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  • Undergraduate Poster Abstracts
  • Microbiology

    Room National Harbor 5

    ap028 SCHISTOSOMICIDAL OXAMNIQUINE DERIVATIVE DRUG ACTIVITY AGAINST HUMAN SCHISTOSOMIASIS

    • Anastasia Rugel ;
    • Xiaohang Cao ;
    • Bethany Cruz ;
    • P. John Hart ;
    • Philip LoVerde ;

    n/a

    SCHISTOSOMICIDAL OXAMNIQUINE DERIVATIVE DRUG ACTIVITY AGAINST HUMAN SCHISTOSOMIASIS

    Anastasia Rugel1, Xiaohang Cao1, Xiaohang Cao1, Bethany Cruz2, P. John Hart1, Philip LoVerde1.

    1The University of Texas Health Science Center at San Antonio, San Antonio, TX, 2Texas Biomedical Research Institute, San Antonio, TX.

    Human schistosomiasis is a disease caused by species of the genus Schistosoma, which globally affects over 200 million people. The major species effecting humans are S. mansoni, S. haematobium, and S. japonicum. There is currently only 1 method of treatment (monotherapy): the drug praziquantel (PZQ). Constant selection pressure through mass chemotherapy, over 250 million doses per annum, has yielded evidence of resistance to PZQ. This has been observed in both the laboratory and field. The purpose of this research is to develop a second drug for use in conjunction with PZQ. Previous treatment of S. mansoni included, among others, the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified, described, and crystallized by our laboratory as a sulfotransferase (SmSULT). The focus of this research is to reengineer OXA to be effective against S. haematobium and S. japonicum. Five generations of OXA derivatives have been synthesized and tested against S. mansoni, with a total of 8 derivatives discovered they may potentially be used to treat schistosomiasis. In vitro tests of these 8 derivatives against S. haematobium and S. japonicum have also yielded positive results. This iterative generational process of using structural data to inform chemical synthesis of derivatives, which are then tested in vitro, continues to provide us with novel compounds with improved antischistosomal activity. The information gleaned from these early studies will be used to optimize OXA derivative design.

    ap029 NITROGEN CYCLING IN THE SUBSURFACE: MICROBIAL ROLES

    • Jason Kimble ;
    • Ara Kooser ;
    • Robert L. Sinsabaugh ;
    • Diana E. Northup ;

    n/a

    NITROGEN CYCLING IN THE SUBSURFACE: MICROBIAL ROLES

    Jason Kimble, Ara Kooser, Robert L. Sinsabaugh, Diana E. Northup.

    The University of New Mexico, Albuquerque, NM.

    Many caves are considered extreme environments because of their oligotrophic nature and low biomass productivity. Little is known about how microbes cycle nutrients in subsurface environments. On the ceilings and walls of the arid-land caves Spider and Lechuguilla in Carlsbad Caverns National Park, New Mexico, exist multicolored secondary mineral deposits of low-density, soil-like material known as ferromanganese deposits (FMD) that contain microbial communities driven by chemolithoautotrophic metabolic processes. We hypothesize those microbial communities cycle nitrogen though nitrification and denitrification pathways, which are chemolithotrophic processes that could potentially provide energy and electrons to the cells and through nitrogen fixation and assimilation pathways. A total of 10 samples were collected from FMD found in Spider and Lechuguilla Caves, 7 and 3 samples respectively, and sequenced using Illumina shotgun metagenomics. Raw sequences were processed with USEARCH, Sickle, and Bowtie2, and submitted for annotation using the open-source automated software program MG-RAST. Preliminary results from enzyme gene fragments associated with denitrification pathways suggest that bacterial communities within FMD potentially derive energy from these pathways. Additionally, gene fragments associated with ammonia assimilation were identified. The presence of these gene fragments varied by FMD color and location. Our results suggest that short-term demand for nitrogen is potentially met through various nitrogen cycling pathways within the microbial FMD community. This investigation contributes to our understanding of how organisms acquire and retain essential nutrients in a dark, extreme oligotrophic chemolithotrophically driven ecosystem.

    ap030 THE PREVALENCE OF RICKETTSIA SPP. THE CAUSATIVE AGENT OF ROCKY MOUNTAIN SPOTTED FEVER IN ARIZONA

    • Alma Solis ;
    • Joshua Calvano ;
    • Adam Pilkington ;
    • Nathan Nieto ;

    n/a

    THE PREVALENCE OF RICKETTSIA SPP. THE CAUSATIVE AGENT OF ROCKY MOUNTAIN SPOTTED FEVER IN ARIZONA

    Alma Solis, Joshua Calvano, Adam Pilkington, Nathan Nieto.

    Northern Arizona University, Flagstaff, AZ.

    Rickettsiales are intracellular bacteria that are primarily transmitted by hard ticks (Ixodidae) globally. Rhipicephalus sanguineus, the brown dog tick, is a newly recognized vector of the pathogen in Arizona. This tick is found globally and is associated with high population densities of dogs (Canis familiaris). The pathogen infects a human or dog via the bite of an infected tick and causes Rocky Mountain spotted fever (RMSF). The objective of this research was to calculate the prevalence and diversity of Rickettsii spp. in Arizona. R. sanguineus ticks (N = 481) were collected throughout Arizona with the help of a veterinarian. The ticks were identified to species, as well as sex and life stage. DNA from each tick was extracted and analyzed using both qualitative PCR and nested PCR. qPCR assays for all Rickettsia spp. and specifically R. rickettsii targeted the 16S rRNA sequence. Nested-PCR assays targeted the outer membrane protein gene ompA. We calculated a 13.9% total prevalence of Rickettsia spp. and 0% prevalence of R. rickettsii. After sequencing, we identified R. massiliae in R sanguineus from Arizona. We hypothesize that the diversity of Rickettsia spp. in Arizona may be unrealized and widespread throughout the state.

    ap031 INDUCTION OF PROTECTIVE IMMUNITY AGAINST CRYPTOCOCCOSIS CAUSED BY DISPARATE CRYPTOCOCCUS SEROTYPES

    • Marley Van Dyke ;
    • Ashok Chaturvedi ;
    • Chrissy Leopold Wager ;
    • Camaron Hole ;
    • Karen Wozniak ;
    • Floyd Wormley ;

    n/a

    INDUCTION OF PROTECTIVE IMMUNITY AGAINST CRYPTOCOCCOSIS CAUSED BY DISPARATE CRYPTOCOCCUS SEROTYPES

    Marley Van Dyke, Ashok Chaturvedi, Chrissy Leopold Wager, Camaron Hole, Karen Wozniak, Floyd Wormley.

    The University of Texas at San Antonio, San Antonio, TX.

    Cryptococcosis is a fungal disease caused by Cryptococcus neoformans (serotypes A and D) or Cryptococcus gattii (serotypes B and C). Cryptococcus causes pulmonary infections and can lead to life-threatening infections of the central nervous system. We have shown that mice given an experimental pulmonary inoculation with an IFN-γ producing C. neoformans serotype A strain, designated H99γ, resolve the acute infection and are protected against a subsequent pulmonary challenge with fully pathogenic serotype A strain. However, no study has shown that cross-protection against disparate Cryptococcus serotypes can be achieved. Consequently, we sought to determine the efficacy of immunization with C. neoformans strain H99γ to elicit protection against all serotypes of Cryptococcus. For these studies, we immunized BALB/c mice with either heat killed H99γ (HKγ) or H99γ and 70 days later challenged these mice with Cryptococcus serotype A-D clinical isolates. The mice were monitored for survival or sacrificed at day 7 and day 14 post-challenge to determine pulmonary fungal burden, leukocyte infiltration, and cytokines/chemokine responses. We observed a significant increase in the survival rates of mice immunized with H99γ compared to mice immunized with HKγ following challenge with serotypes A, B, and D. Additionally, the results showed significantly decreased pulmonary fungal burden, a trending Th1-type cytokine profile, and delayed T cell-mediated immune responses with serotypes B-D compared to those challenged with serotype A in H99γ-immunized mice compared to those immunized with HKγ. These studies provide proof-of-concept for development of a cryptococcal vaccine that induces protection across all Cryptococcus serotypes.

    ap032 ADAPTIVE EVOLUTION OF THE SIV ENV PROTEIN REFLECTS VIRUS-SPECIFIC ANTIBODY RESPONSES DURING PERSISTENT INFECTION

    • Sergio Ita ;
    • Welkin Johnson ;

    n/a

    ADAPTIVE EVOLUTION OF THE SIV ENV PROTEIN REFLECTS VIRUS-SPECIFIC ANTIBODY RESPONSES DURING PERSISTENT INFECTION

    Sergio Ita, Welkin Johnson

    1Harvard Medical School, Boston, MA, 2Boston College, Chestnut Hill, MA.

    Primate lentiviruses including HIV-1, 2, and SIVs cause persistent lifelong infections despite the presence of virus-specific adaptive immune responses. The target of antibodies is the viral envelope glycoprotein (Env) which is expressed on the surface of virions and infected cells. SIV env sequence variation arising from evasion of antibodies is well known, yet the evolution of escape within hosts is poorly understood and, whether these adaptations exact a fitness cost, is not well defined. We sequenced longitudinal samples (2 - 29 weeks post infection) from 4 SIV-infected rhesus macaques to reconstruct SIV env sequence evolution starting from a defined stock inoculum using high-resolution next-generation sequencing (NGS). By NGS analysis, we captured a population bottleneck at the point of transmission from the stock into each animal and the subsequent emergence of Env diversity from the initially homogeneous population that correlated with the onset of Env-specific antibodies. We tracked selection of mutations, in-frame insertions, and deletions of the viral populations in each animal and found that changes were highly concentrated at multiple sites in surface-exposed variable loops. The deterministic appearance of changes leading to replacement of the residue present at transmission, polymorphism, and insertion/deletions revealed a remarkable degree of viral protein evolvability. The observation that similar or identical changes occurred in multiple animals suggests SIV Env relies on specific sequence pathways to evade continuous antibody targeting. We engineered selected adaptations into full length virus to now determine whether these mutations incur a replicative fitness cost using a deep sequencing-based viral fitness assay.

    ap033 RNA VIRUSES TARGET EXORIBONUCLEASE XRN1 TO PROMOTE PATHOGENESIS

    • Phillida Charley ;
    • Stephanie Moon ;
    • John Anderson ;
    • Jeffery Wilusz ;

    n/a

    RNA VIRUSES TARGET EXORIBONUCLEASE XRN1 TO PROMOTE PATHOGENESIS

    Phillida Charley, Stephanie Moon, John Anderson, Jeffery Wilusz.

    Colorado State University, Fort Collins, CO.

    All eukaryotic cells rely on RNA decay pathways for the removal of unwanted RNA and the regulation of 20 - 50% of cellular gene expression. The 5’-3’ exoribonuclease Xrn1 is an essential part of a major cellular RNA decay pathway. Xrn1 is located in the cytoplasm; thus we hypothesized that certain RNA viruses may target Xrn1 to dysregulate cellular gene expression and disarm the cell defense mechanisms. To support this model, we demonstrated that several members of Flaviviridae (e.g., dengue, West Nile, hepatitis C, and bovine diarrhea viruses) have the ability to stall and suppress Xrn1 through complex RNA structures located in the 3’ or 5’ untranslated regions (UTRs) of their transcripts. As a result, many cellular mRNAs are stabilized in flavivirus-infected cells. This accumulation of mRNAs leads to dysregulation of cellular gene expression and likely contributes to viral-induced cytopathology and pathogenesis. We have now turned our attention to another RNA virus family to see if they also target Xrn1 during infection. The Arenaviridae (e.g., junín virus) family have a segmented genome and employ an ambisense transcription strategy using a very large stable stem-loop structure in their intergenic region that we hypothesize may also stall Xrn1. Consistent with this hypothesis, we identified decay intermediates produced by Xrn1 stalling in the 3’ UTRs of junín virus mRNAs. Our data indicate that the stalling and repression of Xrn1 may be a common strategy shared by several RNA virus families to dysregulate cellular gene expression during infection.

    ap034 ELUCIDATING THE ROLE OF A MEMBER OF THE MBTH-LIKE PROTEIN SUPERFAMILY IN MYXOCOCCUS XANTHUS

    • Karla Esquilin-Lebron ;
    • Michael G. Thomas ;

    n/a

    ELUCIDATING THE ROLE OF A MEMBER OF THE MBTH-LIKE PROTEIN SUPERFAMILY IN MYXOCOCCUS XANTHUS

    Karla Esquilin-Lebron, Michael G. Thomas.

    University of Wisconsin-Madison, Madison, WI.

    One of the main ways bacteria and fungi produce bioactive natural products such as antibiotics and siderophores is through nonribosomal peptide synthetase (NRPS) multimodular assembly lines. Some NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for solubility and function. The role MLPs play in NRPS enzymology has yet to be elucidated; however, MLPs are known to interact with adenylation domains of NRPSs. MLPs are nearly always encoded within NRPS-encoding gene clusters. Interestingly, the only exemptions are found in the Myxococcales order. Myxococcus xanthus has 14 NRPS-encoding gene clusters and only one MLP-encoding gene. Importantly, this gene (MXAN_3118) is not encoded within any of the NRPS-encoding gene clusters. These observations lead us to hypothesize that MXAN_3118 may interact with one or more NRPSs, may play some NRPS-independent role, or plays some role in both NRPS-enzymology and other physiological processes in M. xanthus. Using a comparative genomics approach, we identified 3 potential NRPS partners for MXAN_3118. The NRPSs were determined to require MXAN_3118 for solubility and activity. These findings suggest that MXAN_3118 can functionally interact with NRPSs and influence their solubility and function. Ongoing efforts to identify the associated metabolite are being pursued. Interestingly, protein profile analysis shows that the M. xanthus proteome profile changes when MXAN_3118 is absent. The findings of our research will allow the development of a model for the role that MXAN_3118 plays in the physiology of M. xanthus and other members of the Myxococcales order and will provide insights into the function of MLPs.

    ap035 CHARACTERIZATION OF MEMBRANE PROTEIN COMPLEXES IN THE BIOFUEL MICROBE CLOSTRIDIUM PHYTOFERMENTANS

    • Jesús Alvelo-Maurosa ;
    • Susan Leschine ;

    n/a

    CHARACTERIZATION OF MEMBRANE PROTEIN COMPLEXES IN THE BIOFUEL MICROBE CLOSTRIDIUM PHYTOFERMENTANS

    Jesús Alvelo-Maurosa, Susan Leschine.

    University of Massachusetts Amherst, Amherst, MA.

    The anaerobic cellulosic microbe Clostridium phytofermentans is capable of breaking down and fermenting multiple carbohydrates to ethanol as its main product, making it attractive for biofuels production. However, this process by itself would appear to be energetically limited due to a lack of acetate production which would result in additional ATP formation. Here, we identify various membrane-bound proteins that may play a role in generating ATP using an electrochemical gradient. Sequence alignment and protein modeling analyses have shown the presence of 3 ATPases: a Na+ F-type ATPase, Na+ V-type ATPase, and an unknown ATPase; as well as a dual Na+/H+ pyrophosphatase and a NAD+ ferredoxin oxidoreductase (Rnf) with unknown ion use. However, it was found that the Rnf D subunit of C. phytofermentans has a 43.86% sequence similarity to Rnf D of Acetobacter woodii, an Na+ complex, and a 37.05% sequence similarity to the H+ Rnf D subunit of Clostridium ljungdahlii. Surprisingly, cell growth of C. phytofermentans was significantly reduced in the presence of either 25 µM ETH2120, a Na+ ionophore, or 25 µM TCS, a protonophore. Effects of the Na+ ionophore, ETH2120, and the protonophore TCS on cell growth and ATPase assays using inverted membrane vesicles suggested that both Na+ and H+ gradients may be involved in membrane energetics in C. phytofermentans.

    ap036 GLUTAMINE METABOLISM IS REQUIRED DURING INFECTION WITH AN ONCOGENIC VIRUS

    • Erica Sanchez ;
    • Patrick Carroll ;
    • Angel Thalhofer ;
    • Michael Lagunoff ;

    n/a

    GLUTAMINE METABOLISM IS REQUIRED DURING INFECTION WITH AN ONCOGENIC VIRUS

    Erica Sanchez1, Patrick Carroll2, Angel Thalhofer1, Michael Lagunoff1.

    1University of Washington, Seattle, WA, 2Fred Hutchinson Cancer Research Center, Seattle, WA.

    Human viruses cause approximately 10 to 15% of cancers worldwide. To better understand how viruses cause cancer, we study how the oncogenic virus, Kaposi’s sarcoma-associated herpesvirus (KSHV), alters host cellular metabolism. KSHV is the infectious agent of Kaposi’s sarcoma (KS), the most commonly reported tumor in parts of Africa and the most common tumor of AIDS patients worldwide. Our lab previously published that KSHV infection induces and requires both glycolysis and fatty acid synthesis, 2 pathways commonly perturbed in cancer cells and essential for their survival. A metabolomics screen detected elevated levels of glutamine in KSHV-infected cells. Therefore, we hypothesized that glutamine metabolism may also be important during KSHV infection. We found that KSHV-infected cells exhibit increased glutamine uptake and, moreover, starving infected cells of exogenous glutamine leads to a significant increase in apoptosis compared to controls. We also show that supplementing infected cells starved of glutamine with downstream metabolic intermediates can prevent cell death, suggesting that glutamine is a required metabolic carbon source during KSHV infection. Additionally, we found that KSHV infection induces the expression of the cancer metabolism regulators c-Myc and MondoA. Knockdown of MondoA by siRNA induces cell death of KSHV-infected cells, which is also rescued by the addition of downstream metabolites, indicating that MondoA is playing a role in the regulation of glutamine metabolism during KSHV infection. Overall, these findings expand our understanding of the required metabolic alterations established during KSHV infection, and may provide novel therapeutic targets for the inhibition of KSHV infection and ultimately KS tumors.