Phagocytic Superoxide Specifically Damages an Extracytoplasmic Target to Inhibit or Kill Salmonella

The phagocytic oxidative burst is a primary effector of innate immunity that protects against bacterial infection. However, the mechanism by which reactive oxygen species (ROS) kill or inhibit bacteria is not known. It is often assumed that DNA is a primary target of oxidative damage, consistent with known effects of endogenously produced ROS in the bacterial cytoplasm. But most studies fail to distinguish between effects of host derived ROS versus damage caused by endogenous bacterial sources. We took advantage of both the ability of Salmonella enterica serovar Typhimurium to survive in macrophages and the genetic tractability of the system to test the hypothesis that phagocytic superoxide damages cytoplasmic targets including DNA.SodCI is a periplasmic Cu-Zn superoxide dismutase (SOD) that contributes to the survival of Salmonella Typhimurium in macrophages. Through competitive virulence assays, we asked if sodCI has a genetic interaction with various cytoplasmic systems. We found that SodCI acts independently of cytoplasmic SODs, SodA and SodB. In addition, SodCI acts independently of the base excision repair system and RuvAB, involved in DNA repair. Although sodCI did show genetic interaction with recA, this was apparently independent of recombination and is presumably due to the pleiotropic effects of a recA mutation.Taken together, these results suggest that bacterial inhibition by phagocytic superoxide is primarily the result of damage to an extracytoplasmic target

HilE regulates HilD by blocking DNA binding in Salmonella enterica serovar Typhimurium

ABSTRACT The Salmonella type three secretion system (T3SS), encoded in the Salmonella pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and hilA , which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an in vivo reporter system and an in vitro gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of hilE does not confer a phenotype, the hilE mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system. IMPORTANCE Salmonella is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for Salmonella to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output. </jats:p

The Role of Coupled Positive Feedback in the Expression of the SPI1 Type Three Secretion System in Salmonella

Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics

SdiA, an N-Acylhomoserine Lactone Receptor, Becomes Active during the Transit of Salmonella enterica through the Gastrointestinal Tract of Turtles

encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species..We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals

Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa

RtsA Coordinately Regulates DsbA and the Salmonella Pathogenicity Island 1 Type III Secretion System

Salmonella serovars cause a wide variety of diseases ranging from mild gastroenteritis to life-threatening systemic infections. An important step in Salmonella enterica serovar Typhimurium infection is the invasion of nonphagocytic epithelial cells, mediated by a type III secretion system (TTSS) encoded on Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS forms a needle complex through which effector proteins are injected into the cytosol of host cells, where they promote actin rearrangement and engulfment of the bacteria. We previously identified the Salmonella-specific regulatory protein RtsA, which induces expression of hilA and, thus, the SPI1 genes. Here we show that the hilA regulators RtsA, HilD, and HilC can each induce transcription of dsbA, which encodes a periplasmic disulfide bond isomerase. RtsA induces expression of dsbA independent of either the SPI1 TTSS or the only known regulator of dsbA, the CpxRA two-component system. We show that DsbA is required for both the SPI1 and SPI2 TTSS to translocate effector proteins into the cytosol of host cells. DsbA is also required for survival during the systemic stages of infection. We also present evidence that production of SPI1 effector proteins is coupled to assembly of the TTSS. This feedback regulation is mediated at either the transcriptional or posttranscriptional level, depending on the particular effector. Loss of DsbA leads to feedback inhibition, which is consistent with the hypothesis that disulfide bond formation plays a role in TTSS assembly or function

Salmonella enterica Serovar Typhimurium Periplasmic Superoxide Dismutase SodCI Is a Member of the PhoPQ Regulon and Is Induced in Macrophages

Salmonella enterica serovar Typhimurium replicates within host macrophages during the systemic stage of infection. In the macrophage, the bacteria must survive the respiratory burst that produces superoxide. Serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases, SodCI and SodCII, but only SodCI contributes to virulence. Although we have shown that this is primarily due to differences in the two proteins, evidence suggests differential regulation of the two genes. Using transcriptional sodCI- and sodCII-lac fusions, we show that sodCII is under the control of the RpoS sigma factor, as was known for the Escherichia coli ortholog, sodC. In contrast, we show that sodCI is transcriptionally controlled by the PhoPQ two-component regulatory system, which regulates an array of virulence genes required for macrophage survival. Introduction of a phoP-null mutation into the sodCI fusion strain resulted in a decrease in transcription and loss of regulation. The sodCI-lac fusion showed high-level expression in a background containing a phoQ constitutive allele. The sodCI gene is induced 15-fold in bacteria recovered from either the tissue culture macrophages or the spleens of infected mice. Induction in macrophages is dependent on PhoP. The sodCII fusion was induced three- to fourfold in macrophages and animals; this induction was unaffected by loss of PhoP. Thus, sodCI, which is horizontally transferred by the Gifsy-2 phage, is regulated by PhoPQ such that it is induced at the appropriate time and place to combat phagocytic superoxide

Fur Regulates Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System through HilD▿

The invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is mediated by a type III secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). Expression of the SPI1 T3SS is tightly regulated by the combined action of HilC, HilD, and RtsA, three AraC family members that can independently activate hilA, which encodes the direct regulator of the SPI1 structural genes. Expression of hilC, hilD, and rtsA is controlled by a number of regulators that respond to a variety of environmental signals. In this work, we show that one such signal is iron mediated by Fur (ferric uptake regulator). Fur activates hilA transcription in a HilD-dependent manner. Fur regulation of HilD does not appear to be simply at the transcriptional or translational level but rather requires the presence of the HilD protein. Fur activation of SPI1 is not mediated through the Fur-regulated small RNAs RfrA and RfrB, which are the Salmonella ortholog and paralog of RyhB that control expression of sodB. Fur regulation of HilD is also not mediated through the known SPI1 repressor HilE or the CsrABC system. Although understanding the direct mechanism of Fur action on HilD requires further analysis, this work is an important step toward elucidating how various global regulatory systems control SPI1