Incremental increases in physiological fluid shear progressively alter pathogenic phenotypes and gene expression in multidrug resistant Salmonella.

The ability of bacteria to sense and respond to mechanical forces has important implications for pathogens during infection, as they experience wide fluid shear fluctuations in the host. However, little is known about how mechanical forces encountered in the infected host drive microbial pathogenesis. Herein, we combined mathematical modeling with hydrodynamic bacterial culture to profile transcriptomic and pathogenesis-related phenotypes of multidrug resistant S. Typhimurium (ST313 D23580) under different fluid shear conditions relevant to its transition from the intestinal tract to the bloodstream. We report that D23580 exhibited incremental changes in transcriptomic profiles that correlated with its pathogenic phenotypes in response to these progressive increases in fluid shear. This is the first demonstration that incremental changes in fluid shear forces alter stress responses and gene expression in any ST313 strain and offers mechanistic insight into how forces encountered by bacteria during infection might impact their disease-causing ability in unexpected ways.

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.

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.

Microevolution of Salmonella 4,[5],12:i:- derived from Salmonella enterica serovar Typhimurium through complicated transpositions.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-), derived from S. Typhimurium, has become the dominant serotype causing human salmonellosis. In this study, we define the genetic mechanism of the generation of Salmonella 4,[5],12:i:- from S. Typhimurium through complicated transpositions and demonstrate that Salmonella 4,[5],12:i:- displays more efficient colonization and survival abilities in mice than its parent S. Typhimurium strain. We identified intermediate strains carrying both resistance regions (RRs) and the fljAB operon for the generation of Salmonella 4,[5],12:i:-. The insertion of RR3 into the chromosomal hin-iroB site of S. Typhimurium produced RR3-S. Typhimurium as a primary intermediate. Salmonella 4,[5],12:i:- was then produced by replacing the fljAB operon and/or its flanking sequences through intramolecular transpositions mediated by IS26 and/or IS1R elements in RR3-S. Typhimurium, which was further confirmed both in vitro and in vivo. Overall, we demonstrate the molecular mechanism underlying the origin, generation, and advantage of RRs-Salmonella 4,[5],12:i:- from S. Typhimurium.

Phenotypic Characterization and Comparative Genomic Analysis of Novel Salmonella Bacteriophages Isolated from a Tropical Rainforest.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

Bidirectional regulation between AP-1 and SUMOylation pathway genes modulates inflammatory signaling during Salmonella infection.

Post-translational modifications (PTMs), such as SUMOylation, are known to modulate fundamental processes of a cell. Infectious agents such as Salmonella Typhimurium (STm), which causes gastroenteritis, utilize the PTM mechanism SUMOylation to hijack the host cell. STm suppresses host SUMO pathway genes UBC9 (also known as UBE2I) and PIAS1 to perturb SUMOylation for an efficient infection. In the present study, the regulation of SUMO pathway genes during STm infection was investigated. A direct binding of c-Fos (encoded by FOS), a component of activator protein-1 (AP-1), to promoters of both UBC9 and PIAS1 was observed. Experimental perturbation of c-Fos led to changes in the expression of both UBC9 and PIAS1. STm infection of fibroblasts with SUMOylation-deficient c-Fos (c-FOS-KOSUMO-def-FOS) resulted in uncontrolled activation of target genes, leading to massive immune activation. Infection of c-FOS-KOSUMO-def-FOS cells favored STm replication, indicating misdirected immune mechanisms. Finally, chromatin immunoprecipitation assays confirmed a context-dependent differential binding and release of AP-1 to and from target genes due to its phosphorylation and SUMOylation, respectively. Overall, our data point towards the existence of a bidirectional cross-talk between c-Fos and the SUMO pathway and highlight their importance in AP-1 function in STm infection and beyond. This article has an associated First Person interview with the first author of the paper.

Genome-wide characterization of Salmonella Typhimurium genes required for the fitness under iron restriction.

BACKGROUND: Iron is a crucial element for bacterial survival and virulence. During Salmonella infection, the host utilizes a variety of mechanisms to starve the pathogen from iron. However, Salmonella activates distinctive defense mechanisms to acquire iron and survive in iron-restricted host environments. Yet, the comprehensive set of the conditionally essential genes that underpin Salmonella survival under iron-restricted niches has not been fully explored. RESULTS: Here, we employed transposon sequencing (Tn-seq) method for high-resolution elucidation of the genes in Salmonella Typhimurium (S. Typhimurium) 14028S strain required for the growth under the in vitro conditions with four different levels of iron restriction achieved by iron chelator 2,2'-dipyridyl (Dip): mild (100 and 150 µM), moderate (250 µM) and severe iron restriction (400 µM). We found that the fitness of the mutants reduced significantly for 28 genes, suggesting the importance of these genes for the growth under iron restriction. These genes include sufABCDSE, iron transport fepD, siderophore tonB, sigma factor E ropE, phosphate transport pstAB, and zinc exporter zntA. The siderophore gene tonB was required in mild and moderate iron-restricted conditions, but it became dispensable in severe iron-restricted conditions. Remarkably, rpoE was required in moderate and severe iron restrictions, leading to complete attenuation of the mutant under these conditions. We also identified 30 genes for which the deletion of the genes resulted in increased fitness under iron-restricted conditions. CONCLUSIONS: The findings broaden our knowledge of how S. Typhimurium survives in iron-deficient environments, which could be utilized for the development of new therapeutic strategies targeting the pathways vital for iron metabolism, trafficking, and scavenging.

Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross.

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection.

Stabilization but No Functional Influence of HIF-1α Expression in the Intestinal Epithelium during Salmonella Typhimurium Infection.

Hypoxia-inducible transcription factor 1 (HIF-1) has been shown to enhance microbial killing and ameliorate the course of bacterial infections. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the gastrointestinal tract remains largely elusive. With the help of a publicly available gene expression data set, we inferred significant activation of HIF-1 after oral infection of mice with Salmonella enterica serovar Typhimurium. Immunohistochemistry and Western blot analyses confirmed marked HIF-1α protein stabilization, especially in the intestinal epithelium. This prompted us to analyze conditional Hif1a-deficient mice to examine cell type-specific functions of HIF-1 in this model. Our results demonstrate enhanced noncanonical induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells did not impact inflammatory gene expression, bacterial spread, or disease outcomes. In contrast, Hif1a deletion in myeloid cells enhanced intestinal Cxcl2 expression and reduced the cecal Salmonella load. In vitro, HIF-1α-deficient macrophages showed overall impaired transcription of mRNA encoding proinflammatory factors; however, the intracellular survival of Salmonella was not impacted by HIF-1α deficiency.

Isolation and Sequencing of the First Case of Symptomatic MDR Salmonella enterica Subsp. Enterica ST40 in Human in Italy, July 2020.

Strains of drug-resistant nontyphoidal Salmonella spp. are emerging in livestock worldwide. We describe the first case of symptomatic multidrug-resistant (MDR) Salmonella enterica subsp. enterica in human and the genetic mechanisms at the basis of its antibiotic resistance. To control outbreaks, rapid identification and sequencing are necessary. Proactive research and notification are needed to evaluate the routes of transmission from livestock to humans and risk-management strategies of MDR Salmonella strains.

The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses.

Host engulfment protein ELMO1 generates intestinal inflammation following internalization of enteric bacteria. In Shigella, bacterial effector IpgB1 interacts with ELMO1 and promotes bacterial invasion. IpgB1 belongs to the WxxxE effector family, a motif found in several effectors of enteric pathogens. Here, we have studied the role of WxxxE effectors, with emphasis on Salmonella SifA and whether it interacts with ELMO1 to regulate inflammation. In-silico-analysis of WxxxE effectors was performed using BLAST search and Clustal W program. The interaction of ELMO1 with SifA was assessed by GST pulldown assay and co-immunoprecipitation. ELMO1 knockout mice, and ELMO1-depleted murine macrophage J774 cell lines were challenged with WT and SifA mutant Salmonella. Bacterial effectors containing the WxxxE motif were transfected in WT and ELMO1-depleted J774 cells to assess the inflammatory cytokines. ELMO1 generates differential pro-inflammatory cytokines between pathogenic and nonpathogenic bacteria. WxxxE motif is present in pathogens and in the TIR domain of host proteins. The C-terminal part of ELMO1 interacts with SifA where WxxxE motif is important for interaction. ELMO1-SifA interaction affects bacterial colonization, dissemination, and inflammatory cytokines in vivo. Moreover, ELMO1-SifA interaction increases TNF-α and IL-6 production from the macrophage cell line and is associated with enhanced Rac1 activity. ELMO1 also interacts with WxxxE effectors IpgB1, IpgB2, and Map and induces inflammation after challenge with microbes or microbial ligands. ELMO1 generates a differential response through interaction with the WxxxE motif, which is absent in commensals. ELMO1-WxxxE interaction plays a role in bacterial pathogenesis and induction of inflammatory response.

Activation of epigenetic regulator KDM6B by Salmonella Typhimurium enables chronic infections.

Non-typhoidal Salmonella (NTS) infections result in self limiting gastroenteritis except in rare cases wherein manifestations of chronic infections can occur. Strategies employed by Salmonella to thrive in hostile environments of host during chronic infections are complex and multifaceted. In chronic state, a coordinated action of bacterial effectors allows reprogramming of macrophages to M2 subtype and thereby creating a permissible replicative niche. The mechanistic details of these processes are not fully known. In the current study we identified, histone H3-lysine 27 trimethylation (H3K27me3)-specific demethylase, KDM6B to be upregulated in both cell culture and in murine model of Salmonella infection. KDM6B recruitment upon infection exhibited an associated loss of overall H3K27me3 in host cells and was Salmonella SPI1 effectors coordinated. ChIP-qRT-PCR array analysis revealed several new gene promoter targets of KDM6B demethylase activity including PPARδ, a crucial regulator of fatty acid oxidation pathway and Salmonella-persistent infections. Furthermore, pharmacological inhibition of KDM6B demethylase activity with GSKJ4 in chronic Salmonella infection mice model led to a significant reduction in pathogen load and M2 macrophage polarization in peripheral lymphoid organs. The following work thus reveals Salmonella effector-mediated epigenetic reprogramming of macrophages responsible for its long-term survival and chronic carriage.

A 3,000-year-old, basal S. enterica lineage from Bronze Age Xinjiang suggests spread along the Proto-Silk Road.

Salmonella enterica (S. enterica) has infected humans for a long time, but its evolutionary history and geographic spread across Eurasia is still poorly understood. Here, we screened for pathogen DNA in 14 ancient individuals from the Bronze Age Quanergou cemetery (XBQ), Xinjiang, China. In 6 individuals we detected S. enterica. We reconstructed S. enterica genomes from those individuals, which form a previously undetected phylogenetic branch basal to Paratyphi C, Typhisuis and Choleraesuis-the so-called Para C lineage. Based on pseudogene frequency, our analysis suggests that the ancient S. enterica strains were not host adapted. One genome, however, harbors the Salmonella pathogenicity island 7 (SPI-7), which is thought to be involved in (para)typhoid disease in humans. This offers first evidence that SPI-7 was acquired prior to the emergence of human-adapted Paratyphi C around 1,000 years ago. Altogether, our results show that Salmonella enterica infected humans in Eastern Eurasia at least 3,000 years ago, and provide the first ancient DNA evidence for the spread of a pathogen along the Proto-Silk Road.

Salmonella Typhimurium ST313 isolated in Brazil revealed to be more invasive and inflammatory in murine colon compared to ST19 strains.

Salmonella Typhimurium (ST313) has caused an epidemic of invasive disease in sub-Saharan Africa and has been recently identified in Brazil. As the virulence of this ST is poorly understood, the present study aimed to (i) perform the RNA-seq in vitro of S. Typhimurium STm30 (ST313) grown in Luria-Bertani medium at 37°C; (ii) compare it with the RNA-seq of the S. Typhimurium SL1344 (ST19) and S. Typhimurium STm11 (ST19) strains under the same growing conditions; and (iii) examine the colonization capacity and expression of virulence genes and cytokines in murine colon. The STm30 (ST313) strain exhibited stronger virulence and was associated with a more inflammatory profile than the strains SL1344 (ST19) and STm11 (ST19), as demonstrated by transcriptome and in vivo assay. The expression levels of the hilA, sopD2, pipB, and ssaS virulence genes, other Salmonella pathogenicity islands SPI-1 and SPI-2 genes or effectors, and genes of the cytokines IL-1ß, IFN-γ, TNF-α, IL-6, IL-17, IL-22, and IL-12 were increased during ST313 infection in C57BL/6J mice. In conclusion, S. Typhimurium STm30 (ST313) isolated from human feces in Brazil express higher levels of pathogenesis-related genes at 37°C and has stronger colonization and invasion capacity in murine colon due to its high expression levels of virulence genes, when compared with the S. Typhimurium SL1344 (ST19) and STm11 (ST19) strains. STm30 (ST313) also induces stronger expression of pro-inflammatory cytokines in this organ, suggesting that it causes more extensive tissue damage.

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice.

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26's role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.

Global Transcriptomics Uncovers Distinct Contributions From Splicing Regulatory Proteins to the Macrophage Innate Immune Response.

Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells.

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.