hydrogen peroxide, less motile, and faster to colonize an artificial surface than the wild-type strain. Collectively, these data provide evidence for pleiotropic effects of indigoidine production in this strain. Gene expression assays support phenotypic observations and demonstrate thatigiDgene expression is upregulated during growth on surfaces. Furthermore, competitive cocultures ofV. fischeriand Y4I show that the production of indigoidine by Y4I significantly inhibits colonization ofV. fischerion surfaces. This study is the first to characterize a secondary metabolite produced by an NRPS in roseobacters.

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.

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.

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.

bCoinfection 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 Larginine bioavailability via oral supplementation can ameliorate malaria-induced intestinal pathology, providing a basis for testing nutritional interventions to reduce malaria-associated mortality in humans. Half of the global population is at risk for malaria, which results in nearly 1 million deaths annually, 86% of which are of children (1). The majority of cases are in sub-Saharan Africa, where there is a high prevalence of coinfection with nontyphoidal Salmonella serotypes (NTS) during the rainy season (2-5). While infections with NTS are normally self-limiting in immunocompetent children, coinfection with malaria can predispose to the development of deadly NTS bacteremia (6-9). During malaria infection, sequestration of parasitized red blood cells (RBCs) and capillary blockage are prominent in intestinal villi (10) and are associated with ischemia, malabsorption, and increased gastrointestinal (GI) permeability (11,12). These phenomena are not restricted to severe malaria; up to 50% of Nigerian children with uncomplicated malaria have GI disturbances (13). The mechanisms that underlie malaria-associated GI pathology and enhance the risk of bacteremia are incompletely understood (14), although recent data indicate that malaria-induced heme oxygenase-1 (HO-1) contributes to impaired resistance to NTS by reducing the production of reactive oxygen species (15). Beyond these findings, knowledge is limited and therapeutic options for coinfection are few in the face of high antibiotic resistance in areas of malaria endemicity (16). To support the development of novel therapeutic interventions for invasive bacterial disease, we developed a murine model of coinfection with Plasmodium yoelii and the NTS strain Salmonella enterica serotype Typhimurium ATCC 14028. In this...

SUMMARYCo-infection can markedly alter the response to a pathogen, thereby changing its clinical presentation. For example, non-typhoidal Salmonella (NTS) serotypes are associated with gastroenteritis in immunocompetent individuals. In contrast, individuals with severe pediatric malaria can develop bacteremic infections with NTS, during which symptoms of gastroenteritis are commonly absent. Here, we report that in both a ligated ileal loop model and a mouse colitis model, malaria parasites caused a global suppression of gut inflammatory responses and blunted the neutrophil influx that is characteristic of NTS infection. Further, malaria parasite infection led to increased recovery of S. Typhimurium from the draining mesenteric lymph node of mice. In the mouse colitis model, blunted intestinal inflammation during NTS infection was independent of anemia, but instead required parasite-induced synthesis of IL-10. Blocking of IL-10 in co-infected mice reduced dissemination of S. Typhimurium to the mesenteric lymph node, suggesting that induction of IL-10 contributes to development of disseminated infection. Thus, IL-10 produced during the immune response to malaria in this model contributes to suppression of mucosal inflammatory responses to invasive NTS, which may contribute to differences in the clinical presentation of NTS infection in the setting of malaria.

Objective-To identify an HIV epitope suitable for vaccine development.Design-Diverse HIV-1 strains express few structurally constant regions on their surface vulnerable to neutralizing antibodies. The mostly-conserved CD4 binding site (CD4BS) of gp120 is essential for host cell binding and infection by the virus. Antibodies that recognize the CD4BS are rare, and one component of the CD4BS, the 421-433 peptide region, expresses B cell superantigenic character, a property predicted to impair the anti-CD4BS adaptive immune response.Methods-IgA samples purified from the plasma of subjects with HIV infection were analyzed for the ability to bind synthetic mimetics containing the 416-433 gp120 region and full-length gp120. Infection of peripheral blood mononuclear cells by clinical HIV isolates was measured by p24 ELISA.Results-IgA preparations from 3 subjects with subtype B infection for 19-21 years neutralized heterologous, coreceptor CCR5-dependent subtype A, B, C, D and AE strains with exceptional potency. The IgAs displayed specific binding of a synthetic 416-433 peptide mimetics dependent on recognition of the CD4 binding residues located in this region. Immunoadsorption, affinity chromatography and mutation procedures indicated that HIV neutralization occurred by IgA recognition of the CD4BS. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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