Elucidation of innate immune responses using C. elegans as a model host.
Mechanisms of gene regulation in the human pathogen Enterococcus faecalis.
The emergence of untreatable bacterial infection in modern medicine is due to several factors. The hospital patient population is increasingly elderly and immune-compromised, creating a pool of susceptible hosts. The overuse of antibiotics has provided the necessary selective pressure for the development of resistance. Pathogenic bacteria have seemingly endless versatility in creating and sharing mechanisms of resistance. As the development of antibiotic resistance continues to erode one of the greatest advances in modern health care, it is crucial to identify alternative strategies that can form the basis of novel anti-infective therapies. One approach is to target the immune response to more effectively dispel infection.
To this end, one focus of my laboratory’s research includes studies of the host response to pathogens using a tiny worm called C. elegans as a model host. C. elegans has favorable characteristics that include a short 3-day lifecycle during which hundreds of progeny are produced, small size and ease of laboratory cultivation, a fully sequenced genome and a vast array of molecular and genetic tools and resources. Interestingly, C. elegans become sick and die when fed on many human pathogens, making this approach possible. Importantly, many of the same immune defense signaling pathways and mechanisms employed by higher animals appear to also be at play in the worm. For example, my laboratory discovered that C. elegans produces reactive oxygen species (ROS) in response to pathogens, a defense mechanism analogous to the oxidative burst that occurs in human phagocytic cells. We are in the process of identifying the machinery and the regulators that generate this response, characterizing its role in C. elegans immunity, and ultimately applying our knowledge to better understand this response in humans.
Our studies of host immune response to human bacterial infection are particularly focused on Enterococcus faecalis now the second or third most common hospital-acquired infection, but amenable to laboratory studies due to the existence of complete array of molecular tools. In a screen for mutants attenuated in killing C. elegans we discovered that mutations in an operon encoding for ethanolamine utilization affected virulence. Although possible connections between ethanolamine utilization and bacterial pathogenesis are documented in the literature (Garsin 2010), we have focused our initial studies on dissecting the regulation of this operon, as it contains several novel features associated with post-transcriptional regulation. These include an AdoCbl-binding riboswitch and a series of transcription terminators regulated by an RNA-binding two-component system. With the growing realization of the importance of regulatory RNAs to the control of biological systems, we are well poised to make significant discoveries investigating this system.
Armand O. Brown
Postdoctoral Research Fellow
Description of Project:
In previous work investigating the interactions between the polymorphic commensal fungus Candida albicans and E. faecalis, we identified a novel bacteriocin produced by E. faecalis known as EntV. Preliminary characterization has revealed that it inhibits C. albicans hyphal morphogenesis and biofilm formation, and is likely post-translationally modified by the combined activity of gelatinase (GelE) and serine protease (SprE). Recently, we identified a predicted thioredoxin known as DsbA within the E. faecalis OG1RF genome that we believe also contributes to the post-translational processing of EntV. Since EntV has potential as an antifungal therapy, it is important to understand these post-translational modifications and how they contribute to EntV’s activity.
- Brown AO, Mann B, Gao G, Hankins JS, Humann J, Giardina J, Faverio P, Restrepo MI, Halade GV, Mortensen EM, Lindsey ML, Hanes M, Happel KI, Nelson S, Bagby GJ, Lorent JA, Cardinal P, Granados R, Esteban A, LeSaux CJ, Tuomanen EI, Orihuela CJ. (2014) Streptococcus pneumoniae translocates into the myocardium and forms unique microlesions that disrupt cardiac function. PLoS Pathogens, Vol. 10, No. 9, pp. 1-14
- Brown AO, Millet E.R.C., Quint J, Orihuela CJ. (2015) S. pneumoniae mediated cardiotoxicity during community acquired pneumonia. American Journal of Respiratory and Critical Care Medicine, Vol. 191, No. 7, pp. 739-745.
- Brown AO and Orihuela CJ. (2015) Visualization of Streptococcus pneumoniae within cardiac microlesions and cardiac remodeling. Journal of Visualized Experiments.DOI:10.3791/52590.
- Soong G, Paulino F, Wachtel S, Parker D, Wickersham M, Brown AO, Zhang D, Lauren C, West E, Planet P, Prince A. (2015) MRSA adaption to keratinocytes. Mbio, Vol. 6, No. 2, pp. 1-14.
- Kitur K, Wachtel S, Brown AO, Wickersham M, Paulino F, Penaloza HF, Soong G, Bueno S, Parker D, Prince A. (2016) Necroptosis Promotes Staphylococcus aureus Clearance by Inhibiting Excessive Inflammatory Signaling. Cell Reports, Vol. 16, No. 8, pp.2219-2230.
I enjoy outdoor activities (hiking, camping, and fishing), exercising, playing sports, reading, cooking, and spending time with friends and family. In addition, I am a foodie/food blogger and avid gamer.
Description of Project:
Enterococcus faecalis and Candida albicans are found as commensals in the human body. Both are classified as opportunistic pathogens and have been found in polymicrobial, nosocomial infections. Our previous work identified the Enterococcus faecalis bacteriocin, EntV, as having activity against Candida albicans virulence and biofilm formation. Our current studies are focused on the impact of Candida albicans on E. faecalis biofilm formation and virulence.
I am additionally involved in supporting other ongoing projects in the Garsin laboratory and facilitating major collaborations.
- Graham CE, Cruz MR, Garsin DA, Lorenz MC. Enterococcus faecalisbacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc Natl Acad Sci U S A. 2017 Apr 25;114(17):4507-4512. doi: 10.1073/pnas.1620432114. Epub 2017 Apr 10. PubMed PMID: 28396417; PubMed Central PMCID: PMC5410809.
- Mayo SA, Song YK, Cruz MR, Phan TM, Singh KV, Garsin DA, Murray BE, Dial EJ, Lichtenberger LM. Indomethacin injury to the rat small intestine is dependent upon biliary secretion and is associated with overgrowth of enterococci.Physiol Rep. 2016 Mar;4(6). pii: e12725. doi: 10.14814/phy2.12725. Epub 2016 Mar 31. PubMed PMID: 27033447; PubMed Central PMCID: PMC4814884.
- Reyes J, Panesso D, Tran TT, Mishra NN, Cruz MR, Munita JM, Singh KV, Yeaman MR, Murray BE, Shamoo Y, Garsin D, Bayer AS, Arias CA. A liaR deletion restores susceptibility to daptomycin and antimicrobial peptides in multidrug-resistant Enterococcus faecalis.J Infect Dis. 2015 Apr 15;211(8):1317-25. doi: 10.1093/infdis/jiu602. Epub 2014 Oct 31. PubMed PMID: 25362197; PubMed Central PMCID: PMC4402337.
- Bhatty M, Cruz MR, Frank KL, Gomez JA, Andrade F, Garsin DA, Dunny GM, Kaplan HB, Christie PJ. Enterococcus faecalis pCF10-encoded surface proteins PrgA, PrgB (aggregation substance) and PrgC contribute to plasmid transfer, biofilm formation and virulence.Mol Microbiol. 2015 Feb;95(4):660-77. doi: 10.1111/mmi.12893. Epub 2014 Dec 30. PubMed PMID: 25431047; PubMed Central PMCID: PMC4329047.
- van der Hoeven R, Cruz MR, Chávez V, Garsin DA. Localization of the Dual Oxidase BLI-3 and Characterization of Its NADPH Oxidase Domain during Infection of Caenorhabditis elegans.PLoS One. 2015 Apr 24;10(4):e0124091. doi: 10.1371/journal.pone.0124091. eCollection 2015. PubMed PMID: 25909649; PubMed Central PMCID: PMC4409361.
- DebRoy S, Gebbie M, Ramesh A, Goodson JR, Cruz MR, van Hoof A, Winkler WC, Garsin DA. A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator.Science. 2014 Aug 22;345(6199):937-40. doi: 10.1126/science.1255091. PubMed PMID: 25146291; PubMed Central PMCID: PMC4356242.
Crochet, sewing, piñata maker, volunteer clinic assistant at a homeless clinic, manna chess club leader.
6thYear Graduate Research Assistant
Description of Project:
As a collaboration between the Garsin Lab and Lorenz Lab, using different in vivo and in vitro models I am investigating the interactions between Candida albicans and Enterococcus faecalis. E. faecalis and C. albicans occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species are also among the most important and problematic, opportunistic nosocomial pathogens. We have demonstrated antagonist interaction between C. albicans and E. faecalis, whereby a secreted bacterial factor inhibits hyphal growth in biofilms and in the Caenorhabditis elegans infection model, abrogating virulence. Using an in vitro biofilm model, I identified the secreted bacterial factor EntV, a putative bacteriocin, as both necessary and sufficient for the reduction of C. albicans virulence and biofilm formation through the inhibition of hyphal formation, a critical virulence trait. EntV is protective against C. albicans in a C. elegans infection model, murine macrophage infection model, and murine oropharyngeal candidiasis (OPC) model. Our findings demonstrate that EntV has potential as an antifungal agent that targets virulence rather than viability.
2017, Presidents’ Research Scholarship, Graduate School of Biomedical Sciences
2016 – 2018, NIH Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship to Promote Diversity in Health-Related Research
2015, Investing in Student Futures Scholarship, Graduate School of Biomedical Sciences
- Graham, C.E., Cruz, M.R., Garsin, D.A., and Lorenz, M.C. 2017. The Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. PNAS. 114(17):4507-4512. PMID: 28396417.
- Cruz, M.R., Graham, C.E., Gagliano, B.C., Lorenz, M.C., and Garsin, D.A. 2013. Enterococcus faecalis inhibits hyphal morphogenesis and virulence of Candida albicans. Infect. Immun. 81(1):189. PMID: 23115035.
In my free time I like making mixed media art, refurbishing furniture, making jewelry, hiking, canoeing, bird watching, and playing with my son.
Postdoctoral Research Fellow
Description of Project:
Generation of reactive oxygen species (ROS) as a mechanism of innate immunity against bacterial infections is well conserved across nematodes to mammals. Besides their anti-microbial effects, ROS produced by the host have the potential to damage host’s own cells. Oxidative stress response enzymes are activated through mechanisms triggered by ROS production as a response to protect the host cells from “friendly-fire”. We study the regulation of the oxidative stress response pathways in the well-studied host model Caenorhabditis elegans in the context of ROS generation against infectious bacteria.
Our lab and others discovered that intestinal infections trigger the generation of ROS by a dual oxidase Ce-DUOX-1/BLI-3 as an anti-bacterial response. ROS generated by Ce-DUOX-1/BLI-3 also induces an oxidative stress response for self-protection. SKN-1 is one of the key transcription factors that mediates the oxidative stress response in C. elegans, similar to its ortholog NRF2 in mammals. SKN-1/NRF2 upregulates Phase 2 genes of the three-phase detoxification system.
It is well documented that ROS generated by Ce-DUOX-1/BLI-3 during infection activates SKN-1 through p38 MAPK signaling pathway. However, infection-specific sensors of ROS and regulators of SKN-1 activity are not well-studied. I aim to discover novel regulators of SKN-1 activity during bacterial infections. I plan to utilize reverse genetic screen using interfering RNA (RNAi) to identify potential candidates. I plan to perform genetic and biochemical experiments for validation of candidates and elucidation of their mechanisms of action.
- Ming Zhang M, Yilmaz T, Boztas AO, Karakuzu O, Bang WY, Yegin Y, Luo Z, Lenox M, Cisneros-Zevallos L, Akbulut M. A Multifunctional Nanoparticulate Theranostic System with Simultaneous Chemotherapeutic, Photothermal Therapeutic, and MRI Contrast Capabilities. RSC Adv., 2016, 6, 27798.
- Boztas AO, Karakuzu O, Galante G, Ugur Z, Kocabas F, Altuntas CZ, Yazaydin AO. Synergistic interaction of paclitaxel and curcumin with cyclodextrin polymer complexation in human cancer cells. Mol Pharm. 2013 Jul 1;10(7):2676-83.
- Karakuzu O, Wang DP, Cameron S. MIG-32 and SPAT-3A are PRC1 homologs that control neuronal migration in Caenorhabditis elegans. Development. 2009 Mar;136(6):943-53.
- Prasad B, Karakuzu O, Reed RR, Cameron S. unc-3-dependent repression of specific motor neuron fates in Caenorhabditis elegans. Dev Biol. 2008 Nov 15;323(2):207-15.
- Erbel PJ, Card PB, Karakuzu O, Bruick RK, Gardner KH. Structural basis for PAS domain heterodimerization in the basic helix–loop–helix-PAS transcription factor hypoxia-inducible factor. Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15504-9.
Travel, gardening, cooking, history and archaeology, playing with my twin daughters.
Karan Kaval (K.K.)
Postdoctoral Research Fellow
Description of Project:
Our studies revolve around the utilization of ethanolamine in the gut by Enterococcus faecalis. Ethanolamine, a breakdown product of the cell membrane phospholipid, phosphatidylethanolamine, can serve as a valuable source of carbon and nitrogen to bacteria like E. faecalis that possess an ethanolamine utilization (eut) locus. The eut genes encode metabolic enzymes as well as structural components that enclose the enzymes within a proteinaceous shell, forming capsid-like structures called bacterial microcompartments (BMCs). Eut BMCs function to catabolize ethanolamine in a manner that separates the toxic intermediates from the rest of the cytoplasm. Gene expression of both the enzymatic as well as the shell proteins is under the control of a 2-component regulatory system consisting of the EutV and EutW proteins. Our studies are currently focused on ascertaining the spatio-temporal regulation of these BMCs and defining their structural features.
- Kaval, K. G., Rismondo, J., Hauf, S., Hahn, B. and Halbedel, S. (2017). Genetic dissection of DivIVA functions in Listeria monocytogenes. J Bacteriol. re-submitted after review.
- Kaval, K. G., Hahn, B., Tusamda, N., Albrecht, D. and Halbedel, S. (2015). The PadR-like transcriptional regulator LftR ensures efficient invasion of Listeria monocytogenes into human host cells. Microbiol. 6:772.
- Kaval, K. G., Rismondo, J. & Halbedel, S. (2014). A function of DivIVA in Listeria monocytogenes division site selection. Mol Microbiol, 94, 637-54.
- van Baarle, S., Celik, I. N., Kaval, K. G., Bramkamp, M., Hamoen, L. W. & Halbedel, S. (2013). Protein-protein interaction domains of Bacillus subtilis J Bacteriol, 195, 1012-21.
- Kaval, K. G. & Halbedel, S. (2012). Architecturally the same, but playing a different game: the diverse species-specific roles of DivIVA proteins. Virulence, 3, 406-7.
Hiking, playing the guitar, photography and painting.
Graduate Research Assistant
Description of project:
Using C. elegans as a model system, we are studying the mechanism of Reactive Oxygen Species production, specifically, the role of putative peroxidases, in innate immunity against pathogen. Previous studies in our lab have demonstrated that a dual oxidase (DUOX), BLI-3, is responsible for the production of H202 and pathogen resistance upon microbial infection. H2O2 is a weak ROS that in some cases can be converted into more potent antimicrobial agents by heme-containing peroxidases. Although there is recognition of the synergistic relationships between DUOX and peroxidases in recent years, the mechanisms by which immune-associated peroxidases use H2O2 to contribute to the response are incompletely understood. My project focuses on identifying and characterizing putative peroxidases required for pathogen resistance, and investigating their potential interaction with BLI-3.
2017 Elevator speech Contest (1st place and People’s choice), Graduate School of Biomedical Sciences
2016 GSBS travel award, UTHealth
2015 Ralph H. & Ruth McCullough Foundation Award, Graduate School of Biomedical Sciences
1. Hu, H, Yu, Z., Liu, Y., Wang, T., Wei, Y., and Li, Z. (2014) The Aurora B kinase in Trypanosoma brucei undergoes post-translational modifications and is targeted to various subcellular locations by binding to TbCPC1. Molecular Microbiology, 91: 256-274
2. Liu, Y., Hu, H., and Li, Z. (2013) The cooperative roles of PHO80-like cyclins in regulating the G1/S transition and posterior cytoskeletal morphogenesis in Trypanosoma brucei. Molecular Microbiology, 90: 130-146
3. Yu, Z., Liu, Y., & Li, Z. (2012). Structure–function relationship of the Polo-like kinase in Trypanosoma brucei. Journal of Cell Science, 125(6), 1519-1530.
4. Yang, S., Gong, H., Zhang, L., Liu, Y., He, Z. (2010). Characterization of physical and functional interactions between eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase in the hyperthermophilic archaeon Sulfolobus solfataricus. Biochem Biophys Res Commun, 396(3), 755-762
5. Zhang, L.*, Zhang, L.*, Liu, Y.*, Yang, S., Gao, C., Gong, H., Feng, Y., He, Z. (2009). Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions. Proc Natl Acad Sci U S A, 106(19), 7792-7797. * Co-first authors
Traveling and experiencing new cultures, playing volleyball, gardening