Salmonella enterica is an important foodborne pathogen that uses secreted effector proteins to manipulate host pathways to facilitate survival and dissemination. Different S. enterica serovars cause disease syndromes ranging from gastroenteritis to typhoid fever and vary in their effector repertoire. We leveraged this natural diversity to identify stm2585, here designated sarA (Salmonella anti-inflammatory response activator), as a Salmonella effector that induces production of the anti-inflammatory cytokine IL-10. RNA-seq of cells infected with either ΔsarA or wild-type S. Typhimurium revealed that SarA activates STAT3 transcriptional targets. Consistent with this, SarA is necessary and sufficient for STAT3 phosphorylation, STAT3 inhibition blocks IL-10 production, and SarA and STAT3 interact by co-immunoprecipitation. These effects of SarA contribute to intracellular replication in vitro and bacterial load at systemic sites in mice. Our results demonstrate the power of using comparative genomics for identifying effectors and that Salmonella has evolved mechanisms for activating an important anti-inflammatory pathway.

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Retrons are genetic retroelements, commonly found in bacterial genomes and recently repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate.Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems..

Each drug caused predictable changes in sedation and vital signs, but consistent anti-nociceptive effects were not evident.

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19Retrons are genetic retroelements, commonly found in bacterial genomes and recently 20 repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a 21 multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex 22 biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has 23 remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a 24 toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when 25 msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during 26 anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, 27 and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that 28 this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate. 29Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems. 30 31 1A), do not affect msDNA biosynthesis 13,18 , and neither their sequence nor the position in 54 retron elements are conserved. 55 56The bottleneck to understand the natural function of retrons has been the absence of 57 phenotypes associated with retron deletions. The first retron-deletion phenotype was reported 58 for retron-Sen2 of Salmonella enterica subsp. enterica ser. Typhimurium str. 14028s (STm), 59wherein the RT-Sen2 was found to be essential for STm survival in calves 18 . This was because 60 it allows STm to grow in anaerobic conditions, present in calf intestines 19 . Here, we report that 61 retron-Sen2 deletion mutants are also unable to grow at lower temperatures. By exploiting the 62 retron cold-sensitivity phenotype, we show that the retron-Sen2 accessory gene STM14_4640 63 (rcaT) encodes a bona fide toxin. Perturbing msDNA biosynthesis at any stage results in toxin 64 activation, and thereby, growth inhibition in anaerobic conditions and cold. Although 65 reminiscent of Toxin/Antitoxin (TA) systems, which are composed of a protein or RNA antitoxin 66 and cognate toxin 20 , retron-Sen2 forms a novel tripartite TA system: RcaT is the toxin, and 67 the RT-msDNA complex is the antitoxin. Using another retron encoded by E. coli NILS-16 21 , 68 3 retron-Eco9, we demonstrate that this TA function is conserved and that the RT provides 69 specificity to the TA system. We propose that bacterial retrons function as TA systems, where 70 the RT-msDNA antitoxins directly inhibit retron-encoded toxins, by forming inactive msDNA-71 RT-toxin complexes. 72 73 RESULTS 74Perturbations in msDNA-Sen2 biosynthesis inhibit STm growth in cold 75As part of a larger chemical-genetics effort, we profiled the fitness of a single-gene deletion 76STm library 22 across hundreds of conditions (unpublished data). The two gene deletions that 77 led to the highest growth sensitivity at room temperature (cold-sensitivity) were ΔrrtT (ΔRT-78 Sen2) and ΔxseA (Fig. 1C). Both RT-Sen2 and Exo VII are involved in msDNA-Sen2 126Retron-Sen2 i...

Nontyphoidal salmonellae (NTS) are exposed to reactive oxygen species (ROS) during their residency in the gut. To survive oxidative stress encountered during infection, salmonellae employ several mechanisms. One of these mechanisms involves the multidrug efflux pump MacAB, although the natural substrate of this pump has not been identified. MacAB homologs in pseudomonads secrete products of nonribosomal peptide synthesis (NRPS). In Salmonella enterica serovar Typhimurium, the siderophore enterobactin is produced by NRPS in response to iron starvation and this molecule can be processed into salmochelin and several linear metabolites. We found that Salmonella mutants lacking the key NRPS enzyme EntF are sensitive to peroxide mediated killing and cannot detoxify extracellular H2O2. Moreover, EntF and MacAB function in a common pathway to promote survival of Salmonella during oxidative stress. We further demonstrated that S. Typhimurium secretes siderophores in iron-rich media when peroxide is present and that these MacAB-secreted metabolites participate in protection of bacteria against H2O2. We showed that secretion of anti-H2O2 molecules is independent of the presence of the known siderophore efflux pumps EntS and IroC, well-described efflux systems involved in secretion of enterobactin and salmochelin. Both salmochelin and enterobactin are dispensable for S. Typhimurium protection against ROS; however, linear metabolites of enterobactin produced by esterases IroE and Fes are needed for bacterial survival in peroxide-containing media. We determined that linearized enterobactin trimer protects S. Typhimurium against peroxide-mediated killing in a MacAB-dependent fashion. Thus, we suggest that linearized enterobactin trimer is a natural substrate of MacAB and that its purpose is to detoxify extracellular reactive oxygen species. IMPORTANCE Nontyphoidal Salmonella bacteria induce a classic inflammatory diarrhea by eliciting a large influx of neutrophils, producing a robust oxidative burst. Despite substantial progress understanding the benefits to the host of the inflammatory response to Salmonella, little is known regarding how Salmonella can simultaneously resist the damaging effects of the oxidative burst. The multidrug efflux pump MacAB is important for survival of oxidative stress both in vitro and during infection. We describe a new pathway used by Salmonella Typhimurium to detoxify extracellular reactive oxygen species using a multidrug efflux pump (MacAB) to secrete a linear siderophore, a metabolite of enterobactin. The natural substrates of many multidrug efflux pumps are unknown, and functional roles of the linear metabolites of enterobactin are unknown. We bring two novel discoveries together to highlight an important mechanism used by Salmonella to survive under the oxidative stress conditions that this organism encounters during the classic inflammatory diarrhea that it also induces.

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846, recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle.