Browsing by Subject "Antigens, Bacterial"
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Item Biochemical Characterization of IpaH E3 Ubiquitin Ligase Effector Proteins and Their Host Substrates(August 2021) Hansen, Justin Mark; Orth, Kim; Sperandio, Vanessa; Reese, Michael L.; Alto, NealShigella flexneri is a gram negative pathogen that utilizes its type 3 secretion system (T3SS) to inject effector proteins in the cytoplasm of host cells to manipulate host cells processes. T3SS effectors are able to post translationally modify host proteins to reprogram intracellular signaling pathways, actin dynamics, membrane trafficking, and innate immune pathways. This allows Shigella to modify the intracellular environment to be conducive to bacterial replication and dissemination to neighboring cells. Shigella flexneri and other bacteria including Salmonella and Yersinia secreted E3 ubiquitin ligases into the host cell cytoplasm via the Type III secretion system (T3SS) apparatus. The invasion plasmid antigen Hs (IpaHs) are a novel family of bacterial E3 ubiquitin ligases that are secreted by Shigella, Salmonella, and Yersinia. These bacterial enzymes highjack the host ubiquitin conjugation machinery by binding to ubiquitin-charged E2 conjugating enzymes and facilitating direct transfer of ubiquitin onto host substrates. IpaH effectors induce polyubiquitination and subsequent proteasomal degradation of their substrates during bacterial infection. The effector substrate interaction of IpaH1.4/2/5 and HOIP was previously characterized. I went on to identify that IpaH2.5 is able to inhibit the in vitro catalytic activity of HOIP via mono-ubiquitination of catalytic lysine residues in the HOIP ring-between-ring domain (RBR-C). Subsequent to this Ubiquitin activated interactive trapping (UBAIT) screening was then utilized to identify the host substrate of IpaH7.8, Gasdermin B (GSDMB). GSDMB belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to inflammatory disease, its function in human physiology remains poorly understood. I investigated a previously unrecognized host-pathogen conflict between GSDMB and the IpaH7.8 effector protein encoded by Shigella flexneri. Through extensive biochemical and cellular characterization, I show that IpaH7.8 ubiquitinates and targets GSDMB for proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing infected cells. Rather, GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system.Item Mycobacterium Tuberculosis Virulence Factor Mpt64 Targets the Endoplasmic Reticulum(2019-04-10) Stamm, Chelsea Elizabeth; Sperandio, Vanessa; Shiloh, Michael; Alto, Neal; Winter, Sebastian E.Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that M. tuberculosis can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, M. tuberculosis is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. I hypothesized that M. tuberculosis secreted proteins that target host membranes are vital for M. tuberculosis to adapt to and manipulate the host environment for survival. Thus, I characterized nearly 200 secreted proteins from M. tuberculosis for their ability to associate with eukaryotic membranes using a live-dead, temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. I identified five M. tuberculosis secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during M. tuberculosis infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in M. tuberculosis pathogenesis and provide a basis for further investigation into their molecular mechanisms.Item Trimming Fat Upon Infection: Proteolytic Demyristoylation as a Novel Bacterial Pathogenic Strategy(2014-08-28) Burnaevskiy, Nikolay; Seemann, Joachim; Chook, Yuh Min; Orth, Kim; Alto, NealHost-pathogen interaction is a complex process that involves an array of molecular tools on both sides of the conflict. In the light of growing antibiotics resistance, understanding of virulence factors utilized by pathogenic bacteria is crucial for developing better treatment for infectious diseases. Shigella spp are food-borne bacterial pathogens and are one of the leading causes of bacterial dysentery worldwide. Similarly to other gram-negative pathogens, Shigella injects effector proteins into an infected cell to control its responses. Dedicated type III secretion system delivers more than twenty effectors which highjack normal cell signaling by modulating activity of host proteins. By interfering with immune signaling, cytoskeleton, and other pathways, effector proteins promote survival of the bacterial pathogen. Inability to secrete effectors severely attenuates Shigella spp, pointing to the crucial role of these proteins in pathogenesis. Therefore, gaining an insight into the mechanism of action of secreted effectors may hold a key to better understanding and treatment of infectious diseases. Here I characterized the Shigella flexneri secreted virulence factor IpaJ. I found that IpaJ is a cysteine protease with novel specificity. IpaJ cleaves a myristoylated amino-terminal glycine of host proteins and therefore irreversibly delipidates them. Although IpaJ can target multiple host proteins, it strongly favors members of ADP-ribosylation factors family (ARF) and related ARF-like (ARL) proteins. By inactivating ARF and ARL proteins, IpaJ disrupts the Golgi structure and prevents secretion through general secretory pathway. I also found that by blocking protein export from the endoplasmic reticulum, IpaJ suppresses the activation of immune signaling pathways. In summary, I characterized a novel virulence factor of Shigella spp, IpaJ. I have shown that IpaJ promotes bacterial pathogenesis and therefore may be of potential interest for therapeutic interventions. The biochemical analysis of the enzyme provides the first look at the mechanism of substrate recognition and highlights the potential use of IpaJ as a research tool to study protein N-myristoylation and the mechanisms of membrane trafficking.