Salmonella Typhimurium exploits host polyamines for assembly of the type 3 secretion machinery.

Bacterial pathogens utilize the factors of their hosts to infect them, but which factors they exploit remain poorly defined. Here, we show that a pathogenic Salmonella enterica serovar Typhimurium (STm) exploits host polyamines for the functional expression of virulence factors. An STm mutant strain lacking principal genes required for polyamine synthesis and transport exhibited impaired infectivity in mice. A polyamine uptake-impaired strain of STm was unable to inject effectors of the type 3 secretion system into host cells due to a failure of needle assembly. STm infection stimulated host polyamine production by increasing arginase expression. The decline in polyamine levels caused by difluoromethylornithine, which inhibits host polyamine production, attenuated STm colonization, whereas polyamine supplementation augmented STm pathogenesis. Our work reveals that host polyamines are a key factor promoting STm infection, and therefore a promising therapeutic target for bacterial infection.

The Salmonella Effector SspH2 Facilitates Spatially Selective Ubiquitination of NOD1 to Enhance Inflammatory Signaling.

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the so-called novel E3 ubiquitin ligase family, that interacts with and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling by targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture via the NF-κB pathway. Moreover, purified SspH2 and NOD1 directly interact, where NOD1 potentiates SspH2 E3 ubiquitin ligase activity. Mass spectrometry and mutational analyses identified four key lysine residues in NOD1 that are required for its enhanced activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on the receptor that appears to be targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin and uncovers a unique mechanism of spatially selective ubiquitination to enhance the activation of an archetypal NLR.

Quantitative Proteomics of the Intracellular Bacterial Pathogen Salmonella enterica Serovar Typhimurium.

Mass spectrometry-based proteomics provides a wealth of information about changes in protein production and abundance under diverse conditions, as well as mechanisms of regulation, signaling cascades, interaction partners, and communication patterns across biological systems. For profiling of intracellular pathogens, proteomic profiling can be performed in the absence of a host to singularly define the pathogenic proteome or during an infection-like setting to identify dual perspectives of infection. In this chapter, we present techniques to extract proteins from the human bacterial intracellular pathogen, Salmonella enterica serovar Typhimurium, in the presence of macrophages, an important innate immune cell in host defense. We outline sample preparation, including protein extraction, digestion, and purification, as well as mass spectrometry measurements and bioinformatics analysis. The data generated from our dual perspective profiling approach provides new insight into pathogen and host protein modulation under infection-like conditions.

Intestinal lysozyme engagement of Salmonella Typhimurium stimulates the release of barrier-impairing InvE and Lpp1.

Lysozyme is a ß-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium and then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella in vitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.

Commensal E. coli limits Salmonella gut invasion during inflammation by producing toxin-bound siderophores in a tonB-dependent manner.

The gastrointestinal tract is densely colonized by a polymicrobial community known as the microbiota which serves as primary line of defence against pathogen invasion. The microbiota can limit gut-luminal pathogen growth at different stages of infection. This can be traced to specific commensal strains exhibiting direct or indirect protective functions. Although these mechanisms hold the potential to develop new approaches to combat enteric pathogens, they remain far from being completely described. In this study, we investigated how a mouse commensal Escherichia coli can outcompete Salmonella enterica serovar Typhimurium (S. Tm). Using a salmonellosis mouse model, we found that the commensal E. coli 8178 strain relies on a trojan horse trap strategy to limit S. Tm expansion in the inflamed gut. Combining mutants and reporter tools, we demonstrated that inflammation triggers the expression of the E. coli 8178 antimicrobial microcin H47 toxin which, when fused to salmochelin siderophores, can specifically alter S. Tm growth. This protective function was compromised upon disruption of the E. coli 8178 tonB-dependent catecholate siderophore uptake system, highlighting a previously unappreciated crosstalk between iron intake and microcin H47 activity. By identifying the genetic determinants mediating S. Tm competition, our work not only provides a better mechanistic understanding of the protective function displayed by members of the gut microbiota but also further expands the general contribution of microcins in bacterial antagonistic relationships. Ultimately, such insights can open new avenues for developing microbiota-based approaches to better control intestinal infections.

Alleviating effect of vagus nerve cutting in Salmonella-induced gut infections and anxiety-like behavior via enhancing microbiota-derived GABA.

The vagus nerve, a pivotal link within the gut-brain axis, plays a critical role in maintaining homeostasis and mediating communication between the gastrointestinal tract and the brain. It has been reported that gastrointestinal infection by Salmonella typhimurium (S. typhimurium) triggers gut inflammation and manifests as anxiety-like behaviors, yet the mechanistic involvement of the vagus nerve remains to be elucidated. In this study, we demonstrated that unilateral cervical vagotomy markedly attenuated anxiety-like behaviors induced by S. typhimurium SL1344 infection in C57BL/6 mice, as evidenced by the open field test and marble burying experiment. Furthermore, vagotomy significantly diminished neuronal activation within the nucleus of the solitary tract and amygdala, alongside mitigating aberrant glial cell activation in the hippocampus and amygdala. Additionally, vagotomy notably decreases serum endotoxin levels, counters the increase in splenic Salmonella concentration, and modulates the expression of inflammatory cytokines-including IL-6, IL-1ß, and TNF-α-in both the gastrointestinal tract and brain, with a concurrent reduction in IL-22 and CXCL1 expression. This intervention also fostered the enrichment of beneficial gut microbiota, including Alistipes and Lactobacillus species, and augmented the production of gamma-aminobutyric acid (GABA) in the gut. Administration of GABA replicated the vagotomy's beneficial effects on reducing gut inflammation and anxiety-like behavior in infected mice. However, blockade of GABA receptors with picrotoxin abrogated the vagotomy's protective effects against gut inflammation, without influencing its impact on anxiety-like behaviors. Collectively, these findings suggest that vagotomy exerts a protective effect against infection by promoting GABA synthesis in the colon and alleviating anxiety-like behavior. This study underscores the critical role of the vagus nerve in relaying signals of gut infection to the brain and posits that targeting the gut-brain axis may offer a novel and efficacious approach to preventing gastrointestinal infections and associated behavioral abnormalities.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Functional Divergence of the Paralog Salmonella Effector Proteins SopD and SopD2 and Their Contributions to Infection.

Salmonella enterica is a leading cause of bacterial food-borne illness in humans and is responsible for millions of cases annually. A critical strategy for the survival of this pathogen is the translocation of bacterial virulence factors termed effectors into host cells, which primarily function via protein-protein interactions with host proteins. The Salmonella genome encodes several paralogous effectors believed to have arisen from duplication events throughout the course of evolution. These paralogs can share structural similarities and enzymatic activities but have also demonstrated divergence in host cell targets or interaction partners and contributions to the intracellular lifecycle of Salmonella. The paralog effectors SopD and SopD2 share 63% amino acid sequence similarity and extensive structural homology yet have demonstrated divergence in secretion kinetics, intracellular localization, host targets, and roles in infection. SopD and SopD2 target host Rab GTPases, which represent critical regulators of intracellular trafficking that mediate diverse cellular functions. While SopD and SopD2 both manipulate Rab function, these paralogs display differences in Rab specificity, and the effectors have also evolved multiple mechanisms of action for GTPase manipulation. Here, we highlight this intriguing pair of paralog effectors in the context of host-pathogen interactions and discuss how this research has presented valuable insights into effector evolution.

Novel Synergistic Probiotic Intervention: Transcriptomic and Metabolomic Analysis Reveals Ameliorative Effects on Immunity, Gut Barrier, and Metabolism of Mice during Salmonella typhimurium Infection.

Salmonella typhimurium (S. typhimurium), a prevalent cause of foodborne infection, induces significant changes in the host transcriptome and metabolome. The lack of therapeutics with minimal or no side effects prompts the scientific community to explore alternative therapies. This study investigates the therapeutic potential of a probiotic mixture comprising Lactobacillus acidophilus (L. acidophilus 1.3251) and Lactobacillus plantarum (L. plantarum 9513) against S. typhimurium, utilizing transcriptome and metabolomic analyses, a novel approach that has not been previously documented. Twenty-four SPF-BALB/c mice were divided into four groups: control negative group (CNG); positive control group (CPG); probiotic-supplemented non-challenged group (LAPG); and probiotic-supplemented Salmonella-challenged group (LAPST). An RNA-sequencing analysis of small intestinal (ileum) tissue revealed 2907 upregulated and 394 downregulated DEGs in the LAPST vs. CPG group. A functional analysis of DEGs highlighted their significantly altered gene ontology (GO) terms related to metabolism, gut integrity, cellular development, and immunity (p ≤ 0.05). The KEGG analysis showed that differentially expressed genes (DEGs) in the LAPST group were primarily involved in pathways related to gut integrity, immunity, and metabolism, such as MAPK, PI3K-Akt, AMPK, the tryptophan metabolism, the glycine, serine, and threonine metabolism, ECM-receptor interaction, and others. Additionally, the fecal metabolic analysis identified 1215 upregulated and 305 downregulated metabolites in the LAPST vs. CPG group, implying their involvement in KEGG pathways including bile secretion, propanoate metabolism, arginine and proline metabolism, amino acid biosynthesis, and protein digestion and absorption, which are vital for maintaining barrier integrity, immunity, and metabolism. In conclusion, these findings suggest that the administration of a probiotic mixture improves immunity, maintains gut homeostasis and barrier integrity, and enhances metabolism in Salmonella infection.

Salmonella Typhimurium employs spermidine to exert protection against ROS-mediated cytotoxicity and rewires host polyamine metabolism to ameliorate its survival in macrophages.

Salmonella infection entails a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by macrophages, where the bacteria encounter multiple stresses, to which it employs relevant countermeasures. Our study shows that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which prevents the oxidation of proteins in E. coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro and reduced organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. We found that Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which addresses the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D, L-α-difluoromethylornithine (DFMO), reduces Salmonella colonisation and tissue damage in the mouse model of infection while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a bacterial strategy in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

Salmonella-driven intestinal edema in mice is characterized by tensed fibronectin fibers.

Intestinal edema is a common manifestation of numerous gastrointestinal diseases and is characterized by the accumulation of fluid in the interstitial space of the intestinal wall. Technical advances in laser capture microdissection and low-biomass proteomics now allow us to specifically characterize the intestinal edema proteome. Using advanced proteomics, we identify peptides derived from antimicrobial factors with high signal intensity, but also highlight major contributions from the blood clotting system, extracellular matrix (ECM) and protease-protease inhibitor networks. The ECM is a complex fibrillar network of macromolecules that provides structural and mechanical support to the intestinal tissue. One abundant component of the ECM observed in Salmonella-driven intestinal edema is the glycoprotein fibronectin, recognized for its structure-function interplay regulated by mechanical forces. Using mechanosensitive staining of fibronectin fibers reveals that they are tensed in the edema, despite the high abundance of proteases able to cleave fibronectin. In contrast, fibronectin fibers increasingly relax in other cecal tissue areas as the infection progresses. Co-staining for fibrin(ogen) indicates the formation of a provisional matrix in the edema, similar to what is observed in response to skin injury, while collagen staining reveals a sparse and disrupted collagen fiber network. These observations plus the absence of low tensional fibronectin fibers and the additional finding of a high number of protease inhibitors in the edema proteome could indicate a critical role of stretched fibronectin fibers in maintaining tissue integrity in the severely inflamed cecum. Understanding these processes may also provide valuable functional diagnostic markers of intestinal disease progression in the future.

The NLRP3 molecule is responsible for mediating the pyroptosis of intestinal mucosa cells and plays a crucial role in salmonellosis enteritis in chicks.

Salmonella enteritis in poultry can result in reduced immune function, decreased growth rate, and increased mortality. Many farm salmonella strains have developed severe drug resistance and are less susceptible to multiple antibiotics. In the post-antibiotic era, it is of great significance to identify the mechanism of salmonella-induced enteritis in chicks to protect their health and ensure food safety. This article will elucidate the activation mechanism of NOD-like receptor protein 3 (NLRP3) inflammasomes in Salmonella enteritis and review the research on interventions targeting NLRP3 inflammasomes.

Salmonella manipulates the host to drive pathogenicity via induction of interleukin 1ß.

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1ß (IL-1ß). However, the role of IL-1ß in intestinal defense against Salmonella remains unclear. Here, we show that IL-1ß production is detrimental during Salmonella infection. Mice lacking IL-1ß (IL-1ß -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1ß -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1ß induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1ß loss prevented mortality in Salmonella-infected IL-1ß -/- mice. Finally, we found that IL-1ß expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1ß signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1ß signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis.

HilE represses the activity of the Salmonella virulence regulator HilD via a mechanism distinct from that of intestinal long-chain fatty acids.

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long-chain fatty acids, which are highly abundant throughout the host intestine, directly bind to and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerization, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. Long-chain fatty acids bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerization interface. Our results highlight two distinct, mutually exclusive mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

Lactate promotes Salmonella intracellular replication and systemic infection via driving macrophage M2 polarization.

IMPORTANCE: The important enteropathogen Salmonella can cause lethal systemic infection via survival and replication in host macrophages. Lactate represents an abundant intracellular metabolite during bacterial infection, which can also induce macrophage M2 polarization. In this study, we found that macrophage-derived lactate promotes the intracellular replication and systemic infection of Salmonella. During Salmonella infection, lactate via the Salmonella type III secretion system effector SteE promotes macrophage M2 polarization, and the induction of macrophage M2 polarization by lactate is responsible for lactate-mediated Salmonella growth promotion. This study highlights the complex interactions between Salmonella and macrophages and provides an additional perspective on host-pathogen crosstalk at the metabolic interface.

RhoB promotes Salmonella survival by regulating autophagy.

Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and -E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella, RhoB is specifically required for Salmonella-induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella-induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB.

Human neutrophil IL1ß directs intestinal epithelial cell extrusion during Salmonella infection.

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.

Salmonella enterica Infections Are Disrupted by Two Small Molecules That Accumulate within Phagosomes and Differentially Damage Bacterial Inner Membranes.

Gram-negative bacteria have a robust cell envelope that excludes or expels many antimicrobial agents. However, during infection, host soluble innate immune factors permeabilize the bacterial outer membrane. We identified two small molecules that exploit outer membrane damage to access the bacterial cell. In standard microbiological media, neither compound inhibited bacterial growth nor permeabilized bacterial outer membranes. In contrast, at micromolar concentrations, JAV1 and JAV2 enabled the killing of an intracellular human pathogen, Salmonella enterica serovar Typhimurium. S. Typhimurium is a Gram-negative bacterium that resides within phagosomes of cells from the monocyte lineage. Under broth conditions that destabilized the lipopolysaccharide layer, JAV2 permeabilized the bacterial inner membrane and was rapidly bactericidal. In contrast, JAV1 activity was more subtle: JAV1 increased membrane fluidity, altered reduction potential, and required more time than JAV2 to disrupt the inner membrane barrier and kill bacteria. Both compounds interacted with glycerophospholipids from Escherichia coli total lipid extract-based liposomes. JAV1 preferentially interacted with cardiolipin and partially relied on cardiolipin production for activity, whereas JAV2 generally interacted with lipids and had modest affinity for phosphatidylglycerol. In mammalian cells, neither compound significantly altered mitochondrial membrane potential at concentrations that killed S. Typhimurium. Instead, JAV1 and JAV2 became trapped within acidic compartments, including macrophage phagosomes. Both compounds improved survival of S. Typhimurium-infected Galleria mellonella larvae. Together, these data demonstrate that JAV1 and JAV2 disrupt bacterial inner membranes by distinct mechanisms and highlight how small, lipophilic, amine-substituted molecules can exploit host soluble innate immunity to facilitate the killing of intravesicular pathogens. IMPORTANCE Innovative strategies for developing new antimicrobials are needed. Combining our knowledge of host-pathogen interactions and relevant drug characteristics has the potential to reveal new approaches to treating infection. We identified two compounds with antibacterial activity specific to infection and with limited host cell toxicity. These compounds appeared to exploit host innate immunity to access the bacterium and differentially damage the bacterial inner membrane. Further, both compounds accumulated within Salmonella-containing and other acidic vesicles, a process known as lysosomal trapping, which protects the host and harms the pathogen. The compounds also increased host survival in an insect infection model. This work highlights the ability of host innate immunity to enable small molecules to act as antibiotics and demonstrates the feasibility of antimicrobial targeting of the inner membrane. Additionally, this study features the potential use of lysosomal trapping to enhance the activities of compounds against intravesicular pathogens.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B. subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B. subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B. subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B. subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B. subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination.

The intestinal mucosa exists in a state of "physiologic hypoxia," where oxygen tensions are markedly lower than those in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain homeostasis in this austere environment through oxygen-sensitive transcription factors, including hypoxia-inducible factors (HIFs). Using an unbiased chromatin immunoprecipitation (ChIP) screen for HIF-1 targets, we identify autophagy as a major pathway induced by hypoxia in IECs. One important function of autophagy is to defend against intracellular pathogens, termed "xenophagy." Analysis reveals that HIF is a central regulator of autophagy and that in vitro infection of IECs with Salmonella Typhimurium results in induction of HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Work in vivo demonstrates that IEC-specific deletion of HIF compromises xenophagy and exacerbates bacterial dissemination. These results reveal that the interaction between hypoxia, HIF, and xenophagy is an essential innate immune component for the control of intracellular pathogens.