Dataset Information

FlgM is required to evade NLRC4-mediated host protection against flagellated Salmonella.

ABSTRACT: Salmonella enterica serovar Typhimurium is a leading cause of gastroenteritis worldwide and a deadly pathogen in children, immunocompromised patients, and the elderly. Salmonella induces innate immune responses through the NLRC4 inflammasome, which has been demonstrated to have distinct roles during systemic and mucosal detections of flagellin and non-flagellin molecules. We hypothesized that NLRC4 recognition of Salmonella flagellin is the dominant protective pathway during infection. To test this hypothesis, we used wild-type, flagellin-deficient, and flagellin-overproducing Salmonella to establish the role of flagellin in mediating NLRC4-dependent host resistance during systemic and mucosal infections in mice. We observed that during the systemic phase of infection, Salmonella efficiently evades NLRC4-mediated innate immunity. During mucosal Salmonella infection, flagellin recognition by the NLRC4 inflammasome pathway is the dominant mediator of protective innate immunity. Deletion of flgM results in constitutive expression of flagellin and severely limits systemic and mucosal Salmonella infections in an NLRC4 inflammasome-dependent manner. These data establish that recognition of Salmonella's flagellin by the NLRC4 inflammasome during mucosal infection is the dominant innate protective pathway for host resistance against the enteric pathogen and that FlgM-mediated evasion of the NLRC4 inflammasome enhances virulence and intestinal tissue destruction.

SUBMITTER: Lopez-Yglesias AH

PROVIDER: S-EPMC10501211 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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FlgM is required to evade NLRC4-mediated host protection against flagellated Salmonella.

López-Yglesias Américo Harry AH Lu Chun-Chi CC Lai Marvin A MA Quarles Ellen K EK Zhao Xiaodan X Hajjar Adeline M AM Smith Kelly D KD

Infection and immunity 20230828 9

Salmonella enterica serovar Typhimurium is a leading cause of gastroenteritis worldwide and a deadly pathogen in children, immunocompromised patients, and the elderly. Salmonella induces innate immune responses through the NLRC4 inflammasome, which has been demonstrated to have distinct roles during systemic and mucosal detections of flagellin and non-flagellin molecules. We hypothesized that NLRC4 recognition of Salmonella flagellin is the dominant protective pathway during ...[more]

PMID: 37638725

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Project description:Salmonella infection entails a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by macrophages, where the bacteria encounter multiple stresses, to which it employs relevant countermeasures. Our study shows that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which prevents the oxidation of proteins in E. coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro and reduced organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. We found that Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which addresses the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D, L-α-difluoromethylornithine (DFMO), reduces Salmonella colonisation and tissue damage in the mouse model of infection while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a bacterial strategy in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

Project description:Salmonella is a common and important pathogen for both human and animals. All Salmonella except Salmonella pullorum and Salmonella gallinarum have flagellum. Flagellin (FliC) is the main subunit protein forming the bacterial filament, which is present in large amounts on the surface of all flagellated Salmonella. After bioinformatics analysis, the most highly conserved region (locates position from 1 to 102 amino acid residue of FliC, we named it as FliC') was selected, and corresponding recombinant FliC' (rFliC') protein was tailored as an immunogen to generate monoclonal antibodies (MAbs) against Salmonella flagellin. BALB/c mice were immunized with the purified recombinant protein rFliC', which were prepared by prokaryotic expression system pET22b (+) expressing FliC'. After fusion of spleen cells from the immunized mice and SP2/0 cells, three hybridoma cells (1D6, 2G6 and 3E2) producing MAbs against targeted flagellate Salmonella FliC' were generated and screened. The ability of MAb 3E2 to recognize and bind to Salmonella flagella was demonstrated by immunogold electron microscopy (IEM) method. Western blot (WB) analysis demonstrated that MAb 3E2 could specifically recognize flagellated Salmonella strains. Moreover, MAb 3E2 has a direct agglutination activity against Salmonella strains with visible agglutination reaction. To further verify this agglutination activity, a total of 52 flagellated Salmonella strains (23 serovars), 8 non-flagellate Salmonella strains (2 serovars) and 16 other non-Salmonella bacteria strains were used to evaluate the specificity of the MAb by direct Slide Agglutination Test (SAT). Results showed that MAb 3E2 reacted with all Salmonella strains possessing flagellum and had no cross-reaction with non-flagellate Salmonella strains or other non-Salmonella bacteria strains. Sequentially, the ability to detect the presence of Salmonella in raw samples of the MAb 3E2-based SAT method was evaluated. The conventional culture-based detection method was performed as the standard reference method for detection of Salmonella. Altogether, 369 samples collected from laying hens were tested, and the results indicated that the MAb 3E2-based SAT method could specifically detect Salmonella. Furthermore, the SAT results were obtained more quickly, as compared with the standard method. As a whole, the MAbs against the tailored conserved region of Salmonella flagellin were prepared in this study, and MAb 3E2-based SAT is a promising candidate for the flagellated Salmonella spp. rapid detection.

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FlgM is required to evade NLRC4-mediated host protection against flagellated Salmonella.

Publications

FlgM is required to evade NLRC4-mediated host protection against flagellated <i>Salmonella</i>.

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