Ph.D. - Microbiology
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Item CHARACTERIZATION OF MACROPHAGE RESPONSES TO ZINC-LIMITED MYCOBACTERIUM TUBERCULOSIS(2024) Marcantonio, Endrei; Prišić, Sladjana; MicrobiologyItem Characterization Of A Novel Attachment Virulence Factor From The Intracellular Bacterial Pathogen Burkholderia pseudomallei(University of Hawaii at Manoa, 2022) Sun, Zhenxin; Hoang, Tung T.; MicrobiologyBurkholderia pseudomallei (Bp) is an intracellular bacterial pathogen causing melioidosis, a life-threatening disease affecting humans with a high mortality rate. Bp causes disease in estimated 165,000 people each year with approximately 89,000 fatalities. The innovative single-cell transcriptomic approach has made it possible to scrutinize differential gene expression of Bp at each stage during the infection of eukaryotic cells. Bp intracellular “TRANSITome” has led to the identification of a novel surface attachment protein BPSS0097 (Sap1) that was specifically turned on at the vacuole entry stage. A Sap1 mutant showed a significant decrease in attachment to host cells and was completely attenuated in the BALB/c mouse intranasal model of acute Bp infection. This is the first description of an attachment deficient mutant that is completely attenuated in a lethal challenge mouse model. We hypothesized that Sap1 recognizes specific host cell surface receptor molecules that facilitate the binding of Bp to the surface of host cells to initiate its intracellular infection. To test this central hypothesis, I first optimized Sap1 protein production and determined Sap1 crystal structure via collaboration. The structure of Sap1 was successfully determined at 1.30Å resolution revealing a significant similarity to Src- Homology 3 (SH3) protein family, a very abundant protein-protein interactions module in eukaryotes. The knowledge gained in the functional analysis based on Sap1 structural information laid a solid foundation for me to further identify the receptor protein from host cells to be the glycoprotein non-metastatic melanoma protein b (GPNMB). The interaction between GPNMB and Sap1 was validated to be specific and strong. Overall, I identified a new ligand/receptor interaction pair of Sap1/GPNMB and my study is the first to show that GPNMB is used by a bacterial pathogen as a gateway to attach and invade host cells. iv Furthermore, a transcriptional activator of Sap1 was identified, which also acts as a global regulator of other membrane-associated virulence factors in Bp. In summary, I accomplished a comprehensive characterization of a novel Bp attachment factor Sap1 that leads to a further understanding of Bp attachment mechanisms at the molecular level. The findings of my dissertation project will aid in future vaccine development and therapeutics for melioidosis treatment.Item Describing The Role Of Zinc And Zinc-independent Ribosomal Proteins In Mycobacterial Physiology(University of Hawaii at Manoa, 2021) Burger, Allexa Dow; Prisic, Sladjana; MicrobiologyMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has plagued humanity for millennia and remains the world’s deadliest bacterium today. Bacterial heterogeneity complicates TB treatment, however specific cues from the host leading to development of Mtb subpopulations are not well understood. Access to zinc ion (Zn2+) may be a relevant cue, considering microenvironments developed during TB create a perpetual cycle exposing Mtb to high and low concentrations of Zn2+. In this dissertation I investigate the relevance of Zn2+ as a cue in mycobacterial physiology. I start by describing the role of Zn2+ and Zn2+-independent ribosomal proteins using the model mycobacterium, M. smegmatis (Msm). I found that Msm undergoes a unique morphogenesis in Zn2+-limiting conditions but interestingly the altRP deletion mutant does not, implicating a role for Zn2+-independent ribosomal proteins in mycobacterial physiology. Using a multiomics approach, I demonstrated that Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress and exhibit increased replication in vivo. The results provide a novel link to the oxidative stress response in Mtb and suggest host-pathogen interactions are influenced by the pre-exposure status of Mtb to Zn2+. Mycobacteria that transit through a Zn2+-depleted microenvironment, a prerequisite for host-to-host transmission, have anticipatory adaptations and are primed to withstand impending oxidative stress upon subsequent contact with immune cells. Considering that standard mycobacterial media recapitulates a Zn2+-replete environment, the Zn2+-dependent phenotype of the pathogen may confound our fundamental understanding of initial interactions between Mtb and the human host.Item Functional Characterization Of Essential Burkholderia Pseudomallei Virulence Regulators(University of Hawaii at Manoa, 2020) McMillan, Ian Andrew; Hoang, Tung T.; MicrobiologyBurkholderia pseudomallei (Bp) is a Gram-negative facultative intracellular pathogen that causes the disease melioidosis. Bp is endemic to tropical and subtropical regions around the globe with an expanding reach due to increased awareness and better diagnostics. Melioidosis is predicted to infect 165,000 people and cause 89,000 deaths annually marking this disease as a significant threat to public health. The CDC has classified Bp as a Tier 1 select agent due to potential malicious use and there is currently no licensed vaccine to protect against infection. Bp has one of the most complex bacterial genomes with ~7.24 mega base-pairs of genetic material across two highly plastic chromosomes. The genetic determinants encoded on the Bp genome allow for growth in various environments including many cell types within the human body. Bp has a complex intracellular lifecycle that starts by attaching to host cells, followed by invasion of the cytoplasm and replication within, and finally protrusion towards neighboring cells to continue the spread of infection. Approximately 1,953 genes are differentially regulated in a stage-specific manner indicating that a sophisticated regulation system is in place to coordinate intracellular infection. In order for Bp to sustain a productive infection, it relies on tight control and coordination of virulence factors and metabolic pathways by transcriptional regulators in a stage specific manner. The following dissertation investigates this hypothesis by characterizing three yet to be described transcriptional regulators: i) BP1026B_II1198; ii) BP1026B_II1561; and iii) BP1026B_II2312. Mutants of these transcriptional regulators are attenuated in cell culture and BALB/c mouse models of infection indicating a role during Bp pathogenesis. To characterize these transcriptional regulators I have: i) elucidated the regulation networks using an RNA-seq strategy; ii) determined the direct DNA binding sequences to discover the genes directly controlled by each transcriptional regulator with a ChIP-seq approach; and iii) determined what genes within each regulon contribute to pathogenesis. In addition to the characterization of three novel transcriptional regulators, two potential vaccine candidates were tested showing significant promise towards the development of a protective melioidosis vaccine.Item Intercellular Signaling Activity Encoded by hetN in the Cyanobacterium Anabaena sp. strain PCC 7120.(University of Hawaii at Manoa, 2017-08) Rivers, Orion S.; MicrobiologyDevelopmental regulators coordinate cellular differentiation in many organisms. Regulators can be small molecules or proteins. Developmental regulators called morphogens are produced in source cell(s) that determine the developmental fate of cells adjacent to the source in a concentration dependent manner. The filamentous cyanobacterium Anabaena sp. Strain PCC 7120 is a model organism used to study cellular differentiation. When Anabaena filaments are supplied a source of fixed nitrogen a single cell type, vegetative cells, comprise the filaments. However, removal of fixed nitrogen from the medium induces differentiation of one in every 10- 15 cells into a heterocyst. Heterocysts are terminally differentiated cells that are the sites of atmospheric nitrogen fixation. Differentiation within Anabaena requires 24 hours and can be divided into four stages: induction, patterning, commitment, and morphogenesis. The periodic pattern of heterocyst is initially determined by the interplay of HetR, the primary activator of differentiation within Anabaena, and PatS, a diffusible inhibitor expressed during the patterning stage. The initial pattern of heterocysts in maintained during growth by a secondary inhibitor, HetN, which is expressed in mature heterocysts. The pentapeptide sequence RGSGR is conserved in the amino acid sequences of both inhibitors and has been shown to inhibit differentiation and induce HetR degradation when added to the medium, bind directly to HetR in vitro, and is required for the inhibitory function of PatS. In this work HetN was found to require the RGSGR sequence for inhibitory function and did not require predicted ketoacyl reductase activity. Full-length HetN was found to be confined to source cell(s) membranes, but a hetNdependent inhibitory signal was shown to move away from source heterocysts in a manner similar to a paracrine-type intercellular signal. The hetN-dependent inhibitory signal was found not to require the intercellular channel forming protein SepJ. However, mutation of sepJ reduced the signal range of the HetN-dependent inhibitory signal, suggesting its involvement in signal transport. Finally, evidence supporting the use of M119 of HetN as the developmentally regulated translational start site is presented. This work contributes to our knowledge of morphogen signals and supports the role of HetN as an inhibitory morphogen within Anabaena.Item Quorum Sensing in the Hawaiian Coral Pathogen Vibrio coralliilyticus strain OCN008(University of Hawaii at Manoa, 2017-08) Burger, Andrew H.; MicrobiologyCoral disease represents a serious threat to reefs worldwide. Reef ecosystems have been reshaped by coral disease in the Caribbean, Florida Keys, and the Great Barrier Reef. Reefs represent millions of dollars in economic value as well as contribute heavily to marine primary production, thus efforts to minimize such damage have become crucial. Coral disease in Hawaiʻi has presented less of a problem when compared to the Caribbean, but emerging diseases over the past decade have brought the potential for similar destruction. One disease of concern in Kāneʻohe Bay, Oahu, is Acute Montipora White Syndrome (aMWS), a rapidly progressing tissue-loss disease affecting the reef-building coral Montipora capitata. Early efforts studying this disease identified Vibrio coralliilyticus strain OCN008 as an etiological agent of aMWS. Interestingly, OCN008 produces and utilizes the antibiotic andrimid as a novel virulence factor. This discovery represented one of only a handful (<5) of known virulence factors in the field of coral disease, and potentially provides a point at which to begin investigations into preventative and/or curative strategies. This work describes the quorum sensing (cell-density dependent bacterial communication/behavior) mechanics of strain OCN008. The main objectives were the identification and characterization of putative quorum sensing circuits and the role they play in the infection of M. capitata. Despite possessing homologs of four known Vibrio quorum sensing pathways, OCN008 requires only one, the LuxPQ/S pathway (AI-2), to activate the high cell density response. Quorum sensing also contributes to the virulence of OCN008 at both low and high cell densities. Since the novel virulence factor andrimid was shown here to be under regulation of AI-2 mediated quorum sensing, a scenario is presented in which OCN008 can initiate andrimid production in response to a quorum of AI-2 producing bacteria, regardless of taxonomy. Interestingly, the two most severe outbreaks of aMWS were recorded following periods of heavy rain, events known to cause increased bacterial abundance and perturbations in coastal microbial communities. Shifts in coral microbiota have been linked to increased incidents of coral disease. This work offers one potential mechanism behind this phenomenon. In addition, this work offers the first direct evidence that quorum sensing is involved in coral disease.Item Investigating Acute Montipora White Syndrome in Kāne‘ohe Bay, O‘ahu: Causative Agents, Putative Environmental Drivers, and the Importance of Host Health(University of Hawaii at Manoa, 2017-05) Beurmann, Silvia; MicrobiologyReports of disease-related coral mortality have increased over the last few decades. Coral diseases contribute to the decline of coral reefs globally and threaten the health and future of coral reef communities. There is an imminent need to develop our understanding of the biotic and abiotic drivers of coral disease outbreaks on an ecological and molecular level. Montipora white syndrome (MWS) is a tissue loss disease that affects populations of the coral Montipora capitata in Kāne‘ohe Bay, Hawai‘i. Two types of MWS have been documented; a chronic progressive tissue loss disease termed chronic MWS (cMWS), and a comparatively faster infection termed acute MWS (aMWS). Colonies exhibiting cMWS have been observed to spontaneously switch to aMWS in the field. This dissertation describes analysis of coral-associated bacterial communities, causative agents of disease, the importance of host health, and putative environmental drivers that may be promoting outbreaks of aMWS and the switching of chronic infections to acute lesions. This investigation of aMWS is described over four chapters. First, the bacterial communities between healthy and diseased M. capitata colonies were compared during an ongoing aMWS outbreak versus a non-outbreak period to identify whether a specific shift in bacterial community structure is associated with this disease. The bacterial communities were analyzed using high-throughput sequencing and all health states shared different community compositions with an overall high abundance of Escherichia spp. possibly originating from sewage contamination. Second, a coral disease treatment method was assessed to determine whether the removal of cMWS lesions from M. capitata colonies could reduce morbidity and prevent re-infections. The treatment resulted in an overall reduction in morbidity and prevented lesions from switching from chronic to acute tissue loss. Third, to further describe potential causative agents of this disease, bacterial isolates from diseased M. capitata were screened for virulence using controlled infection experiments. Isolate OCN003 was identified as an etiological agent of aMWS, and more readily infected cMWS-afflicted coral fragments than healthy fragments, which is the first coral pathogen demonstrated to act as a secondary pathogen. Lastly, OCN003 genome was sequenced and was identified as a novel bacterial species and named Pseudoalteromonas piratica.Item Viral vector construction, production and vector-mediated gene transduction(University of Hawaii at Manoa, 2013-05) Wu, ChengxiangUntil now, viral vectors are considered necessary for gene therapy, and current approaches are prohibited from wide applications mainly due to low efficiency and genotoxicity. The use of optimized vector production systems, the right choice of target cells, and improved transduction protocols may overcome these obstacles. To improve viral vector production, I initially optimized a calcium phosphate-mediated transfection method through inclusion of dextran and combined use of polybrene, and significantly improved the quality and quantity of the produce. Following that, multiple strategies, including a novel E. coli-based recombination system, Taq DNA polymerase treatment, and introduction of a bacteria toxic gene, were established and significantly improved the efficiency of generation of recombinant adenovirus vector. Moreover, multiple molecular manipulative strategies tested to a prototype retroviral vector system improved vector titers by 2-3 logs and led to enhanced transduction of a broad variety of cell types, especially cells of human and mouse haematopoietic and lymphocytic lineages that hold potential for gene therapy against a wide range of inherited and acquired diseases. Furthermore, a series of mutant tRNALys3 genes were constructed and expressed using the optimized viral vector production systems, and showed potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication through improved priming of HIV-1 reverse transcription from their targeting sites. Transduction of multiple copies of mutant tRNALys3 further enhanced the anti-HIV-1 potency. Lastly, a soluble tumor necrosis factor-α receptor (sTNFR)-Fc fusion protein was designed and expressed to meliorate neurons through neutralizing TNF-α. TNF-α-binding activity of secreted sTNFR-Fc from transduced cells was demonstrated and conditioned medium containing sTNFR-Fc was shown to be protective to neuronal cells from TNF-α-, HIV-1 Tat-, and gp120-mediated neurotoxicity. Overall, this study established multiple strategies and methods for improved viral vector production to facilitate gene therapy tests against HIV/AIDS and other diseases. The mutant tRNALys3-and sTNFR-Fc-based anti-HIV/NeuroAIDS strategies laid the groundwork for development of novel therapeutics against HIV and NeuroAIDS. Particularly, high efficiency transduction of cells of haematopoietic and lymphocytic lineages hold potential of using the genetically modified cells as noninvasive vehicles to deliver therapeutic substances across the blood-brain barrier into the central nervous system.Item Utilization of invasive algal biomass for bioethanol production and the dynamics of planktonic fungi in the West Pacific(University of Hawaii at Manoa, 2013-08) Wang, XinAlgae represent the most promising feedstock for biomass derived biofuel production. Certain invasive algae in Hawaii can form dense biomass and are potential feedstocks for bioethanol production. In this study, the biomass from the invasive algae Gracilaria salicornia was used as feedstock for ethanol production using the ethanologenic strain Escherichia coli KO11. The algal hydrolysates were successfully utilized in a two-stage saccharification and fermentation platform, showing no inhibition of its bacterial fermenting ability, and producing 79.1 g ethanol from one kilogram of dry algal mass. Algae contain large quantities of species-dependent polysaccharides that cannot be readily metabolized by current ethanologenic bacteria. To fully explore the potential of microbial conversion of algal biomass and increase the systematic efficiency for ethanol production, culture-dependent and independent methods were applied to identify bacterial candidates fulfilling these purposes. The microbial communities profile associated with selected native and invasive algae were determined, which supplied valuable information in searching for candidates for polysaccharides utilization. Furthermore, microbes that can facilitate consolidated bioprocessing (CBP)--a process that can potentially optimize the systematic efficiency of biomass derived ethanol production--are isolated from various sources. Two bacteria FNP1 and TF2 showed great potential in further engineering for CBP platform development. Collectively, this study supplied valuable information in developing an efficient bioethanol production platform using invasive algal biomass. The dynamics of planktonic fungi in the west Pacific was investigated in part II of the dissertation. This study revealed that planktonic fungi are molecularly diverse and the fungal distribution was related to major phytoplankton taxa and various nutrients including nitrate, nitrite, orthophosphate and silicic acid. Over 400 fungal phylotypes were recovered and nearly half of them grouped into two major novel lineages. Ascomycota and Basidiomycota were found to be dominant groups at majority of the investigated stations. These results suggest that planktonic fungi are an integral component of the marine microbial community and should be included in future marine microbial ecosystem models.Item Molecular epidemiology of seasonal and pandemic influenza A (H1N1) in Hawaiʻi(University of Hawaii at Manoa, 2013-05) Nelson, Denise CynthiaInfluenza is a viral infection causing seasonal outbreaks, periodic epidemics and global pandemics in humans, the latest being the 2009 pandemic. The State of Hawaiʻi is particularly vulnerable to the spread of influenza due to its unique geographic position in the Pacific Ocean with heavily trafficked passenger and freight patterns. By combining epidemiological data on case occurrences with their laboratory-derived viral sequences, we are able to trace viral strain origins based on phylogenetic relationships between isolates. In collaboration with the Hawaiʻi Department of Health State Laboratories Division, we present a study in which seasonal, or pandemic, H1N1 influenza A viral isolates collected from infected individuals in Hawaiʻi were extracted, hemagglutinin and neuraminidase genes were amplified and sequenced, and examined for evolutionary relationships and spatio-temporal patterns. Implications of molecular data are also supported by epidemiologic information and statistical support of summary transmission data. Phylogenetic analysis suggests that Hawaiʻi acts as both a source and sink population for type A influenza virus: in some instances Hawaiʻi isolates represented the earliest instance of a strain subsequently seen elsewhere; in other instances Hawaiʻi isolates clustered with strains observed earlier in other countries or geographic regions. Through the continued usage of molecular methods, we hope to develop an improved understanding of influenza dynamics in Hawaiʻi. Targeting an area of geographic importance additionally assists in depicting how location and population distribution play a role in the spread of infectious disease. Enhanced comprehension as a result of these analyses may help to improve efficiency and effectiveness of preparation and response efforts, and reduce the impact of influenza on Hawaiʻi and the continental United States.