Codependent and Independent Effects of Nitric Oxide-Mediated Suppression of PhoPQ and Salmonella Pathogenicity Island 2 on Intracellular Salmonella enterica Serovar Typhimurium Survival▿

Here we show that the Salmonella enterica serovar Typhimurium PhoQ sensor kinase lessens the cytotoxicity of reactive nitrogen species (RNS) generated by inducible nitric oxide synthase (iNOS) in the innate response of mononuclear phagocytic cells. This observation is consistent with the expression patterns of PhoP-activated genes during moderate nitrosative stress in the innate host response. In contrast, RNS synthesized during high-NO fluxes of gamma interferon (IFN-γ)-activated macrophages repress PhoP-activated lpxO, pagP, and phoP gene transcription. Because PhoP-regulated Salmonella pathogenicity island 2 (SPI2) genes are also repressed by high-order RNS (39), we investigated whether the NO-mediated inhibition of PhoPQ underlies the repression of SPI2. Our studies indicate that a third of the expression of the SPI2 spiC gene recorded in nonactivated macrophages depends on PhoQ. Transcription of spiC is repressed in IFN-γ-primed macrophages in an iNOS-dependent manner, irrespective of the phoQ status of the bacteria. Transcription of spiC is restored in IFN-γ-treated, iNOS-deficient macrophages to levels sustained by a phoQ mutant in nonactivated phagocytes, suggesting that most NO-dependent repression of spiC is due to the inhibition of PhoPQ-independent targets. Comparison of the intracellular fitness of spiC, phoQ, and spiC phoQ mutants revealed that PhoPQ and SPI2 have codependent and independent effects on S. Typhimurium survival during innate nitrosative stress. However, the intracellular survival of most S. Typhimurium bacteria is conferred by the PhoPQ two-component regulator, and the SPI2 type III secretion system is repressed by high-order RNS of IFN-γ-activated macrophages

Antioxidant Defense by Thioredoxin Can Occur Independently of Canonical Thiol-Disulfide Oxidoreductase Enzymatic Activity

The thiol-disulfide oxidoreductase CXXC catalytic domain of thioredoxin contributes to antioxidant defense in phylogenetically diverse organisms. We find that although the oxidoreductase activity of thioredoxin-1 protects Salmonella enterica serovar Typhimurium from hydrogen peroxide in vitro, it does not appear to contribute to Salmonella’s antioxidant defenses in vivo. Nonetheless, thioredoxin-1 defends Salmonella from oxidative stress resulting from NADPH phagocyte oxidase macrophage expression during the innate immune response in mice. Thioredoxin-1 binds to the flexible linker, which connects the receiver and effector domains of SsrB, thereby keeping this response regulator in the soluble fraction. Thioredoxin-1, independently of thiol-disulfide exchange, activates intracellular SPI2 gene transcription required for Salmonella resistance to both reactive species generated by NADPH phagocyte oxidase and oxygen-independent lysosomal host defenses. These findings suggest that the horizontally acquired virulence determinant SsrB is regulated post-translationally by ancestrally present thioredoxin

Salmonella enterica Serovar Gallinarum Requires ppGpp for Internalization and Survival in Animal Cells ▿

To elucidate the pathogenic mechanism of Salmonella enterica serovar Gallinarum, we examined the expression of the genes encoded primarily in Salmonella pathogenicity island 1 (SPI-1) and SPI-2. These genes were found to be induced as cultures entered stationary phase under high- and low-oxygen growth conditions, as also observed for Salmonella serovar Typhimurium. In contrast, Salmonella serovar Gallinarum in the exponential growth phase most efficiently internalized cultured animal cells. Analysis of mutants defective in SPI-1 genes, SPI-2 genes, and others implicated in early stages of infection revealed that SPI-1 genes were not involved in the internalization of animal cells by Salmonella serovar Gallinarum. Following entry, however, Salmonella serovar Gallinarum was found to reside in LAMP1-positive vacuoles in both phagocytic and nonphagocytic cells, although internalization was independent of SPI-1. A mutation that conferred defects in ppGpp synthesis was the only one found to affect animal cell internalization by Salmonella serovar Gallinarum. It was concluded that Salmonella serovar Gallinarum internalizes animal cells by a mechanism independent of SPI-1 genes but dependent on ppGpp. Intracellular growth also required ppGpp for the transcription of genes encoded in SPI-2

DNA looping-mediated repression by histone-like protein H-NS: specific requirement of Eσ(70) as a cofactor for looping

Transcription initiation by RNA polymerase (RNP) carrying the house-keeping σ subunit, σ(70) (Eσ(70)), is repressed by H-NS at a number of promoters including hdeABp in Escherichia coli, while initiation with RNP carrying the stationary phase σ, σ(38) (Eσ(38)), is not. We investigated the molecular mechanism of selective repression by H-NS to identify the differences in transcription initiation by the two forms of RNPs, which show indistinguishable promoter selectivities in vitro. Using hdeABp as a model promoter, we observed with purified components that H-NS, acting at a sequence centered at -118, selectively repressed transcription by Eσ(70). This selective repression is attributed to the differences in the interactions between hdeABp and the two forms of RNPs, since no other factor is required for the repression. We observed that the two forms of RNPs could form an open initiation complex (RP(O)) at hdeABp, but that Eσ(70) failed to initiate transcription in the presence of H-NS. Interestingly, KMnO(4) assays and high-resolution atomic force microscopy (AFM) revealed that hdeABp DNA wrapped around Eσ(70) more tightly than around Eσ(38), resulting in the potential crossing over of the DNA arms that project out of Eσ(70) · RP(O) but not out of Eσ(38) · RP(O). Based on these observations, we postulated that H-NS bound at -118 laterally extends by the cooperative recruitment of H-NS molecules to the promoter-downstream sequence joined by wrapping of the DNA around Eσ(70) · RP(O), resulting in effective sealing of the DNA loop and trapping of Eσ(70). Such a ternary complex of H-NS · Eσ(70) hdeABp was demonstrated by AFM. In this case, therefore, Eσ(70) acts as a cofactor for DNA looping. Expression of this class of genes by Eσ(38) in the stationary phase is not due to its promoter specificity but to the architecture of the promoter · Eσ(38) complex

Control of type III protein secretion using a minimal genetic system

Gram-negative bacteria secrete proteins using a type III secretion system (T3SS), which functions as a needle-like molecular machine. The many proteins involved in T3SS construction are tightly regulated due to its role in pathogenesis and motility. Here, starting with the 35 kb Salmonella pathogenicity island 1 (SPI-1), we eliminated internal regulation and simplified the genetics by removing or recoding genes, scrambling gene order and replacing all non-coding DNA with synthetic genetic parts. This process results in a 16 kb cluster that shares no sequence identity, regulation or organizational principles with SPI-1. Building this simplified system led to the discovery of essential roles for an internal start site (SpaO) and small RNA (InvR). Further, it can be controlled using synthetic regulatory circuits, including under SPI-1 repressing conditions. This work reveals an incredible post-transcriptional robustness in T3SS assembly and aids its control as a tool in biotechnology

Control of type III protein secretion using a minimal genetic system

Gram-negative bacteria secrete proteins using a type III secretion system (T3SS), which functions as a needle-like molecular machine. The many proteins involved in T3SS construction are tightly regulated due to its role in pathogenesis and motility. Here, starting with the 35 kb Salmonella pathogenicity island 1 (SPI-1), we eliminated internal regulation and simplified the genetics by removing or recoding genes, scrambling gene order and replacing all non-coding DNA with synthetic genetic parts. This process results in a 16 kb cluster that shares no sequence identity, regulation or organizational principles with SPI-1. Building this simplified system led to the discovery of essential roles for an internal start site (SpaO) and small RNA (InvR). Further, it can be controlled using synthetic regulatory circuits, including under SPI-1 repressing conditions. This work reveals an incredible post-transcriptional robustness in T3SS assembly and aids its control as a tool in biotechnology

The hepcidin-ferroportin axis controls the iron content of Salmonella-containing vacuoles in macrophages

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for Salmonella

Lipocalin2 Induced by Bacterial Flagellin Protects Mice against Cyclophosphamide Mediated Neutropenic Sepsis

Neutropenic sepsis is a fatal consequence of chemotherapy, and septic complications are the principal cause of mortality. Chemotherapy-induced neutropenia leads to the formation of microscopic ulcers in the gastrointestinal epithelium that function as a portal of entry for intraluminal bacteria, which translocate across the intestinal mucosal barrier and gain access to systemic sites, causing septicemia. A cyclophosphamide-induced mouse model was developed to mimic the pathophysiologic sequence of events that occurs in patients with neutropenic sepsis. The TLR5 agonist bacterial flagellin derived from Vibrio vulnificus extended the survival of cyclophosphamide-treated mice by reducing the bacterial load in internal organs. The protective effect of flagellin was mediated by the antimicrobial protein lipocalin 2 (Lcn2), which is induced by TLR5-NF-κB activation in hepatocytes. Lcn2 sequestered iron from infecting bacteria, particularly siderophore enterobactin-dependent members of the Enterobacteriaceae family, thereby limiting their proliferation. Lcn2 should be considered for the treatment of neutropenic sepsis and gastrointestinal damage during chemotherapy to prevent or minimize the adverse effects of cancer chemotherapy