• Login
    View Item 
    •   etd@IISc
    • Division of Biological Sciences
    • Microbiology and Cell Biology (MCB)
    • View Item
    •   etd@IISc
    • Division of Biological Sciences
    • Microbiology and Cell Biology (MCB)
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Deciphering the mechanism behind the division of the Salmonella containing vacuole (SCV) and the immunomodulation strategy employed by Salmonella Typhimurium

    Thumbnail
    View/Open
    Thesis full text (7.628Mb)
    Author
    Chopra, Umesh
    Metadata
    Show full item record
    Abstract
    The genus Salmonella represents flagellated, Gram-negative, rod-shaped anaerobic bacteria which is a leading cause of food-borne illness and causes enteric disease in a wide range of animals. In humans and several warm-blooded animals, it is known to cause various diseases ranging from mild gastroenteritis to life-threatening systemic infections like typhoid. The route of transmission of Salmonella occurs through the fecal-oral route, following which it survives the acidic pH of the stomach and finally reaches the intestinal epithelial cells. For establishing successful infection, Salmonella disseminates to the secondary sites of infection, such as the liver, spleen, and MLN, through the reticuloendothelial system (RES). Inside the host cell, Salmonella resides in a membrane-bound compartment called Salmonella containing vacuole (SCV). Post-maturation of SCV, the bacteria start dividing and proliferating. Our lab previously demonstrated that the SCV membrane simultaneously divides along with the bacterial division, leading to one bacterium per vacuole. The vacuolar state of Salmonella is evident at all stages of infection (positive for LAMP-1), but the mechanism involved in executing the division remains elusive. Further, upon encountering the pathogen, the host immune system employs several strategies to eliminate the pathogen. Nevertheless, Salmonella, being a successful pathogen, has evolved several mechanisms to combat the innate and adaptive immune responses. It uses several effector proteins, which help in protection from various reactive oxygen species/ reactive nitrogen species (ROS/RNS) and antimicrobial peptides, inhibit lysosomal fusion, and inhibit CD4-T cell activation by downregulation of MHC-Ⅱ. Salmonella, by employing unknown mechanisms, also upregulates the expression of immune-modulatory molecules such as Programmed death ligand 1 (PDL1) on the surface of infected cells, which helps in attenuating the adaptive immune response and establishing successful infection in the host. In this regard, this study aims to understand: Objective 1: Elucidating the mechanism behind the division of Salmonella containing vacuole (SCV) The fission of several intracellular organelles is regulated by the dynamic nature of the tubular endoplasmic reticulum (ER). In this study, we have evaluated the role of ER in controlling SCV division. Interestingly, Salmonella-infected cells show the activation of unfolded protein response (UPR) with expanded ER tubules compared to the uninfected cells. Further, changing the expression of ER morphology regulators, such as reticulon-4a (Rtn4a) and CLIMP63, affected bacterial proliferation significantly, suggesting a potential role for tubular ER in facilitating the SCV division. Live-cell imaging analysis shows the marking of tubular ER precisely at the center of the majority of SCV division (78%) sites. We have investigated the role of SteA, (a known Salmonella effector essential for modulating the membrane dynamics) in coordinating the SCV division. We observed that SteA resides on the SCV membranes and helps in making membrane contact sites between SCV and ER. Moreover, we have observed that the colocalization of ER with SCV enclosing SteA mutant Salmonella (STMΔsteA) was significantly reduced compared to SCV-formed by wild-type Salmonella. The loss of function of steA in Salmonella resulted in profound defects in SCV division, leading to multiple bacteria residing in a single vacuole and attenuated proliferation compared to the wild-type STM in epithelial cells. However, during infection in mice, the STMΔsteA mutant did not show any defect in colonization, but STM WT-infected mice started succumbing to death earlier than STMΔsteA infected mice. Overall, our study suggests a coordinated role of Salmonella effector SteA in promoting the ER contact sites with SCVs and thus regulating the successful division of Salmonella containing vacuole. Objective 2: Investigating the mechanism behind the upregulation of programmed death ligand 1 (PDL1) by Salmonella Typhimurium Recent reports highlight the importance of PDL1 upregulation upon Salmonella infection in culling the T-cell. However, the mechanism behind this upregulation is not known. Our findings suggest that the upregulation of PDL1 is through Salmonella pathogenicity island 2 (SPI-2) encoded effectors since PFA-fixed bacteria and STMΔssaV (which is unable to secrete effector proteins) did not alter PDL1 level. We further observed that the vacuolar state of Salmonella is not essential for PDL1 upregulation. Based on previous literature, we have investigated the role of the SPI-2 effector SseL (a deubiquitinase known to affect the NFĸB pathway) and SpvD (cysteine hydrolase) in PDL1 upregulation. Our study identifies SPI-2 effector SseL to be crucial for upregulating PDL1 in cell culture as well as in vivo mice model. However, the loss of function of SpvD did not alter PDL1 levels in mice. Hence, further, we focused our study on Salmonella effector SseL to unravel the mechanism behind PDL1 upregulation. An increase in the level of PDL1 by STM WT helps in colonization in the secondary sites of infection in C57BL/6 mice, such as the liver and spleen, while STMΔsseL shows defects in colonization. Despite the diminished colonization of STMΔsseL, the infected mice succumb to death earlier with high-grade inflammation. We further delineated the molecular mechanism behind PDL1 upregulation and observed that bacterial effector SseL helps in the stabilization of β-catenin inside the cell. β-catenin thus translocates to the nucleus and directly regulates the transcriptional levels of PDL1, which is abrogated upon using β-catenin/TCF inhibitor FH535. Thus, our study shows the mechanism behind Salmonella mediated immune suppression via PDL1. Overall, our finding suggests the mechanism behind the division of Salmonella containing vacuole (SCV) and, hence, maintaining a single bacterium per vacuole state. Also, we have uncovered the role of bacterial effector SseL essential for PDL1 upregulation and evasion from host immune response.
    URI
    https://etd.iisc.ac.in/handle/2005/6973
    Collections
    • Microbiology and Cell Biology (MCB) [253]

    Related items

    Showing items related by title, author, creator and subject.

    • Typhoidal And Non-Typhoidal Salmonella Serovars - A Comparartive Study 

      Arvindhan, G N (2011-02-08)
      Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes ...
    • Survival Strategies Of SALMONELLA 

      Sandeepa, M E (2010-08-25)
      The genus Salmonella includes facultative intracellular pathogens. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans killing about 2,00,000 people globally every year. Salmonella enterica serovars ...
    • Racemases in Salmonella : Insights into the Dexterity of the Pathogen 

      Iyer, Namrata (2018-02-27)
      Chapter -I Introduction Salmonella is a pathogen well-known for its ability to infect a wide variety of hosts and causes disease ranging from mild gastroenteritis to typhoid fever. During infection, it is exposed to a ...

    etd@IISc is a joint service of SERC & J R D Tata Memorial (JRDTML) Library || Powered by DSpace software || DuraSpace
    Contact Us | Send Feedback | Thesis Templates
    Theme by 
    Atmire NV
     

     

    Browse

    All of etd@IIScCommunities & CollectionsTitlesAuthorsAdvisorsSubjectsBy Thesis Submission DateThis CollectionTitlesAuthorsAdvisorsSubjectsBy Thesis Submission Date

    My Account

    LoginRegister

    etd@IISc is a joint service of SERC & J R D Tata Memorial (JRDTML) Library || Powered by DSpace software || DuraSpace
    Contact Us | Send Feedback | Thesis Templates
    Theme by 
    Atmire NV