Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence.

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages. Here we show that macrophages infected with S. Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates. The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1. The changes in host metabolism promote intracellular replication of S. Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors. Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S. Typhimurium replication or virulence.

PagR mediates the precise regulation of Salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in Salmonella Typhimurium.

To establish systemic infections, Salmonella enterica serovar Typhimurium (S. Typhimurium) requires Salmonella pathogenicity island 2 (SPI-2) to survive and replicate within macrophages. High expression of many SPI-2 genes during the entire intracellular growth period within macrophages is essential, as it contributes to the formation of Salmonella-containing vacuole and bacterial replication. However, the regulatory mechanisms underlying the sustained induction of SPI-2 within macrophages are not fully understood. Here, we revealed a time-dependent regulation of SPI-2 expression mediated by a novel regulator PagR (STM2345) in response to the low Mg2+ and low phosphate (Pi ) signals, which ensured the high induction of SPI-2 during the entire intramacrophage growth period. Deletion of pagR results in reduced bacterial replication in macrophages and attenuation of systemic virulence in mice. The effects of pagR on virulence are dependent on upregulating the expression of slyA, a regulator of SPI-2. At the early (0-4 hr) and later (after 4 hr) stage post-infection of macrophages, pagR is induced by the low Pi via PhoB/R two-component systems and low Mg2+ via PhoP/Q systems, respectively. Collectively, our findings revealed that the PagR-mediated regulatory mechanism contributes to the precise and sustained activation of SPI-2 genes within macrophages, which is essential for S. Typhimurium systemic virulence.

The putative transcriptional regulator STM14_3563 facilitates Salmonella Typhimurium pathogenicity by activating virulence-related genes.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important gram-negative intracellular pathogen that infects humans and animals. More than 50 putative regulatory proteins have been identified in the S. Typhimurium genome, but few have been clearly defined. In this study, the physiological function and regulatory role of STM14_3563, which encodes a ParD family putative transcriptional regulator in S. Typhimurium, were investigated. Macrophage replication assays and mice experiments revealed that S. Typhimurium showed reduced growth in murine macrophages and attenuated virulence in mice owing to deletion of STM14_3563 gene. RNA sequencing (RNA-Seq) data showed that STM14_3563 exerts wide-ranging effects on gene expression in S. Typhimurium. STM14_3563 activates the expression of several genes encoded in Salmonella pathogenicity island (SPI)-6, SPI-12, and SPI-13, which are required for intracellular replication of S. Typhimurium. Additionally, the global transcriptional regulator Fis was found to directly activate STM14_3563 expression by binding to the STM14_3563 promoter. These results indicate that STM14_3563 is involved in the regulation of a variety of virulence-related genes in S. Typhimurium that contribute to its growth in macrophages and virulence in mice.

SoxS is a positive regulator of key pathogenesis genes and promotes intracellular replication and virulence of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important intracellular pathogen, causing gastroenteritis or severe systemic infection in a variety of hosts. During infection, S. Typhimurium must survive and replicate in host macrophages, which produce abundant oxidative compounds. SoxRS regulon is a well-known regulator that is activated in response to oxidative stress and promotes bacterial tolerance to oxidants in E. coli. However, the global regulatory function of SoxS in S. Typhimurium remains poorly characterized. Here, we used an RNA sequencing-based approach to investigate the role of SoxS in the expression of S. Typhimurium virulence genes. Besides the downregulation of genes related to resistance to oxidative stress, we found that in a soxS deletion mutant the expression of Salmonella pathogenicity island (SPI)-2 genes, which are crucial for replication within macrophages, was significantly repressed. Moreover, immunofluorescence and mice infection experiments showed that soxS deletion inhibited replication in macrophages and decreased virulence upon intraperitoneal inoculation in mice, respectively. Collectively, our findings demonstrate that SoxS is a positive regulator of SPI-2 genes and, therefore, plays a crucial role in S. Typhimurium intracellular replication and virulence.

The LysR-type transcriptional regulator STM0030 contributes to Salmonella Typhimurium growth in macrophages and virulence in mice.

Salmonella enterica serovar Typhimurium (S. Tm) is a major intracellular pathogen that infects humans and animals, and its survival and growth in macrophages is essential for its pathogenicity. More than 50 putative regulatory proteins are encoded by the S. Tm genome, but the functions of these regulatory proteins in mediating S. Tm pathogenicity are largely unknown. In this study, we investigated the biological function of the STM0030 gene, which encodes a putative LysR-type transcriptional regulator. We found that STM0030 is upregulated 2.8-5.7-fold during S. Tm growth in macrophages. Further, mutating this gene decreased bacterial growth in macrophages and attenuated virulence in mice. RNA-sequencing to investigate the regulatory function of STM0030 in S. Tm revealed that 447 genes were differentially expressed between the mutant and the wild-type strains; 429 of these genes were downregulated, suggesting that STM0030 mainly acts as a transcriptional activator. Moreover, the expression of gluconate, maltose, and hexose-p transport genes, as well as allantoin utilization genes were downregulated in the STM0030 mutant; this might be associated with the observed decrease in intracellular replication and pathogenicity of the mutant. Our findings suggest that STM0030 is a new pathogenicity-associated regulatory protein that broadens our understanding of the virulence regulatory network of S. Tm.

YaeB, Expressed in Response to the Acidic pH in Macrophages, Promotes Intracellular Replication and Virulence of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that infects humans and animals. Survival and growth in host macrophages represents a crucial step for S. Typhimurium virulence. Many genes that are essential for S. Typhimurium proliferation in macrophages and associated with virulence are highly expressed during the intracellular lifecycle. yaeB, which encodes an RNA methyltransferase, is also upregulated during S. Typhimurium growth in macrophages. However, the involvement of YaeB in S. Typhimurium pathogenicity is still unclear. In this study, we investigated the role of YaeB in S. Typhimurium virulence. Deletion of yaeB significantly impaired S. Typhimurium growth in macrophages and virulence in mice. The effect of yaeB on pathogenicity was related to its activation of pstSCAB, a phosphate (Pi)-specific transport system that is verified here to be important for bacterial replication and virulence. Moreover, qRT-PCR data showed YaeB was induced by the acidic pH inside macrophages, and the acidic pH passed to YeaB through inhibiting global regulator histone-like nucleoid structuring (H-NS) which confirmed in this study can repress the expression of yaeB. Overall, these findings identified a new virulence regulatory network involving yaeB and provided valuable insights to the mechanisms through which acidic pH and low Pi regulate virulence.

Transcriptome analysis of virulence gene regulation by the ATP-dependent Lon protease in Salmonella Typhimurium.

Aim: Determination of the virulence regulatory network controlled by the ATP-dependent Lon protease in Salmonella enterica serovar Typhimurium. Materials & methods: The effect of Lon on S. Typhimurium virulence genes expression was investigated by RNA sequencing, and virulence-associated phenotypes between the wild-type and lon mutant were compared. Results:SPI-1, SPI-4, SPI-9 and flagellar genes were activated, while SPI-2 genes were repressed in the lon mutant. Accordingly, the lon mutant exhibited increased adhesion to and invasion of epithelial cells, increased motility and decreased replication in macrophages. The activation of SPI-2 genes by Lon partially accounts for the replication defect of the mutant. Conclusion: A wide range of virulence regulatory functions are governed by Lon in S. enterica ser. Typhimurium.

LoiA directly represses lon gene expression to activate the expression of Salmonella pathogenicity island-1 genes.

Salmonella enterica serovar Typhimurium uses virulence factors encoded by Salmonella pathogenicity island 1 (SPI-1) to invade the intestinal epithelium. The low oxygen-induced transcriptional regulator LoiA is established to be a positive regulator of SPI-1 genes and can also repress the expression of the ATP-dependent Lon protease, a negative regulator of SPI-1 genes. Whether the repression of lon by LoiA is associated with its regulation of SPI-1 genes, as well as the regulatory mechanism involved, is unknown. In this study, we showed that LoiA directly represses the expression of the lon gene during invasion by binding to the promoter region of lon. The activation of S. Typhimurium SPI-1 gene expression by LoiA is associated with its repression of lon. Moreover, through invasion and mouse infection assays, we found that the repression of lon by LoiA contributes to S. Typhimurium invasion of intestinal epithelial cells and virulence in mice. However, LoiA does not influence lon expression during S. Typhimurium growth in macrophages, which may be associated with the low expression level of loiA in macrophages. Collectively, these findings establish the direct regulation of lon by LoiA and describe a novel mechanism by which LoiA regulates SPI-1 gene expression.

Global regulatory function of the low oxygen-induced transcriptional regulator LoiA in Salmonella Typhimurium revealed by RNA sequencing.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major intestinal pathogen that can infect both humans and a variety of animals. LoiA, a novel virulence-regulating protein encoded in Salmonella pathogenicity island (SPI)-14, has been shown to be induced under low oxygen conditions and contribute to S. Typhimurium invasion into intestinal epithetical cells by activating the SPI-1 invasion genes. However, the global regulatory network of LoiA remains unknown. Here, we used high-throughput RNA sequencing (RNA-seq) technology to investigate the regulatory function of LoiA in S. Typhimurium under low oxygen conditions. A total of 1250 genes were differentially expressed between the loiA mutant and the wild-type strain; 413 genes were up-regulated and 837 were down-regulated. SPI-1 gene expression was down-regulated in the loiA mutant, consistent with previous results. SPI-2 gene expression was not affected by deletion of loiA; the expression of most genes involved in flagellar basal body and hook biosynthesis was up-regulated in the loiA mutant, while the expression of genes associated with flagellin, motility, and chemotaxis was down-regulated; the expression of lon, encoding an ATP-dependent protease, was up-regulated in the mutant. This study indicates that LoiA regulates a variety of virulence-associated genes in S. Typhimurium. The negative regulation of Lon protease by LoiA indicates that LoiA can regulates several virulence-associated genes in S. Typhimurium via the Lon protease.