RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.

sRNAs and the virulence of Salmonella enterica serovar Typhimurium.

The combination of genomics and high-throughput cDNA sequencing technologies has facilitated the identification of many small RNAs (sRNAs) that play a central role in the post-transcriptional gene regulation of Salmonella enterica serovar Typhimurium. To date, most of the functionally characterized sRNAs have been involved in the regulation of processes which are not directly linked to virulence. Just five sRNAs have been found to affect the ability of Salmonella to replicate within mammalian cells, but the precise regulatory mechanisms that are used by sRNAs to control Salmonella pathogenicity at the post-transcriptional level remain to be identified. It is anticipated that an improved understanding of sRNA biology will shed new light on the virulence of Salmonella.

The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium.

More than 50 y of research have provided great insight into the physiology, metabolism, and molecular biology of Salmonella enterica serovar Typhimurium (S. Typhimurium), but important gaps in our knowledge remain. It is clear that a precise choreography of gene expression is required for Salmonella infection, but basic genetic information such as the global locations of transcription start sites (TSSs) has been lacking. We combined three RNA-sequencing techniques and two sequencing platforms to generate a robust picture of transcription in S. Typhimurium. Differential RNA sequencing identified 1,873 TSSs on the chromosome of S. Typhimurium SL1344 and 13% of these TSSs initiated antisense transcripts. Unique findings include the TSSs of the virulence regulators phoP, slyA, and invF. Chromatin immunoprecipitation revealed that RNA polymerase was bound to 70% of the TSSs, and two-thirds of these TSSs were associated with σ(70) (including phoP, slyA, and invF) from which we identified the -10 and -35 motifs of σ(70)-dependent S. Typhimurium gene promoters. Overall, we corrected the location of important genes and discovered 18 times more promoters than identified previously. S. Typhimurium expresses 140 small regulatory RNAs (sRNAs) at early stationary phase, including 60 newly identified sRNAs. Almost half of the experimentally verified sRNAs were found to be unique to the Salmonella genus, and <20% were found throughout the Enterobacteriaceae. This description of the transcriptional map of SL1344 advances our understanding of S. Typhimurium, arguably the most important bacterial infection model.

Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst.

The oxidative burst produced by the NADPH oxidase (Phox) is an essential weapon used by host cells to eradicate engulfed pathogens. In Salmonella typhimurium, oxidative stress resistance has been previously proposed to be mediated by the pathogenicity island 2 type III secretion system (T3SS-2), periplasmic superoxide dismutases and cytoplasmic catalases/peroxidases. Here, we fused an OxyR-dependent promoter to the gfp to build the ahpC-gfp transcriptional fusion. This reporter was used to monitor hydrogen peroxide levels as sensed by Salmonella during the course of an infection. We showed that the expression of this fusion was under the exclusive control of reactive oxygen species produced by the host. The ahpC-gfp expression was noticeably modified in the absence of bacterial periplasmic superoxide dismutases or cytoplasmic catalases/peroxidases. Surprisingly, inactivation of the T3SS-2 had no effect on the ahpC-gfp expression. All together, these results led to a model in which Salmonella resistance relies on its arsenal of detoxifying enzymes to cope with Phox-mediated oxidative stress.

The challenge of relating gene expression to the virulence of Salmonella enterica serovar Typhimurium.

The first decade of transcriptomic studies of Salmonella enterica serovar Typhimurium focused upon gene expression in vitro, and during the infection of mammalian cells. The published regulons and stimulons show that the three Type Three Secretion Systems of S. Typhimurium respond to a diverse range of environmental conditions, and are controlled by a hierarchy of regulatory proteins. The integration of in vitro generated transcriptomic data with global gene expression of S. Typhimurium during infection is beginning to yield valuable information. The coordinated regulation of Salmonella gene expression is a key process for survival, adaptation and virulence capacities of the pathogen.

Redundant hydrogen peroxide scavengers contribute to Salmonella virulence and oxidative stress resistance.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H(2)O(2)) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H(2)O(2) degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF(-) mutant, which exhibited a high sensitivity to exogenous H(2)O(2) and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of katG or tsaA in the HpxF(-) background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H(2)O(2) in rich medium. The HpxF(-) mutant also showed an attenuated virulence in a mouse model. These data indicate that Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H(2)O(2) and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.