Laboratory of Intracellular Parasites

Hamilton, MT, United States

Laboratory of Intracellular Parasites

Hamilton, MT, United States
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de Jong M.F.,University of California at Davis | de Jong M.F.,University of Groningen | Starr T.,Laboratory of Intracellular Parasites | Winter M.G.,University of California at Davis | And 7 more authors.
mBio | Year: 2013

Host cytokine responses to Brucella abortus infection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required for B. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses during B. abortus infection. Importance Brucella species are pathogens that require a type IV secretion system (T4SS) to survive in host cells and to maintain chronic infection. By as-yet-unknown pathways, the T4SS also elicits inflammatory responses in infected cells. Here we show that inflammation caused by the T4SS results in part from the sensing of a T4SS substrate, VceC, that localizes to the endoplasmic reticulum (ER), an intracellular site of Brucella replication. Possibly via binding of the ER chaperone BiP, VceC causes ER stress with concomitant expression of proinflammatory cytokines. Thus, induction of the unfolded protein response may represent a novel pathway by which host cells can detect pathogens deploying a T4SS. © 2013 de Jong et al.

Larson C.L.,Laboratory of Intracellular Parasites | Beare P.A.,Laboratory of Intracellular Parasites | Howe D.,Laboratory of Intracellular Parasites | Heinzen R.A.,Laboratory of Intracellular Parasites
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/ Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXФ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ?cvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrincoated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms.

Bridge D.R.,West Virginia University | Novotny M.J.,West Virginia University | Moore E.R.,Laboratory of Intracellular Parasites | Olson J.C.,West Virginia University
Microbiology | Year: 2010

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration. © 2010 SGM.

Ireland R.,Laboratory of Intracellular Parasites | Wang R.,Laboratory of Intracellular Parasites | Alinger J.B.,Laboratory of Intracellular Parasites | Small P.,University of Tennessee at Knoxville | Bosio C.M.,Laboratory of Intracellular Parasites
Journal of Immunology | Year: 2013

Induction of innate immunity is essential for host survival of infection. Evasion and inhibition of innate immunity constitute a strategy used by pathogens, such as the highly virulent bacterium Francisella tularensis, to ensure their replication and transmission. The mechanism and bacterial components responsible for this suppression of innate immunity by F. tularensis are not defined. In this article, we demonstrate that lipids enriched from virulent F. tularensis strain SchuS4, but not attenuated live vaccine strain, inhibit inflammatory responses in vitro and in vivo. Suppression of inflammatory responses is associated with IkBa-independent inhibition of NF-kBp65 activation and selective inhibition of activation of IFN regulatory factors. Interference with NF-kBp65 and IFN regulatory factors is also observed following infection with viable SchuS4. Together these data provide novel insight into how highly virulent bacteria selectively modulate the host to interfere with innate immune responses required for survival of infection. © 2013 by The American Association of Immunologists, Inc.

Mital J.,Laboratory of Intracellular Parasites | Miller N.J.,Laboratory of Intracellular Parasites | Fischer E.R.,National Institute of Allergy and Infectious Diseases | Hackstadt T.,Laboratory of Intracellular Parasites
Cellular Microbiology | Year: 2010

Summary: Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein-dependent manner to the microtubule-organizing centre (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain-like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis-infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability. Published 2010. This article is a US Government work and is in the public domain in the USA.

Howe D.,Rocky Mountain Laboratories | Shannon J.G.,Rocky Mountain Laboratories | Winfree S.,Laboratory of Intracellular Parasites | Dorward D.W.,National Institute of Allergy and Infectious Diseases | Heinzen R.A.,Rocky Mountain Laboratories
Infection and Immunity | Year: 2010

Coxiella burnetii infects mononuclear phagocytes, where it directs biogenesis of a vacuolar niche termed the parasitophorous vacuole (PV). Owing to its lumenal pH (∼5) and fusion with endolysosomal vesicles, the PV is considered phagolysosome-like. However, the degradative properties of the mature PV are unknown, and there are conflicting reports on the maturation state and growth permissiveness of PV harboring virulent phase I or avirulent phase II C. burnetii variants in human mononuclear phagocytes. Here, we employed infection of primary human monocyte-derived macrophages (HMDMs) and THP-1 cells as host cells to directly compare the PV maturation kinetics and pathogen growth in cells infected with the Nine Mile phase I variant (NMI) or phase II variant (NMII) of C. burnetii. In both cell types, phase variants replicated with similar kinetics, achieving roughly 2 to 3 log units of growth before they reached stationary phase. HMDMs infected by either phase variant secreted similar amounts of the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha. In infected THP-1 cells, equal percentages of NMI and NMII PVs decorate with the early endosomal marker Rab5, the late endosomal/lysosomal markers Rab7 and CD63, and the lysosomal marker cathepsin D at early (8 h) and late (72 h) time points postinfection (p.i.). Mature PVs (2 to 4 days p.i.) harboring NMI or NMII contained proteolytically active cathepsins and quickly degraded Escherichia coli. These data suggest that C. burnetii does not actively inhibit phagolysosome function as a survival mechanism. Instead, NMI and NMII resist degradation to replicate in indistinguishable digestive PVs that fully mature through the endolysosomal pathway. Copyright © 2010, American Society for Microbiology.

Griffin A.J.,Laboratory of Intracellular Parasites | Crane D.D.,Laboratory of Intracellular Parasites | Wehrly T.D.,Laboratory of Intracellular Parasites | Scott D.P.,Rocky Mountain Laboratories | Bosio C.M.,Laboratory of Intracellular Parasites
PLoS ONE | Year: 2013

Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS) nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.

Larson C.L.,Washington State University | Larson C.L.,Laboratory of Intracellular Parasites | Samuelson D.R.,Washington State University | Eucker T.P.,Washington State University | And 2 more authors.
Emerging Microbes and Infections | Year: 2013

Campylobacter jejuni is a gram-negative, curved and rod-shaped bacterium that causes human gastroenteritis. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Epithelial cells infected with C. jejuni strains containing mutations in the FlpA and CadF fibronectin (Fn)-binding proteins exhibit reduced invasion of host cells and a C. jejuni CadF FlpA double mutant is impaired in the activation of epidermal growth factor receptor (EGFR) and Rho GTPase Rac1. Although these observations establish a role for Fn-binding proteins during C. jejuni invasion, their mechanistic contributions to invasion-associated signaling are unclear. We examined FlpA, a C. jejuni Fn-binding protein composed of three FNIII-like repeats D1, D2 and D3, to identify the interactions required for cellular adherence on pathogen-induced host cell signaling. We report that FlpA binds the Fn gelatin-binding domain via a motif within the D2 repeat. Epithelial cells infected with a flpA mutant exhibited decreased Rac1 activation and reduced membrane ruffling that coincided with impaired delivery of the secreted Cia proteins and reduced cell association. Phosphorylation of the Erk1/2 kinase, a downstream effector of EGFR signaling, was specifically associated with FlpA-mediated activation of β 1-integrin and EGFR signaling. In vivo experiments revealed that FlpA is necessary for C. jejuni disease based on bacterial dissemination to the spleen of IL-10-/-germ-free mice. Thus, a novel Fn-binding motif within FlpA potentiates activation of Erk1/2 signaling via β 1-integrin during C. jejuni infection. © 2013 SSCC.

Olivares-Zavaleta N.,Laboratory of Intracellular Parasites | Whitmire W.,Laboratory of Intracellular Parasites | Gardner D.,U.S. National Institutes of Health | Caldwell H.D.,Laboratory of Intracellular Parasites
Vaccine | Year: 2010

Here we report on the safety, immunogenicity, and vaccine efficacy of the naturally occurring plasmid-free attenuated Chlamydia trachomatis L2-25667R (L2R) strain in a murine infection model. Intravaginal immunization induced both chlamydial specific serum antibody and systemic CD4+ Th1 biased immune responses but failed to induce local IgA antibodies. Immunization induced no pathological changes in the urogenital tract. Protective immunity was evaluated by vaginal challenge with a natural occurring non-attenuated plasmid positive C. trachomatis urogenital strain (serovar D). Vaccinated mice were not protected from colonization/infection but exhibited a reduction in infectious burden at early time periods (1-2 weeks) post-challenge. Partial protective immunity did not protect against inflammatory disease. Thus, intravaginal vaccination with the live-attenuated L2R stain is safe, induces a systemic antibody and CD4+ Th1 biased immune response, but its protective efficacy is limited to reducing chlamydial burden at early time periods post-infection.

Chase J.C.,Laboratory of Intracellular Parasites | Bosio C.M.,Laboratory of Intracellular Parasites
Infection and Immunity | Year: 2010

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.

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