Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut.

Zinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. However, the probiotic bacterium Escherichia coli Nissle 1917 (or "Nissle") exhibits appreciable growth in zinc-limited media even when these transporters are deleted. Here, we show that Nissle utilizes the siderophore yersiniabactin as a zincophore, enabling Nissle to grow in zinc-limited media, to tolerate calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. We also show that yersiniabactin's affinity for iron or zinc changes in a pH-dependent manner, with increased relative zinc binding as the pH increases. Thus, our results indicate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to commensal and pathogenic Enterobacteriaceae.

Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages.

Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1ß (IL-1ß). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1ß signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B Streptococcus (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1ß release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1ß release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, Streptococcus pneumoniae, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating cis- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1ß secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1ß production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a SIGLEC5/14 fusion polymorphism, and we found increased IL-1ß expression in primary macrophages from SIGLEC14+/+ individuals compared to those with the SIGLEC14-/+ and SIGLEC14-/- genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation.

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera.

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.

Innate Immune Interactions between Bacillus anthracis and Host Neutrophils.

Bacillus anthracis, the causative agent of anthrax, has been a focus of study in host-pathogen dynamics since the nineteenth century. While the interaction between anthrax and host macrophages has been extensively modeled, comparatively little is known about the effect of anthrax on the immune function of neutrophils, a key frontline effector of innate immune defense. Here we showed that depletion of neutrophils significantly enhanced mortality in a systemic model of anthrax infection in mice. Ex vivo, we found that freshly isolated human neutrophils can rapidly kill anthrax, with specific inhibitor studies showing that phagocytosis and reactive oxygen species (ROS) generation contribute to this efficient bacterial clearance. Anthrax toxins, comprising lethal toxin (LT) and edema toxin (ET), are known to have major roles in B. anthracis macrophage resistance and systemic toxicity. Employing isogenic wild-type and mutant toxin-deficient B. anthracis strains, we show that despite previous studies that reported inhibition of neutrophil function by purified LT or ET, endogenous production of these toxins by live vegetative B. anthracis failed to alter key neutrophil functions. The lack of alteration in neutrophil function is accompanied by rapid killing of B. anthracis by neutrophils, regardless of the bacteria's expression of anthrax toxins. Lastly, our study demonstrates for the first time that anthrax induced neutrophil extracellular trap (NET) formation.

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Salmonella Mitigates Oxidative Stress and Thrives in the Inflamed Gut by Evading Calprotectin-Mediated Manganese Sequestration.

Neutrophils hinder bacterial growth by a variety of antimicrobial mechanisms, including the production of reactive oxygen species and the secretion of proteins that sequester nutrients essential to microbes. A major player in this process is calprotectin, a host protein that exerts antimicrobial activity by chelating zinc and manganese. Here we show that the intestinal pathogen Salmonella enterica serovar Typhimurium uses specialized metal transporters to evade calprotectin sequestration of manganese, allowing the bacteria to outcompete commensals and thrive in the inflamed gut. The pathogen's ability to acquire manganese in turn promotes function of SodA and KatN, enzymes that use the metal as a cofactor to detoxify reactive oxygen species. This manganese-dependent SodA activity allows the bacteria to evade neutrophil killing mediated by calprotectin and reactive oxygen species. Thus, manganese acquisition enables S. Typhimurium to overcome host antimicrobial defenses and support its competitive growth in the intestine.

The ZupT transporter plays an important role in zinc homeostasis and contributes to Salmonella enterica virulence.

Zinc is an essential metal for cellular homeostasis and function in both eukaryotes and prokaryotes. To acquire this essential nutrient, bacteria employ transporters characterized by different affinity for the metal. Several studies have investigated the role of the high affinity transporter ZnuABC in the bacterial response to zinc shortage, showing that this transporter has a key role in adapting bacteria to zinc starvation. In contrast, the role of the low affinity zinc importer ZupT has been the subject of limited investigations. Here we show that a Salmonella strain lacking ZupT is impaired in its ability to grow in metal devoid environments and that a znuABC zupT strain exhibits a severe growth defect in zinc devoid media, is hypersensitive to oxidative stress and contains reduced levels of intracellular free zinc. Moreover, we show that ZupT also plays a role in the ability of S. Typhimurium to colonize the host tissues. During systemic infections, the single zupT mutant strain was attenuated only in Nramp1(+/+) mice, but competition experiments between znuABC and znuABC zupT mutants revealed that ZupT contributes to metal uptake in vivo independently of the presence of a functional Nramp1 transporter. Altogether, the here reported results show that ZupT plays an important role in Salmonella zinc homeostasis, being involved in metal import both in vitro and in infected animals.

Probiotic bacteria reduce salmonella typhimurium intestinal colonization by competing for iron.

Host inflammation alters the availability of nutrients such as iron to limit microbial growth. However, Salmonella enterica serovar Typhimurium thrives in the inflamed gut by scavenging for iron with siderophores. By administering Escherichia coli strain Nissle 1917, which assimilates iron by similar mechanisms, we show that this nonpathogenic bacterium can outcompete and reduce S. Typhimurium colonization in mouse models of acute colitis and chronic persistent infection. This probiotic activity depends on E. coli Nissle iron acquisition, given that mutants deficient in iron uptake colonize the intestine but do not reduce S. Typhimurium colonization. Additionally, the ability of E. coli Nissle to overcome iron restriction by the host protein lipocalin 2, which counteracts some siderophores, is essential, given that S. Typhimurium is unaffected by E. coli Nissle in lipocalin 2-deficient mice. Thus, iron availability impacts S. Typhimurium growth, and E. coli Nissle reduces S. Typhimurium intestinal colonization by competing for this limiting nutrient.

Circadian clock regulates the host response to Salmonella.

Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut.

Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.

Linking lipid metabolism to the innate immune response in macrophages through sterol regulatory element binding protein-1a.

We show that mice with a targeted deficiency in the gene encoding the lipogenic transcription factor SREBP-1a are resistant to endotoxic shock and systemic inflammatory response syndrome induced by cecal ligation and puncture (CLP). When macrophages from the mutant mice were challenged with bacterial lipopolysaccharide, they failed to activate lipogenesis as well as two hallmark inflammasome functions, activation of caspase-1 and secretion of IL-1ß. We show that SREBP-1a activates not only genes required for lipogenesis in macrophages but also the gene encoding Nlrp1a, which is a core inflammasome component. Thus, SREBP-1a links lipid metabolism to the innate immune response, which supports our hypothesis that SREBPs evolved to regulate cellular reactions to external challenges that range from nutrient limitation and hypoxia to toxins and pathogens.

Th17 cytokines and host-pathogen interactions at the mucosa: dichotomies of help and harm.

The mucosal surfaces are often the first site of interaction between pathogenic microorganisms and the host. Activation of the mucosal immune response has the important function of containing an infection and preventing dissemination of pathogens to systemic sites (barrier function). Numerous lines of evidence suggest that the barrier function is orchestrated by a subset of cytokines (interleukin (IL-)17 and IL-22), which belong to the Th17 family. IL-17 and IL-22 induce expression of antimicrobial peptides and neutrophil chemoattractants at mucosal sites, and thus play an important role in controlling mucosal infections. However, there is increasing evidence that mucosal pathogens achieve greater colonization during inflammation because they are resistant to a subset of these antimicrobial responses. In this review we compare the antimicrobial responses elicited by Th17 cytokines during mucosal infections with four different pathogens: Klebsiella pneumoniae, Citrobacter rodentium, Candida albicans and Salmonella typhimurium. We will then discuss which responses may constitute the mucosal barrier, thus providing a benefit to the host, and which ones may promote the colonization of pathogens, thereby providing a benefit to the microbes.