Browsing by Subject "Salmonella"

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular “bucket lists” to be accomplished before a cell dies. As demonstrated here, engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We engineer S. Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. However, after 48 hours the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFNg-deficient mice this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell’s physiology that make host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.

The relatively simple innate immune system of Caenorhabditis elegans and the number of traits that facilitate genetic and genomic analysis using this organism have nurtured rapid advances into the understanding of C. elegans innate immunity during the last few years. However, traditional methods of isolating and mapping C. elegans mutants exhibiting aberrant immune responses to pathogen infection are often labor intensive and time consuming. Therefore, a simple and rapid means of isolating and mapping C. elegans immune mutants will increase the number of mutants that can be studied. Salmonella enterica, as well as other bacterial pathogens, has been described to cause a significant distension of the C. elegans intestinal lumen, which correlates with death of the nematode. C. elegans mutants which exhibit a weakened immune response would therefore be expected to develop intestinal distension at an earlier time point than wild type. Likewise, mutants which exhibit an enhanced immune response would be expected to develop intestinal distension at a later time point than wild type. Taking advantage of this correlation, we designed a novel approach to isolating C. elegans mutants which exhibit aberrant immune responses to the bacterial pathogen, S. enterica. Furthermore, we validated and optimized the use of Amplifluor®, a high-throughput genotyping system, for use in C. elegans single nucleotide polymorphism (SNP) mapping.

To date, the only known negative regulators of innate immunity in C. elegans are dependent on the FOXO transcription factor, DAF-16 and regulate lifespan in addition to immunity. Therefore, we focused our efforts on identifying additional negative regulators of innate immunity by screening for mutants which display a reduced accumulation of S. enterica at a time point when wild-type nematodes are packed with bacteria. In a genetic screen for C. elegans mutants which display reduced accumulation of S. enterica/GFP, we identified a mutation in nol-6, a nucleolar protein containing a nucleolar RNA-associated protein (Nrap) domain which is conserved across eukaryotic organisms. nol-6 is implicated in ribosomal RNA (rRNA) processing during the early stages of ribosome biogenesis. We show that knockdown of nol-6 as well as other nucleolar genes leads to a reduction of pathogen accumulation and enhanced resistance to killing by pathogen. In addition, we demonstrate that enhanced resistance is dependent on p53/cep-1. Furthermore, microarray analysis shows a significant enrichment of upregulated genes that have previously been shown to be dependent on p53/cep-1 for induction following ultraviolet radiation. These results represent the first evidence that C. elegans innate immunity is regulated by the nucleolus through a p53/cep-1-dependent mechanism.

If nothing else, the 2019 Coronavirus pandemic has made it abundantly clear that understanding the mechanisms of infectious disease is imperative to the survival of our species. While the last fifty years of developments in molecular biology has accelerated our ability to study microbial pathogens, limitations in pathogen tropism, microbial survival in laboratory conditions, uneven sampling of human cohorts across geographical and socioeconomic lines, and heterogeneous complexity during human infection have limited our ability to study complex mechanisms of human susceptibility to infectious disease. In this work, I build on recent developments in utilizing High-throughput Human in vitro Susceptibility Testing (Hi-HOST) to not only (a) identify novel sites in the human genome that contribute to natural variation in infectious disease susceptibility based on highly quantifiable cellular phenotypes, but (b) use these sites as a springboard to understand the entire, complex host-pathogen interaction. From this perspective, I paired the model pathogen Salmonella enterica and the Hi-HOST system to identify that natural variation in the mammalian gene arhgef26 contributes to susceptibility to Salmonella invasion. I used this finding as a starting point to fully explore the role of ARHGEF26 during infection, redefining its role in invasion, inflammation, and its interaction with host and bacterial proteins during the process. Similarly, I used prior Hi-HOST findings that methionine metabolism influences the host response to Salmonella enterica serovar Typhimurium (S. Typhimurium) as a launching point to investigate the impacts of host and bacterial metabolism on the virulence of S. Typhimurium. I found that the metabolite methylthioadenosine is a potent inhibitor of S. Typhimurium type III secretion, motility, and invasion. Finally, I mechanistically explain some of these findings by linking methionine metabolism to DNA methylation using a novel approach to integrate the Salmonella Typhimurium methylome and transcriptome. In sum, these findings demonstrate the ability for cellular GWAS to serve as a launching point to understand complex host-pathogen interactions.