Center for Microbial Interface Biology

Columbus, OH, United States

Center for Microbial Interface Biology

Columbus, OH, United States
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Kulkarni M.M.,Center for Microbial Interface Biology | Barbi J.,Center for Microbial Interface Biology | Barbi J.,Ohio State University | Mcmaster W.R.,University of British Columbia | And 5 more authors.
Cellular Microbiology | Year: 2011

Cathelicidin-type antimicrobial peptides (CAMP) are important mediators of innate immunity against microbial pathogens acting through direct interaction with and disruption of microbial membranes and indirectly through modulation of host cell migration and activation. Using a mouse knock-out model in CAMP we studied the role of this host peptide in control of dissemination of cutaneous infection by the parasitic protozoan Leishmania. The presence of pronounced host inflammatory infiltration in lesions and lymph nodes of infected animals was CAMP-dependent. Lack of CAMP expression was associated with higher levels of IL-10 receptor expression in bone marrow, splenic and lymph node macrophages as well as higher anti-inflammatory IL-10 production by bone marrow macrophages and spleen cells but reduced production of the pro-inflammatory cytokines IL-12 and IFN-γ by lymph nodes. Unlike wild-type mice, local lesions were exacerbated and parasites were found largely disseminated in CAMP knockouts. Infection of CAMP knockouts with parasite mutants lacking the surface metalloprotease virulence determinant resulted in more robust disseminated infection than in control animals suggesting that CAMP activity is negatively regulated by parasite surface proteolytic activity. This correlated with the ability of the protease to degrade CAMP in vitro and co-localization of CAMP with parasites within macrophages. Our results highlight the interplay of antimicrobial peptides and Leishmania that influence the host immune response and the outcome of infection. © 2011 Blackwell Publishing Ltd.

Azad A.K.,Center for Microbial Interface Biology | Knoell D.,Ohio State University | Schlesinger L.S.,Center for Microbial Interface Biology
Genes and Immunity | Year: 2013

Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We used allelic mRNA expression imbalance (AEI) technology for surfactant protein (SP)-A1, SP-A2, SP-D, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), macrophage mannose receptor (MRC1) and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker single-nucleotide polymorphisms (SNPs) and was confirmed by next-generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% minor allele frequency); the only variant consistently associated with an approximately twofold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial linkage disequilibrium with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases. © 2013 Macmillan Publishers Limited.

Soni S.,Center for Microbial Interface Biology | Soni S.,Ohio State University | Ernst R.K.,University of Maryland, Baltimore | Muszynski A.,University of Georgia | And 5 more authors.
Frontiers in Microbiology | Year: 2010

Francisella tularensis is a CDC Category A biological agent and a potential bioterrorist threat. There is no licensed vaccine against tularemia in the United States. A long-standing issue with potential Francisella vaccines is strain phase variation to a gray form that lacks protective capability in animal models. Comparisons of the parental strain (LVS) and a gray variant (LVSG) have identified lipopolysaccharide (LPS) alterations as a primary change. The LPS of the F. tularensis variant strain gains reactivity to F. novicida anti-LPS antibodies, suggesting structural alterations to the O-antigen. However, biochemical and structural analysis of the F. tularensis LVSG and LVS LPS demonstrated that LVSG has less O-antigen but no major O-antigen structural alterations. Additionally, LVSG possesses structural differences in both the core and lipid A regions, the latter being decreased galactosamine modification. Recent work has identified two genes important in adding galactosamine (flmF2 and flmK) to the lipid A. Quantitative real-time PCR showed reduced transcripts of both of these genes in the gray variant when compared to LVS. Loss of flmF2 or flmK caused less frequent phase conversion but did not alter intramacrophage survival or colony morphology. The LVSG strain demonstrated an intramacrophage survival defect in human and rat but not mouse macrophages. Consistent with this result, the LVSG variant demonstrated little change in LD50 in the mouse model of infection. Furthermore, the LVSG strain lacks the protective capacity of F. tularensis LVS against virulent Type A challenge. These data suggest that the LPS of the F. tularensis LVSG phase variant is dramatically altered. Understanding the mechanism of blue to gray phase variation may lead to a way to inhibit this variation, thus making future F. tularensis vaccines more stable and efficacious. © 2010 Soni, Ernst, Muszynski, Mohapatra, Perry, Vinogradov, Carlson and Gunn.

Abdulrahman B.A.,Center for Microbial Interface Biology | Khweek A.A.,Center for Microbial Interface Biology | Akhter A.,Center for Microbial Interface Biology | Caution K.,Center for Microbial Interface Biology | And 12 more authors.
Autophagy | Year: 2011

Cystic fibrosis (CF) is the most common inherited lethal disease in Caucasians which results in multiorgan dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 (ΔF508) mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 macrophages than in WT macrophages. An autophagosome is a compartment that engulfs nonfunctional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagystimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation. © 2011 Landes Bioscience.

Haghighat A.-C.,Center for Microbial Interface Biology | Seveau S.,Center for Microbial Interface Biology
Journal of Immunological Methods | Year: 2010

We describe an automated fluorescence microscopy-based assay that quantifies the invasion of mammalian cells by intracellular pathogens. Pathogens associated with host cell surfaces, intracellular pathogens and mammalian cells are directly counted based on their specific fluorescent labeling. Such approach utilizes automated image acquisition and processing, and is thus ideally suited for high-throughput analyses. This method was validated using Listeria monocytogenes as a model intracellular pathogen.

Torrelles J.B.,Center for Microbial Interface Biology | Schlesinger L.S.,Center for Microbial Interface Biology
Tuberculosis | Year: 2010

Mycobacterium tuberculosis (the causal agent of TB) has co-evolved with humans for centuries. It infects via the airborne route and is a prototypic highly adapted intracellular pathogen of macrophages. Extensive sequencing of the M. tuberculosis genome along with recent molecular phylogenetic studies is enabling us to gain insight into the biologic diversity that exists among bacterial strains that impact the pathogenesis of latent infection and disease. The majority of the M. tuberculosis cell envelope is comprised of carbohydrates and lipids, and there is increasing evidence that these microbial determinants that are readily exposed to the host immune system play critical roles in disease pathogenesis. Studies from our laboratory and others have raised the possibility that M. tuberculosis is adapting to the human host by cloaking its cell envelope molecules with terminal mannosylated (i.e. Man-α-(1 → 2)-Man) oligosaccharides that resemble the glycoforms of mammalian mannoproteins. These mannosylated biomolecules engage the mannose receptor (MR) on macrophages during phagocytosis and dictate the intracellular fate of M. tuberculosis by regulating formation of the unique vesicular compartment in which the bacterium survives. The MR is highly expressed on alveolar macrophages (predominant C-type lectin on human cells) and functions as a scavenger receptor to maintain the healthiness of the lung by clearing foreign particles and at the same time regulating dangerous inflammatory responses. Thus M. tuberculosis exploits MR functions to gain entry into the macrophage and survive. Key biochemical pathways and mycobacterial determinants involved in the development and maintenance of the M. tuberculosis phagosome are being identified. The phylogenetic diversity observed in M. tuberculosis strains that impact its cell wall structure together with the genetic diversity observed in human populations, including those elements that affect macrophage function, may help to explain the extraordinary evolutionary adaptation of this pathogen to the human host. Major developments in these areas are the focus of this review. © 2010 Elsevier Ltd. All rights reserved.

Day J.,Ohio State University | Schlesinger L.S.,Center for Microbial Interface Biology | Friedman A.,Ohio State University
Tuberculosis | Year: 2010

Due to the complexity of the immune response to a Mycobacterium tuberculosis infection, identifying new, effective therapies and vaccines to combat it has been a problematic issue. Although many advances have been made in understanding particular mechanisms involved, they have, to date, proved insufficient to provide real breakthroughs in this area of tuberculosis research. The term "Translational Systems Biology" has been formally proposed to describe the use of experimental findings combined with mathematical modeling and/or engineering principles to understand complex biological processes in an integrative fashion for the purpose of enhancing clinical practice. This opinion piece discusses the importance of using a Translational Systems Biology approach for tuberculosis research as a means by which to go forward with the potential for significant breakthroughs to occur. © 2009 Elsevier Ltd. All rights reserved.

Yang L.,Center for Microbial Interface Biology | Sinha T.,Center for Microbial Interface Biology | Carlson T.K.,Center for Microbial Interface Biology | Carlson T.K.,Biomedical Research Tower | And 4 more authors.
Glycobiology | Year: 2013

One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed. © 2013 The Author 2013.

Khweek A.A.,Center for Microbial Interface Biology | Khweek A.A.,Ohio State University | Caution K.,Center for Microbial Interface Biology | Caution K.,Ohio State University | And 20 more authors.
European Journal of Immunology | Year: 2013

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PubMed | Center for Microbial Interface Biology and Ohio State University
Type: | Journal: Scientific reports | Year: 2015

Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking.

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