Browsing by Subject "Intracellular pathogen"

Now showing 1 - 4 of 4
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    A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion
    (American Society for Microbiology, 2023) Banerjee, Arkaprabha; Sun, Yuan; Muramatsu, Matthew K.; Toh, Evelyn; Nelson, David E.; Microbiology and Immunology, School of Medicine
    Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.
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    Chlamydia muridarum Genital and Gastrointestinal Infection Tropism Is Mediated by Distinct Chromosomal Factors
    (American Society for Microbiology, 2018-06-21) Morrison, Sandra G.; Giebel, Amanda M.; Toh, Evelyn C.; Spencer, Horace J., III; Nelson, David E.; Morrison, Richard P.; Microbiology and Immunology, School of Medicine
    Some members of the genus Chlamydia, including the human pathogen Chlamydia trachomatis, infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437, tc0438, and tc0439, were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439, was highly attenuated for GI infection and had a GI 50% infectious dose (ID50) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses.
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    Coxiella burnetii Sterol-Modifying Protein Stmp1 Regulates Cholesterol in the Intracellular Niche
    (American Society for Microbiology, 2022) Clemente, Tatiana M.; Ratnayake, Rochelle; Samanta, Dhritiman; Augusto, Leonardo; Beare, Paul A.; Heinzen, Robert A.; Gilk, Stacey D.; Microbiology and Immunology, School of Medicine
    Coxiella burnetii replicates in a phagolysosome-like vacuole called the Coxiella-containing vacuole (CCV). While host cholesterol readily traffics to the CCV, cholesterol accumulation leads to CCV acidification and bacterial death. Thus, bacterial regulation of CCV cholesterol content is essential for Coxiella pathogenesis. Coxiella expresses a sterol-modifying protein, Stmp1, that may function to lower CCV cholesterol through enzymatic modification. Using an Stmp1 knockout (Δstmp1), we determined that Stmp1 is not essential for axenic growth. Inside host cells, however, Δstmp1 mutant bacteria form smaller CCVs which accumulate cholesterol, preferentially fuse with lysosomes, and become more acidic, correlating with a significant growth defect. However, in cholesterol-free cells, Δstmp1 mutant bacteria grow similarly to wild-type bacteria but are hypersensitive to cholesterol supplementation. To better understand the underlying mechanism behind the Δstmp1 mutant phenotype, we performed sterol profiling. Surprisingly, we found that Δstmp1 mutant-infected macrophages accumulated the potent cholesterol homeostasis regulator 25-hydroxycholesterol (25-HC). We next determined whether dysregulated 25-HC alters Coxiella infection by treating wild-type Coxiella-infected cells with 25-HC. Similar to the Δstmp1 mutant phenotype, 25-HC increased CCV proteolytic activity and inhibited bacterial growth. Collectively, these data indicate that Stmp1 alters host cholesterol metabolism and is essential to establish a mature CCV which supports Coxiella growth.
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    Host Lipid Transport Protein ORP1 Is Necessary for Coxiella burnetii Growth and Vacuole Expansion in Macrophages
    (American Society for Microbiology, 2023) Schuler, Baleigh; Sladek, Margaret; Gilk, Stacey D.; Microbiology and Immunology, School of Medicine
    Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a type 4B secretion system to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport and metabolism is critical for successful infection. The mammalian lipid transport protein ORP1L (oxysterol binding protein-like protein 1 Long) localizes to the CCV membrane and mediates CCV-endoplasmic reticulum (ER) membrane contact sites. ORP1L functions in lipid sensing and transport, including cholesterol efflux from late endosomes and lysosomes (LELs), and the ER. Its sister isoform, ORP1S (oxysterol binding protein-like protein 1 Short) also binds cholesterol but has cytoplasmic and nuclear localization. In ORP1-null cells, we found that CCVs were smaller than in wild-type cells, highlighting the importance of ORP1 in CCV development. This effect was consistent between HeLa cells and murine alveolar macrophages (MH-S cells). CCVs in ORP1-null cells had higher cholesterol content than CCVs in wild-type cells at 4 days of infection, suggesting ORP1 functions in cholesterol efflux from the CCV. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrated that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol. IMPORTANCE: Coxiella burnetii is an emerging zoonotic pathogen and bioterrorism threat. No licensed vaccine exists in the United States, and the chronic form of the disease is difficult to treat and potentially lethal. Postinfectious sequelae of C. burnetii infection, including debilitating fatigue, place a significant burden on individuals and communities recovering from an outbreak. C. burnetii must manipulate host cell processes in order to promote infection. Our results establish a link between host cell lipid transport processes and C. burnetii’s avoidance of cholesterol toxicity during infection of alveolar macrophages. Elucidating the mechanisms behind bacterial manipulation of the host will yield insight for new strategies to combat this intracellular pathogen.