FOXO inhibition rescues α-defensin expression in human intestinal organoids.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

Cell and Tissue Tropism of Brucella spp.

Brucella spp. are facultatively intracellular bacteria that can infect, survive, and multiply in various host cell types in vivo and/or in vitro. The genus Brucella has markedly expanded in recent years with the identification of novel species and hosts, which has revealed additional information about the cell and tissue tropism of these pathogens. Classically, Brucella spp. are considered to have tropism for organs that contain large populations of phagocytes such as lymph nodes, spleen, and liver, as well as for organs of the genital system, including the uterus, epididymis, testis, and placenta. However, experimental infections of several different cultured cell types indicate that Brucella may actually have a broader cell tropism than previously thought. Indeed, recent studies indicate that certain Brucella species in particular hosts may display a pantropic distribution in vivo. This review discusses the available knowledge on cell and tissue tropism of Brucella spp. in natural infections of various host species, as well as in experimental animal models and cultured cells.

Tumor Necrosis Factor Alpha Contributes to Inflammatory Pathology in the Placenta during Brucella abortus Infection.

Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites, Brucella abortus evades innate immunity, which results in low-level inflammation and chronic infection of phagocytes. In contrast, the host response in the placenta during infection is characterized by severe inflammation and extensive extracellular replication of B. abortus. Despite the importance of reproductive disease caused by Brucella infection, our knowledge of the mechanisms involved in placental inflammation and abortion is limited. To understand the immune responses specifically driving placental pathology, we modeled placental B. abortus infection in pregnant mice. B. abortus infection caused an increase in the production of tumor necrosis factor alpha (TNF-α), specifically in the placenta. We found that placental expression levels of Tnfa and circulating TNF-α were dependent on the induction of endoplasmic reticulum stress and the B. abortus type IV secretion system (T4SS) effector protein VceC. Blockade of TNF-α reduced placental inflammation and improved fetal viability in mice. This work sheds light on a tissue-specific response of the placenta to B. abortus infection that may be important for bacterial transmission via abortion in the natural host species.

Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin.

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

NOD1 and NOD2: Beyond Peptidoglycan Sensing.

NOD1 and NOD2 are pattern recognition receptors of the innate immune system with well-established roles in sensing fragments of bacterial peptidoglycan. In addition to their role as microbial sensors, recent evidence indicates that nucleotide-binding oligomerization domains (NODs) can also recognize a broader array of danger signals. Indeed, recent work has expanded the roles of NOD1 and NOD2 to encompass not only sensing of infections with viruses and parasites but also perceiving perturbations of cellular processes such as regulation of the actin cytoskeleton and maintenance of endoplasmic reticulum homeostasis. This review will comment on recent progress and point out emerging questions in these areas.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

T cell expression of IL-18R and DR3 is essential for non-cognate stimulation of Th1 cells and optimal clearance of intracellular bacteria.

Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.

Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1.

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Colonization resistance: The deconvolution of a complex trait.

Carbapenemase-producing Enterobacteriaceae are an emerging threat to hospitals worldwide, and antibiotic exposure is a risk factor for developing fecal carriage that may lead to nosocomial infection. Here, we review how antibiotics reduce colonization resistance against Enterobacteriaceae to pinpoint possible control points for curbing their spread. Recent work identifies host-derived respiratory electron acceptors as a critical resource driving a post-antibiotic expansion of Enterobacteriaceae within the large bowel. By providing a conceptual framework for colonization resistance against Enterobacteriaceae, these mechanistic insights point to the metabolism of epithelial cells as a possible target for intervention strategies.

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.

Utilization of Host Polyamines in Alternatively Activated Macrophages Promotes Chronic Infection by Brucella abortus.

Treatment of intracellular bacterial pathogens with antibiotic therapy often requires a long course of multiple drugs. A barrier to developing strategies that enhance antibiotic efficacy against these pathogens is our poor understanding of the intracellular nutritional environment that maintains bacterial persistence. The intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs); however, knowledge of the metabolic adaptations promoting exploitation of this niche is limited. Here we show that one mechanism promoting enhanced survival in AAMs is a shift in macrophage arginine utilization from production of nitric oxide (NO) to biosynthesis of polyamines, induced by interleukin 4 (IL-4)/IL-13 treatment. Production of polyamines by infected AAMs promoted both intracellular survival of B. abortus and chronic infection in mice, as inhibition of macrophage polyamine synthesis or inactivation of the putative putrescine transporter encoded by potIHGF reduced both intracellular survival in AAMs and persistence in mice. These results demonstrate that increased intracellular availability of polyamines induced by arginase-1 expression in IL-4/IL-13-induced AAMs promotes chronic persistence of B. abortus within this niche and suggest that targeting of this pathway may aid in eradicating chronic infection.

Type IV Effector Secretion and Subversion of Host Functions by Bartonella and Brucella Species.

Bartonella and Brucella species comprise closely related genera of the order Rhizobiales within the class α-proteobacteria. Both groups of bacteria are mammalian pathogens with a facultative intracellular lifestyle and are capable of causing chronic infections, but members of each genus have evolved broadly different infection and transmission strategies. While Brucella spp. transmit in general via the reproductive tract in their natural hosts, the Bartonella spp. have evolved to transmit via arthropod vectors. However, a shared feature of both groups of pathogens is their reliance on type IV secretion systems (T4SSs) to interact with cells in their mammalian hosts. The genomes of Bartonella spp. encode three types of T4SS, Trw, Vbh/TraG, and VirB/VirD4, whereas those of Brucella spp. uniformly contain a single T4SS of the VirB type. The VirB systems of Bartonella and Brucella are associated with distinct groups of effector proteins that collectively mediate interactions with host cells. This chapter discusses recent findings on the role of T4SS in the biology of Bartonella spp. and Brucella spp. with emphasis on effector repertoires, on recent advances in our understanding of their evolution, how individual effectors function at the molecular level, and on the consequences of these interactions for cellular and immune responses in the host.

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.

Iron acquisition pathways and colonization of the inflamed intestine by Salmonella enterica serovar Typhimurium.

Salmonella enterica serotype Typhimurium is able to expand in the lumen of the inflamed intestine through mechanisms that have not been fully resolved. Here we utilized streptomycin-pretreated mice and dextran sodium sulfate (DSS)-treated mice to investigate how pathways for S. Typhimurium iron acquisition contribute to pathogen expansion in the inflamed intestine. Competitive infection with an iron uptake-proficient S. Typhimurium strain and mutant strains lacking tonB feoB, feoB, tonB or iroN in streptomycin pretreated mice demonstrated that ferric iron uptake requiring IroN and TonB conferred a fitness advantage during growth in the inflamed intestine. However, the fitness advantage conferred by ferrous iron uptake mechanisms was independent of inflammation and was only apparent in models where the normal microbiota composition had been disrupted by antibiotic treatment.

Interactions of the human pathogenic Brucella species with their hosts.

Brucellosis is a zoonotic infection caused primarily by the bacterial pathogens Brucella melitensis and B. abortus. It is acquired by consumption of unpasteurized dairy products or by contact with infected animals. Globally, it is one of the most widespread zoonoses, with 500,000 new cases reported each year. In endemic areas, Brucella infections represent a serious public health problem that results in significant morbidity and economic losses. An important feature of the disease is persistent bacterial colonization of the reticuloendothelial system. In this review we discuss recent insights into mechanisms of intracellular survival and immune evasion that contribute to systemic persistence by the pathogenic Brucella species.

Gut inflammation provides a respiratory electron acceptor for Salmonella.

Salmonella enterica serotype Typhimurium (S. Typhimurium) causes acute gut inflammation by using its virulence factors to invade the intestinal epithelium and survive in mucosal macrophages. The inflammatory response enhances the transmission success of S. Typhimurium by promoting its outgrowth in the gut lumen through unknown mechanisms. Here we show that reactive oxygen species generated during inflammation react with endogenous, luminal sulphur compounds (thiosulphate) to form a new respiratory electron acceptor, tetrathionate. The genes conferring the ability to use tetrathionate as an electron acceptor produce a growth advantage for S. Typhimurium over the competing microbiota in the lumen of the inflamed gut. We conclude that S. Typhimurium virulence factors induce host-driven production of a new electron acceptor that allows the pathogen to use respiration to compete with fermenting gut microbes. Thus the ability to trigger intestinal inflammation is crucial for the biology of this diarrhoeal pathogen.

The flagellar regulator TviA reduces pyroptosis by Salmonella enterica serovar Typhi.

To discern virulent from innocuous microbes, the innate immune system senses events associated with bacterial access to immunoprivileged sites such as the host cell cytosol. One such pathway is triggered by the cytosolic delivery of flagellin, the major subunit of the flagellum, by bacterial secretion systems. This leads to inflammasome activation and subsequent proinflammatory cell death (pyroptosis) of the infected phagocyte. In this study, we demonstrate that the causative agent of typhoid fever, Salmonella enterica serovar Typhi, can partially subvert this critical innate immune recognition event. The transcriptional regulator TviA, which is absent from Salmonella serovars associated with human gastroenteritis, repressed the expression of flagellin during infection of human macrophage-like (THP-1) cells. This mechanism allowed S. Typhi to dampen inflammasome activation, leading to reduced interleukin-1ß (IL-1ß) secretion and diminished cell death. Likewise, the introduction of the tviA gene in nontyphoidal Salmonella enterica serovar Typhimurium reduced flagellin-induced pyroptosis. These data suggest that gene regulation of virulence factors enables S. Typhi to evade innate immune recognition by concealing a pathogen-induced process from being sensed by the inflammasome.

Phospholipase A1 modulates the cell envelope phospholipid content of Brucella melitensis, contributing to polymyxin resistance and pathogenicity.

A subset of bacterial pathogens, including the zoonotic Brucella species, are highly resistant against polymyxin antibiotics. Bacterial polymyxin resistance has been attributed primarily to the modification of lipopolysaccharide; however, it is unknown what additional mechanisms mediate high-level resistance against this class of drugs. This work identified a role for the Brucella melitensis gene bveA (BMEII0681), encoding a predicted esterase, in the resistance of B. melitensis to polymyxin B. Characterization of the enzymatic activity of BveA demonstrated that it is a phospholipase A1 with specificity for phosphatidylethanolamine (PE). Further, lipidomic analysis of B. melitensis revealed an excess of PE lipids in the bacterial membranes isolated from the bveA mutant. These results suggest that by lowering the PE content of the cell envelope, BveA increases the resistance of B. melitensis to polymyxin B. BveA was required for survival and replication of B. melitensis in macrophages and for persistent infection in mice. BveA family esterases are encoded in the genomes of the alphaproteobacterial species that coexist with the polymyxin-producing bacteria in the rhizosphere, suggesting that maintenance of a low PE content in the bacterial cell envelope may be a shared persistence strategy for association with plant and mammalian hosts.