Dataset Information

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

ABSTRACT: In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.

SUBMITTER: Cooper KG

PROVIDER: S-EPMC7806825 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

Cooper Kendal G KG Chong Audrey A Kari Laszlo L Jeffrey Brendan B Starr Tregei T Martens Craig C McClurg Molly M Posada Victoria R VR Laughlin Richard C RC Whitfield-Cargile Canaan C Garry Adams L L Bryan Laura K LK Bryan Laura K LK Little Sara V SV Krath Mary M Lawhon Sara D SD Steele-Mortimer Olivia O

Nature communications 20210113 1

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces s ...[more]

PMID: 33441540

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Project description: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.IMPORTANCESalmonella 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.

Project description:Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give new insights into mechanisms of Salmonella host cell sensing.

Project description:The periplasmic sensor domains GSU0582 and GSU0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens. Both contain one c-type heme group and their crystal structures revealed that these domains form swapped dimers with a PAS fold formed from the two protein chains. The swapped dimerization of these sensors is related to the mechanism of signal transduction and the formation of the swapped dimer involves significant folding changes and conformational rearrangements within each monomeric component. However, the structural changes occurring during this process are poorly understood and lack a mechanistic framework. To address this issue, we have studied the folding and stability properties of two distinct heme-sensor PAS domains, using biophysical spectroscopies. We observed substantial differences in the thermodynamic stability (ΔG = 14.6 kJ.mol(-1) for GSU0935 and ΔG = 26.3 kJ.mol(-1) for GSU0582), and demonstrated that the heme moiety undergoes conformational changes that match those occurring at the global protein structure. This indicates that sensing by the heme cofactor induces conformational changes that rapidly propagate to the protein structure, an effect which is directly linked to the signal transduction mechanism. Interestingly, the two analyzed proteins have distinct levels of intrinsic disorder (25% for GSU0935 and 13% for GSU0582), which correlate with conformational stability differences. This provides evidence that the sensing threshold and intensity of the propagated allosteric effect is linked to the stability of the PAS-fold, as this property modulates domain swapping and dimerization. Analysis of the PAS-domain shows that disorder segments are found either at the hinge region that controls helix motions or in connecting segments of the β-sheet interface. The latter is known to be widely involved in both intra- and intermolecular interactions, supporting the view that it's folding and stability are at the basis of the specificity and regulation of many types of PAS-containing signaling proteins.

Dataset Information

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

Publications

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

Similar Datasets

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