Cell Biology & Molecular Genetics
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Item CHARACTERIZATION OF THE ROLE OF MAPKS IN LEISHMANIA INFECTED MACROPHAGES.(2009) Yang, Ziyan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the current study, we examined the role of the Mitogen Activated Protein Kinases (MAPKs) on the biological responses of macrophages infected with Leishmania. The first section examined the role of MAPK/ERK in IL-10 production by Leishmania-infected macrophages. The macrophage-derived IL-10 has been shown to exacerbate Leishmaniasis. However, the molecular mechanisms whereby Leishmaniasis prompts IL-10 induction are poorly understood. A combination of two signals was necessary for IL-10 induction by the Leishmania amastigotes-infected macrophages. The first signal is mediated by TLR ligation whereas the second signal is mediated by FcgammaR ligation, which yields a population of regulatory macrophages that produce high levels of IL-10. Infection of macrophages with L. amazonensis amastigotes from the lesion sites sparked MAPK/ERK activation, which was required, but not sufficient for IL-10 induction. In combination with an inflammatory stimulus, LMW-HA from the extracellular matrix, these parasites triggered the macrophages to highly produce IL-10. MAPK/ERK activation initiated an epigenetic modification of chromatin at the IL-10 locus, which allowed for transcription factor Sp1 binding to drive IL-10 transcription and subsequent production. U0126, an inhibitor of MAPK/ERK activation, decreased lesion progression in Leishmania infected mice. The second section examined the role of MAPK/p38 in cytokine production and vaccination against Leishmaniasis. TLR agonists activate macrophages to produce pro-inflammatory cytokines and reactive oxygen intermediates. Inhibition of MAPK/p38 reciprocally increased IL-12 but decreased TNFa production from LPS-stimulated macrophages, which also occurred following stimulation by a variety of other TLR agonists, and using different APCs. MAPK/p38 inhibition induced IL-12p40 mRNA accumulation mainly due to enhanced mRNA stability, which was independent of IL-10. Similar results were observed by knocking down MAPK/p38 using specific siRNAs or by targeted deletion of MKK3. IL-12 production following the inhibition of MAPK/p38 skewed antigen-specific T cells to produce more IFN-gamma and less IL-4 in vitro. A MAPK/p38 inhibitor was applied as an adjuvant to vaccine mice against L. major, which resulted in smaller lesions with fewer parasites. Our findings reveal an important role of MAPKs in the Leishmania pathogenesis, and suggest that the manipulation of these kinases may provide novel therapeutics for potential clinical applications.Item Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.Item Phylogenetic analysis of swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan(2009) Padmanabhan, Rangarajan; Perez, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continuous surveillance of influenza becomes important considering the economic, epidemic and pandemic implications of influenza infections. This study details phylogenetic & molecular analysis of the genes of four swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan. Phylogenetic analysis placed the eight segments of the four viruses in the classical H1N1 swine clade, along with the isolate A/sw/Jamesburg/1942, except for the HA of A/Alma-Ata/32/98, which was placed in the human H1N1 lineage, along with the isolate A/WS/1933. On amino acid analysis, the viruses displayed mutations on HA and ribonucleoproteins which putatively disrupt antigenic recognition of the virus by the host immune system. The presence of these viruses relatively unchanged for 6 decades after their initial isolation could be speculated to be a combination of laboratory leaks in southern USSR in 1980s, low divergence of classical H1N1 viruses in pigs, and the low population density of Kazakhstan.Item COMPARING LEVELS OF NDH-1 DEHYDROGENASE ACTIVITY IN DIFFERENT MYCOBACTERIAL SPECIES.(2008) Azogue, Sharon; Briken, Volker; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The nuoG gene of Mycobacterium tuberculosis (Mtb) has the ability to inhibit host cell apoptosis. This ability is a virulence factor and does not exist in facultative pathogenic and non-pathogenic mycobacterial species. NuoG is part of the NDH-1 complex, and this study addressed the potential link between the role of NuoG in apoptosis inhibition and the biochemical activity of the NDH-1 complex. Different mycobacterial species were tested for their NDH-1 activities. Among the bacteria tested were bacteria transformed with the Mtb nuoG plasmid, or with the almost entire NDH-1 coding region. Surprisingly, the levels of NDH-1 activity did not correlate with apoptosis levels, suggesting a potential independent, novel mechanism by which NuoG inhibits host cell apoptosis.Item Binding Interactions in the Bacterial Chemotaxis Signal Transduction Pathway(2008-12-08) Eaton, Anna Kolesar; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The investigation of signal transduction pathways is critical to the basic understanding of cellular processes as these pathways function to regulate diverse processes in both eukaryotes and prokaryotes. This dissertation focuses on understanding some of the biochemical events that take place in the chemotaxis signal transduction pathway of bacteria. In this system, cell-surface receptor proteins regulate a histidine protein kinase, CheA, that autophosphorylates and then transfers its phosphate to an effector protein, CheY. Phospho-CheY, in turn, influences the direction of flagellar rotation. This sequence of biochemical events establishes a chain of communication that ultimately allows the chemotaxis receptor proteins to regulate the swimming pattern of the bacterial cell when it encounters gradients of attractant and repellent chemicals in its environment. The three projects presented in this dissertation sought to fill basic gaps in our current understanding of CheA and CheY function. In the first project, I examined the nucleotide binding reaction of CheA using the fluorescent nucleotide analogue, TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate]. TNP-ATP is an effective inhibitor for CheA. By monitoring the fluorescence of TNP-ATP when it bound to CheA, I examined the affinity of the binding interaction and discovered that the two ATP binding sites of each CheA dimer exhibited negative cooperativity in their interactions with TNP-ATP. This is the first evidence of cooperativity in the histidine protein kinase superfamily. In the second project, I focused on elucidating the binding mechanism that underlies formation of the CheA:TNP-ATP complex. My results indicated a three-step mechanism, including rapid formation of a low-affinity complex, followed by two steps during which conformational changes give rise to the final high-affinity complex. This same basic mechanism applied to CheA from Escherichia coli and from Thermotoga maritima. In the third project, I turned my attention to studying the CheY phosphorylation and binding reactions using fluorescently labeled versions of CheY. The results of this final study indicated that CheY proteins labeled with the fluorophore Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] could be useful tools for investigating CheY biochemistry. However my results also brought to light some of the limitations and difficulties of this approach.Item Diversity of conjugating green algae; phylogenetic studies of a species-rich eukaryotic lineage(2007-12-21) Hall, John David; Delwiche, Charles F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This thesis used molecular phylogenetic techniques to investigate diversity in the conjugating green algae. The conjugating green algae are closely related to land plants and evolution within the group provides a good analogy of how evolution may have proceeded in the lineage that gave rise to land plants. I developed a dataset of the genes coxIII, psaA and rbcL with 109 taxa to determine phylogenetic relationships of the families and genera. I found that the order Zygnematales is not monophyletic and that Spirogyra was the first to branch. The order Desmidiales is monophyletic if one includes the genus Roya. The family Peniaceae is not monophyletic. The genera Cosmarium, Cylindrocystis, Mesotaenium, Penium, Spondylosium, Staurodesmus and in later studies Desmidium and Hyalotheca were found to be paraphyletic or polyphyletic. Investigation of cell division syndromes among filamentous Desmidiaceae revealed greater diversity than was previously reported. Notable among these discoveries is that Spondylosium pulchrum displays the Desmidium-type cell division, Spondylosium pulchellum the Cosmarium-type, and Spondylosium tetragonum the newly described Teilingia-type cell division. The relationship among the syndromes was inferred from phylogenetic analysis of the species that revealed a single lineage comprising filamentous and colonial species and multiple modes of cell division. This suggests that even the fundamental process of cell division can be highly modified. Results from this study also resulted in the taxonomic resurrection of the genus Didymoprium, as well as the creation of the new genus Isthmocatena and the combinations Didymoprium grevillei, Desmidium pulchrum, and Isthmocatena pulchella. Investigations of the Gonatozygaceae revealed unexpected diversity in Gonatozygon brebissonii and G. kinahani. Structural measurements were sufficient to distinguish among strains of Gonatozygon species except for Gonatozygon brebissonii. We have probably underestimated genetic and species diversity in this family. In contrast, the structurally distinct species Triploceras gracile, was found to be closely related to Micrasterias. This relationship was confirmed by sequencing and phylogenetic analysis of the nuclear encoded EF1α, EIF4 and TUA. The results of this study indicate that Triploceras is probably actually bilaterally symmetric, although it has been treated as a radially symmetric species.Item Mechanisms Utilized by Neisseria gonorrhoeae to Persist in the Human Cervical Epithelium: Signaling, Invasion, Intracellular Survival, and Escape(2007-11-07) Bish, Samuel Earl; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The obligate human pathogen Neisseria gonorrhoeae (gonococci) causes the sexually transmitted disease gonorrhea. Interactions between gonococci and human cervical epithelial cells that promote bacterial invasion and post-invasion survival were examined. The data showed that gonococci activated MAPK signaling in cervical epithelial cells prior to invasion. Gonococci triggered prolonged ERK phosphorylation (possibly through EGFR signaling), which could facilitate gonococcal invasion. Furthermore, gonococci expressing lacto-N-neotetraose LOS decreased JNK activation and elicited lower IL-8 production from host cells compared to variants expressing lactosyl LOS. I propose that gonococci can manipulate MAPK signaling to facilitate invasion into cervical epithelial cells and to evade the innate immune response. To investigate gonococcal invasion, I expressed β-lactamase on the outer membrane of gonococcal strain FA1090 to generate the reporter strain FA1090 psi(bla-iga'). Utilizing a β-lactamase reporter assay, I demonstrated that FA1090 psi(bla-iga') only invaded a subpopulation of cervical epithelial cells. Nonviable FA1090 psi(bla-iga') did not invade host cells and failed to recruit F-actin to sites of adherence. Viable gonococci required pili expression but not Opa expression to invade susceptible host cells. These data suggest that viable gonococci elicit invasion-promoting interactions with host cells not triggered by nonviable gonococci. Finally, the data demonstrated that gonococci realize different intracellular fates after entry into cervical epithelial cells. I developed a tannic acid survival assay to show that intracellular gonococci survived within host cells. Intracellular survival did not require catalase or the NfsB nitrogen reductase. A subpopulation of gonococci could escape from cells back into the extracellular milieu. Gonococci utilized an exocytosis pathway to escape after invasion that was blocked by treating cells with tannic acid, but not cytoskeletal inhibitors. I propose a working scenario whereby gonococci undergo cycles of invasion into and escape from a susceptible subpopulation of cervical epithelial cells. Gonococci that survive within host cells may represent bacteria that progress into deeper host tissues to disseminate to secondary sites. Escaping gonococci could invade into other host cells and are well-positioned for transmission to another host. Gonococci interact with the cervical epithelium through invasion, intracellular survival, and escape to establish bacterial persistence in the female host.Item Interferon Gamma Inducible Protein 10 (IP-10) and Regulatory T Cells in Leishmaniasis(2007-08-03) Munoz Roldan, Melba Lucia; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leishmania are intracellular parasites that reside inside macrophages and induce weak innate immune responses. We hypothesize that transgenic parasites that express immune response genes could modify the nature of the host response to the parasite. We previously generated transgenic parasites that produce interferon gamma inducible protein 10 (IP-10). C57BL/6 mice are resistant to wild type L. major infection but when infected with IP-10 transgenic parasites they develop large lesions in the footpad that do not resolve. Recently it was discovered that nTregs express CXCR3, the receptor for IP-10. We hypothesized that IP-10 transgenic parasites could actively recruit nTregs and thereby enhance parasite persistence. We found higher numbers of CD4+CD25+Foxp3+ cells in the draining lymph nodes of IP-10 parasites infected mice compared to wild type infected mice. This work suggests that IP-10 secreting parasites might recruit a population of regulatory T cells that modulates the immune response to the parasite allowing parasite persistence.Item The Influence of Parasite-derived Chemokines in Leishmaniasis(2006-12-13) Conrad, Sean Martin; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Transgenic chemokine-secreting parasites were generated and used to actively recruit immune cells into Leishmania lesions. It was hypothesized that the chemokine induced cell migration would influence the magnitude and character of the immune response and thereby effect the outcome of disease. Two different transgenic chemokine-secreting parasites were engineered. One transgenic parasite secretes murine MCP-1, a CC chemokine primarily responsible for macrophage recruitment. The other transgenic parasite secretes murine IP-10, a CXC chemokine known to attract activated T-cells. Both transgenic parasites transcribed murine chemokine mRNA, translated murine chemokine protein, and infected and replicated inside resting peritoneal macrophages similar to wild-type parasites. However, the two transgenic parasites caused diverse phenotypes in infected mice. The MCP-1 secreting parasites caused little or no detectable lesions, while the IP-10 secreting parasites caused lesions that were significantly larger than the wild-type infected mice. The healing phenotype caused by MCP-1 secreting parasites was further analyzed. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with MCP-1 secreting parasites resulted in minimal lesion development compared to mice infected with wild-type parasites. MCP-1 secreting parasites caused substantial lesions with relatively high numbers of parasites in CCR2 KO mice indicating that the parasites are viable and healthy, and that the lack of lesion development is CCR2- dependent. The enumeration of transgenic MCP-1 parasites in lesions demonstrated a significant reduction in parasite numbers, which coincided with an increase in CCR2-positive macrophage migration on day 7. CCR2-positive macrophages isolated from ears of mice infected with transgenic MCP-1 parasites contained virtually no parasites, whereas infection with wild-type parasites resulted in heavily-infected macrophages in lesions. The lack of parasite survival in mice infected with MCP-1 secreting parasites suggests that parasite-derived MCP-1 is recruiting a population of CCR2-positive macrophages to the lesion that efficiently kill Leishmania parasites. In-vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages followed by stimulation with a combination of both MCP-1 and IFN-g. This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-g to efficiently kill Leishmania parasites.Item A NEW INSIGHT INTO MYCOBACTERIUM RESISTANCE TO REACTIVE OXYGEN INTERMEDIATES(2006-12-22) Kish, Rabab; Gao, Lian-Yong; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genes encoded by the RD1 locus are known to be important for intracellular survival of pathogenic mycobacterium, however their role in counteracting host defense is not known. I hypothesize that RD1 is involved in counteracting host oxidative response by secreting ROI-neutralizing enzymes such as catalase and superoxide dismutase. In support of this hypothesis I have shown that M. marinum RD1 mutants are more sensitive than wild type to ROIs both in vitro and in BMDM. Western blot analysis on the KatG protein levels within the bacterial cells grown in 7H9 rich medium demonstrated that wild type bacteria produced significantly higher amount of KatG than the RD1 mutants. When the bacteria were exposed for 2 hours to H2O2, wild type showed a significant reduction of the KatG level, while the RD1 mutants maintained constant levels of KatG, suggesting that RD1 genes might be involved in the secretion of KatG upon exposure to H2O2. These results together demonstrate an important unknown function of RD1 in resistance to ROIs.