Oral pathobiont Klebsiella chaperon usher pili provide site-specific adaptation for the inflamed gut mucosa.

The ectopic gut colonization by orally derived pathobionts has been implicated in the pathogenesis of various gastrointestinal diseases, including inflammatory bowel disease (IBD). For example, gut colonization by orally derived Klebsiella spp. has been linked to IBD in mice and humans. However, the mechanisms whereby oral pathobionts colonize extra-oral niches, such as the gut mucosa, remain largely unknown. Here, we performed a high-density transposon (Tn) screening to identify genes required for the adaptation of an oral Klebsiella strain to different mucosal sites - the oral and gut mucosae - at the steady state and during inflammation. We find that K. aerogenes, an oral pathobiont associated with both oral and gut inflammation in mice, harbors a newly identified genomic locus named "locus of colonization in the inflamed gut (LIG)" that encodes genes related to iron acquisition (Sit and Chu) and host adhesion (chaperon usher pili [CUP] system). The LIG locus is highly conserved among K. aerogenes strains, and these genes are also present in several other Klebsiella species. The Tn screening revealed that the LIG locus is required for the adaptation of K. aerogenes in its ectopic niche. In particular, we determined K. aerogenes employs a CUP system (CUP1) present in the LIG locus for colonization in the inflamed gut, but not in the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche compared to their native niche.

Microbiota-mediated colonization resistance: mechanisms and regulation.

A dense and diverse microbial community inhabits the gut and many epithelial surfaces. Referred to as the microbiota, it co-evolved with the host and is beneficial for many host physiological processes. A major function of these symbiotic microorganisms is protection against pathogen colonization and overgrowth of indigenous pathobionts. Dysbiosis of the normal microbial community increases the risk of pathogen infection and overgrowth of harmful pathobionts. The protective mechanisms conferred by the microbiota are complex and include competitive microbial-microbial interactions and induction of host immune responses. Pathogens, in turn, have evolved multiple strategies to subvert colonization resistance conferred by the microbiota. Understanding the mechanisms by which microbial symbionts limit pathogen colonization should guide the development of new therapeutic approaches to prevent or treat disease.

Regulation of Citrobacter rodentium colonization: virulence, immune response and microbiota interactions.

Citrobacter rodentium is a mouse-specific pathogen commonly used to model infection by human Enteropathogenic Escherichia coli, an important cause of infant diarrhea and mortality worldwide. In the early phase of infection, C. rodentium overcomes competition by the gut microbiota for successful replication. Then, the pathogen uses a type three secretion system (T3SS) to inject effector proteins into intestinal epithelial cells and induce metabolic and inflammatory conditions that promote colonization of the intestinal epithelium. C. rodentium also elicits highly coordinated innate and adaptive immune responses in the gut that regulate pathogen colonization and eradication. In this review, we highlight recent work on the regulation and function of the C. rodentium T3SS, the mechanisms employed by the pathogen to evade competition by the microbiota, and the function of the host immune response against infection.

The influence of maize genotype on the rhizosphere eukaryotic community.

The microbiota colonizing the rhizosphere contributes to plant growth, productivity, carbon sequestration and phytoremediation. Several studies address plant-associated bacteria; however, few studies analyze the effect of plant genotype on the eukaryotic community. Here, we analyzed the eukaryotic composition of maize rhizosphere from three different plant landraces and one inbred line grown in the same soil (common garden approach). This experimental design, coupled with 18S rDNA gene amplicon sequencing, allowed us to test the influence of maize and its genotype on the rhizosphere's eukaryotic community. We found that plant growth modified the eukaryotic community in soil, as diversity comparisons between maize rhizosphere and unplanted soil revealed significantly different eukaryotic composition. Various genera of nematodes and fungi, predominantly bacterial feeding nematodes and mycorrhizal fungi among other taxa, were increased in the rhizosphere samples. We also observed that maize genotype differentially shaped the relative abundance of the following fungal families in the rhizosphere: Acaulosporaceae, Aspergillaceae, Chaetomiaceae, Claroideoglomeraceae, Corticiaceae, Mortierellaceae, Trichocomaceae and Trichomeriaceae. Thus, plant genotype has a selective influence on establishing fungal communities in the rhizosphere. This study emphasizes the importance of an integrated consideration of plant genetics for future agricultural applications of microbes to crops.

Disrupted Iron Metabolism and Mortality during Co-infection with Malaria and an Intestinal Gram-Negative Extracellular Pathogen.

Individuals with malaria exhibit increased morbidity and mortality when infected with Gram-negative (Gr-) bacteria. To explore this experimentally, we performed co-infection of mice with Plasmodium chabaudi and Citrobacter rodentium, an extracellular Gr- bacterial pathogen that infects the large intestine. While single infections are controlled effectively, co-infection results in enhanced virulence that is characterized by prolonged systemic bacterial persistence and high mortality. Mortality in co-infected mice is associated with disrupted iron metabolism, elevated levels of plasma heme, and increased mitochondrial reactive oxygen species (ROS) production by phagocytes. In addition, iron acquisition by the bacterium plays a key role in pathogenesis because co-infection with a mutant C. rodentium strain lacking a critical iron acquisition pathway does not cause mortality. These results indicate that disrupted iron metabolism may drive mortality during co-infection with C. rodentium and P. chabaudi by both altering host immune responses and facilitating bacterial persistence.

Lipopolysaccharide O structure of adherent and invasive Escherichia coli regulates intestinal inflammation via complement C3.

Gut dysbiosis associated with intestinal inflammation is characterized by the blooming of particular bacteria such as adherent-invasive E. coli (AIEC). However, the precise mechanisms by which AIEC impact on colitis remain largely unknown. Here we show that antibiotic-induced dysbiosis worsened chemically-induced colitis in IL-22-deficient mice, but not in wild-type mice. The increase in intestinal inflammation was associated with the expansion of E. coli strains with genetic and functional features of AIEC. These E. coli isolates exhibited high ability to out compete related bacteria via colicins and resistance to the host complement system in vitro. Mutation of wzy, the lipopolysaccharide O polymerase gene, rendered AIEC more sensitive to the complement system and more susceptible to engulfment and killing by phagocytes while retaining its ability to outcompete related bacteria in vitro. The wzy AIEC mutant showed impaired fitness to colonize the intestine under colitic conditions, but protected mice from chemically-induced colitis. Importantly, the ability of the wzy mutant to protect from colitis was blocked by depletion of complement C3 which was associated with impaired intestinal eradication of AIEC in colitic mice. These studies link surface lipopolysaccharide O-antigen structure to the regulation of colitic activity in commensal AIEC via interactions with the complement system.

An Enteric Pathogen Subverts Colonization Resistance by Evading Competition for Amino Acids in the Gut.

The microbiota confers host protection by limiting the colonization of pathogenic bacteria in the gut, but the mechanisms by which pathogens overcome colonization resistance remain poorly understood. Using a high-density transposon screen in the enteric pathogen Citrobacter rodentium, we find that the bacterium requires amino acid biosynthesis pathways to colonize conventionally raised mice, but not germ-free or antibiotic-treated animals. These metabolic pathways are induced during infection by the presence of the gut microbiota. Reduced amounts of amino acids are found in the guts of conventionally raised mice compared with germ-free animals. Dietary administration of high protein increases amino acid levels in the gut and promotes pathogen colonization. Thus, the depletion of amino acids by the microbiota limits pathogen colonization, and in turn, the pathogen activates amino acid biosynthesis to expand in the presence of the microbiota.

Maternal Immunization Confers Protection to the Offspring against an Attaching and Effacing Pathogen through Delivery of IgG in Breast Milk.

Owing to immature immune systems and impaired colonization resistance mediated by the microbiota, infants are more susceptible to enteric infections. Maternal antibodies can provide immunity, with maternal vaccination offering a protective strategy. We find that oral infection of adult females with the enteric pathogen Citrobacter rodentium protects dams and offspring against oral challenge. Parenteral immunization of dams with heat-inactivated C. rodentium reduces pathogen loads and mortality in offspring but not mothers. IgG, but not IgA or IgM, transferred through breast milk to the intestinal lumen of suckling offspring, coats the pathogen and reduces intestinal colonization. Protective IgG largely recognizes virulence factors encoded within the locus of enterocyte effacement (LEE) pathogenicity island, including the adhesin Intimin and T3SS filament EspA, which are major antigens conferring protection. Thus, pathogen-specific IgG in breast milk induced during maternal infection or immunization protects neonates against infection with an attaching and effacing pathogen.

IL-22 Controls Iron-Dependent Nutritional Immunity Against Systemic Bacterial Infections.

Host immunity limits iron availability to pathogenic bacteria, but whether immunity limits pathogenic bacteria from accessing host heme, the major source of iron in the body, remains unclear. Using Citrobacter rodentium, a mouse enteric pathogen and Escherichia coli, a major cause of sepsis in humans as models, we find that interleukin-22, a cytokine best known for its ability to promote epithelial barrier function, also suppresses the systemic growth of bacteria by limiting iron availability to the pathogen. Using an unbiased proteomic approach to understand the mechanistic basis of IL-22 dependent iron retention in the host, we have identified that IL-22 induces the production of the plasma hemoglobin scavenger haptoglobin and heme scavenger hemopexin. Moreover, the anti-microbial effect of IL-22 depends on the induction of hemopexin expression, while haptogloblin is dispensable. Impaired pathogen clearance in infected Il22-/- mice was restored by hemopexin administration and hemopexin-deficient mice had increased pathogen loads after infection. These studies reveal a previously unrecognized host defense mechanism regulated by IL-22 that relies on the induction of hemopexin to limit heme availability to bacteria leading to suppression of bacterial growth during systemic infections.

Identification and regulation of a novel Citrobacter rodentium gut colonization fimbria (Gcf).

UNLABELLED: The Gram-negative enteric bacterium Citrobacter rodentium is a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. For C. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novel C. rodentium colonization factor, called gut colonization fimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. A gcfA mutant shows a severe colonization defect within the first 10 days of infection. The gcf promoter is not active in C. rodentium under several in vitro growth conditions; however, it is readily expressed in a C. rodentium Δhns1 mutant lacking the closest ortholog of the Escherichia coli histone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region of gcf, further supporting its direct role as a repressor of the gcf promoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. The gcf operon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures. IMPORTANCE: Fimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection by C. rodentium in mice, despite being poorly expressed under in vitro growth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins.

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.

Chromate-resistance genes in plasmids from antibiotic-resistant nosocomial enterobacterial isolates.

The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in México. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (Cr(R)), whereas 36% were resistant to mercury (Hg(R)). Cr(R) levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of Cr(R) isolates), encoding a CHR transporter homologue, and merA gene (74% of Hg(R) isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of Cr(R) isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred Cr(R), as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that Cr(R) genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.