Ph.D. - Microbiology

Permanent URI for this collectionhttps://hdl.handle.net/10125/2103

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CHARACTERIZATION OF MACROPHAGE RESPONSES TO ZINC-LIMITED MYCOBACTERIUM TUBERCULOSIS
(University of Hawai'i at Manoa, 2024) Marcantonio, Endrei; Prišić, Sladjana; Microbiology
Mycobacterium tuberculosis is the causative agent of tuberculosis in humans and one ofthe most successful human pathogens. Tuberculosis killed 1,300,000 million people in 2022 and has killed more than seventy million people over the last 50 years. Despite M. tuberculosis being actively studied since its discovery by Dr. Robert Koch in 1882, there is no effective vaccine to prevent tuberculosis and treatment remains complex. An understudied area that could provide novel insights into anti-tubercular therapy is the host interactions with zinc-limited M. tuberculosis bacteria (Mtb). The host immune system attempts to eradicate Mtb by limiting zinc availability via nutritional immunity, but it is unsuccessful. It has been previously demonstrated that zinc-limited Mtb are physiologically distinct from the standard zinc-replete Mtb used in most research. It is hypothesized that zinc-limited Mtb modulate host responses differently compared to zinc-replete Mtb. In this dissertation, this hypothesis was tested by evaluating the host responses in macrophages, which is one of the main cell types infected with Mtb, when infected with zinc-limited Mtb and zinc-replete Mtb. To determine if zinc-limited Mtb modulate the host response differently, functional assays were undertaken to evaluate macrophages functions and in-depth characterization of macrophage responses was done using transcriptomics. Compared to zinc-replete Mtb, it was found that zinc-limited Mtb are more readily phagocytized by macrophages, increase the production of reactive oxygen species by macrophages, trigger increased macrophage death, and stimulate increased pro-inflammatory responses in macrophages. This dissertation highlights that the distinct physiology of zinclimited Mtb more readily promotes macrophage responses that can be detrimental to the host compared to zinc-replete Mtb.
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.; Microbiology
Burkholderia 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.
Describing The Role Of Zinc And Zinc-independent Ribosomal Proteins In Mycobacterial Physiology
(University of Hawaii at Manoa, 2021) Burger, Allexa Dow; Prisic, Sladjana; Microbiology
Mycobacterium 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.
Functional Characterization Of Essential Burkholderia Pseudomallei Virulence Regulators
(University of Hawaii at Manoa, 2020) McMillan, Ian Andrew; Hoang, Tung T.; Microbiology
Burkholderia 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.
Intercellular Signaling Activity Encoded by hetN in the Cyanobacterium Anabaena sp. strain PCC 7120.
(University of Hawaii at Manoa, 2017-08) Rivers, Orion S.; Microbiology
Developmental 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.
Quorum Sensing in the Hawaiian Coral Pathogen Vibrio coralliilyticus strain OCN008
(University of Hawaii at Manoa, 2017-08) Burger, Andrew H.; Microbiology
Coral 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.
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; Microbiology
Reports 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.
Viral vector construction, production and vector-mediated gene transduction
(University of Hawaii at Manoa, 2013-05) Wu, Chengxiang
Until 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.
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, Xin
Algae 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.
Molecular epidemiology of seasonal and pandemic influenza A (H1N1) in Hawaiʻi
(University of Hawaii at Manoa, 2013-05) Nelson, Denise Cynthia
Influenza 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.
The epidemiology and entomological interactions associated with dengue transmission in Ang Mo Kio GRC, Central Singapore
(University of Hawaii at Manoa, 2013-12) Henry, Amy Beth
Dengue is arguably the most important arboviral disease of humans, having increased dramatically in geographic range and prevalence over the last 25 years. Dengue virus has two main vectors, Aedes aegypti and Aedes albopictus. For decades both vectors have also been increasing their geographic range on regional and global scales. This study took place in Singapore, where dengue fever is a major public health threat despite a successful vector control program. Similar to other hyperendemic countries, local dengue transmission dynamics in Singapore are not well understood: where dengue transmission is occurring, the relative contribution of the two dengue vectors, and the ability to correlate traditional vector surveillance methods to transmission risk remains controversial. In collaboration with the Program of Infectious Diseases at Duke-NUS Graduate Medical School, Singapore Ministry of Health, Singapore National Environmental Agency, and Ang Mo Kio Town Council an adult Aedes female fixed position vector surveillance program was established that detailed temporal and spatial Ae. aegypti and Ae. albopictus distribution and abundance in Ang Mo Kio, Central Singapore. This surveillance method yielded similar results to standard surveillance techniques over a range of habitats and time points. Furthermore, sensitivity of the adult surveillance method presented here is uniquely increased by placing traps on the second floor of Housing Development Board (HDB), government subsidized multistory residential buildings, as opposed to ground level; average Ae. aegypti catch rate of the ground floor was 0.09 and average Ae. aegypti catch rate of the second floor was 0.42. Starting on the second floor a very strong inverse relationship between Ae. aegypti catch rate and floor height (Pearson linear correlation r=-0.91, t=-4.47, df=4, p=0.01) was also identified. In addition, intensive entomological investigations, in focal areas with varying levels of Aedes abundance, identified by the fixed position surveillance system, uncovered details about mosquito ecology and "hotspots" at a local scale that can improve our understanding of dengue transmission dynamics. Dengue transmission is believed to primarily occur in residential units but host seeking Ae. aegypti and Ae. albopcitus were collected at similar frequencies in congregation areas on the ground floors of, HDBs and at greater abundance than inside residential units. Improving knowledge on the focal nature of dengue transmission is critical to designing more targeted and cost-effective surveillance and control strategies in the future, both in Singapore and urban areas elsewhere.
The prevalence and public health significance of human pathogenic Vibrio species (V. cholerae, V. vulnificus, V. parahaemolyticus, V. alginolyticus) in Hawaiʻi's diverse tropical coastal water environments
(University of Hawaii at Manoa, 2011-05) Vithanage, Gayatri
Studies on the prevalence and ecology of Vibrio species in tropical areas, such as Hawaii, is limited, and up to now, there have been no studies conducted in Hawaii to determine the prevalence of these pathogens in our coastal waters. The major goals of this study was to determine the prevalence of the four human pathogenic Vibrio spp. (V. cholerae, V. vulnificus, V. parahaemolyticus, V. alginolyticus) in coastal water environments of Hawaii (islands of Oahu and Hawaii), and to determine the public health significance these pathogens have to people who use these coastal water for recreational purposes. The study showed that water salinity and temperature affected the four human pathogenic Vibrio spp. V. vulnificus and V. parahaemolyticus were prevalent in low salinity sites that were impacted by land run-off but not detectable in high salinity, non-impacted swimming sites. Both species were also prevalent at low salinity swimming ponds on the island of Hawaii. V. alginolyticus was prevalent in all sites regardless of salinity. In addition to low salinity, high water temperature also had an impact. High temperature, low salinity ponds located on the Island of Hawaii were shown to select for V. vulnificus, V. parahaemolyticus and V. alginolyticus. These ponds have shown past evidence of infection and death due to V. vulnificus associated with the use of these ponds. Isolates recovered from these thermal ponds may potentially be more virulent as they have been adapted to survival at temperatures similar to that of human body temperature. V. cholerae was not recovered in either impacted or non-impacted sites. The prevalence of pathogenic Vibrio spp. in sediments followed a similar trend to what was seen with coastal beach samples. V. alginolyticus was prevalent in both primary and secondary beach sediment while V. vulnificus and V. parahaemolyticus were only prevalent in secondary beach sediment. Thus, apparently sediments from secondary coastal waters can spread pathogenic Vibrio species into the water column. Data from this study also showed that V. vulnificus and V. parahaemolyticus were sporadically present in raw and primary treated sewage from three different wastewater treatment plants, while V. cholerae was consistently recovered in raw and primary treated sewage from all three treatment plants. V. vulnificus can cause severe wound infections, which can rapidly lead to death. Thus, this species poses a public health significance. In summary, data gathered from this study was able to provide basic information, that was lacking, regarding the distribution of the four main human Vibrio pathogens in a tropical area such as Hawaii. This data was then used to make a basic assessment of the potential public health significance these pathogens have on humans who use Hawaii's coastal waters for recreational purposes, and to determine if and when warning signs would be warranted to notify the public of the potential risk for infection.
Polyphasic characterization of an epilithic biofilm from a lava cave in Kīlauea Caldera, Hawaiʻi
(University of Hawaii at Manoa, 2012-12) Saw, Jimmy Hser Wah
The microbial community in an epilithic biofilm on an lava cave wall in Kīlauea Caldera, Hawaiʻi, was characterized by a polyphasic approach. Ribosomal-pyrotag and metagenomic sequencing revealed phylogenetic diversity rivaling that in a Guerrero Negro hypersaline microbial mat. Targeted cultivations led to the isolation, characterization, and genome sequencing of a deeply divergent novel cyanobacterium. Diverse Bacteria and Archaea lineages were detected. The most abundant sequences, representing ~24% of the metagenomic reads analyzed, affiliated with Burkholderia. Comparative metagenomic analyses revealed community composition and function most similar to those in soils. Two novel cyanobacteria detected in metagenomic data were cultivated; JS1 is related to Gloeobacter violaceus PCC 7421T , the only cultivated Gloeobacter species. JS2 may represent a new genus in the Oscillatoriales since it shares <95% 16S rRNA gene sequence identity with its nearest neighbor, a Leptolyngbya sp. A third cultivated cyanobacterium (JS3) not detected in clone libraries, ribosomal-pyrotag or metagenomic data sets, belongs in the true-branching filamentous Stigonematales; JS3 shares 98.1% 16S rRNA gene sequence identity with Fischerella muscicola PCC 7414, and may be a new Fischerella sp. Comparing the complete genome sequence of JS1 with that of G. violaceus PCC 7421T revealed JS1 represents a new species, despite sharing 98.7% 16S rRNA gene sequence identity with PCC7421T . The name Candidatus Gloeobacter kilaueaensis is proposed, with JS1T the Type strain. Maximum likelihood phylogenetic trees based on 16S rRNA gene sequences and 43 concatenated ribosomal proteins showed Candidatus Gloeobacter kilaueaensis JS1T places in the deep-branching Gloeobacter clade, but is less basal than G. violaceus. Divergence times based on Bayesian analyses suggested these Gloeobacter species diverged 150-300 MYA. The isolation, characterization, and genome sequencing of a deeply divergent novel Gloeobacter is significant given that for forty years we have known only one species in the entire order. Of broader significance is confirmation that by integrating molecular and cultivation methods we can target for cultivation specific Bacteria and or Archaea only detected in molecular analyses; a range of scripts was also developed to analyze and visualize sequence data.
The ecology of planktonic bacteria in oligotrophic marine systems
(University of Hawaii at Manoa, 2012-05) Hayakawa, Darin Hideo
A broad synthesis regarding bacterioplankton ecology in the oligotrophic ocean was obtained through investigations along three fundamental ecological units--the community, the population and the organism. Community investigations focused on the spatial and temporal dynamics of the bacterioplankton at Station ALOHA, a site representative of the oligotrophic North Pacific subtropical gyre. Microbial DNA was harvested from water samples collected at monthly intervals over a 4-year period from surface to 4000 m depths. A suite of 16S ribosomal RNA gene-based techniques including massively parallel pyrosequencing, terminal restriction fragment length polymorphism and clone library sequencing were used to interrogate the bacterioplankton community. Distinct communities were observed stratified within the water column, with a major distinction near the base of the euphotic zone and finer distinctions through water mass association. These communities appeared structured through K-selection, as they were dominated by a surprisingly few number of specific taxa. Weak seasonality was detected in surface communities that resulted from fluctuations in relative abundances of rare populations. These rare populations were associated with the phytoplankton community and correlated with mild seasonal environmental disturbances. Low diversity was observed in both surface and bottom waters and highest in mesopelagic waters near the oxygen minimum zone. These observations fit an intermediate disturbance model that is based upon high frequency environmental disturbances at the surface and lower frequency disturbances at depth in the form sinking particulate matter, as taxonomic evidence supports. Taken together, the oligotrophic bacterioplankton community is viewed to consist of bulk members that are highly competitive for limited resources and rare opportunists that exploit habitat patchiness. The overall stability of these communities in time and space is dependent upon the integration of autochthonous (water mass) community with the allochthonous (particle associated) community. Major populations representative of the autochthonous community were investigated in further detail, yielding results that suggest their abundances may be controlled by basic physical parameters, such as temperature. Investigations at the organismal level focused on an isolate representative of the allochthonous community at T=0, as its abundances are correlated with diatom blooms. Genomic interrogation uncovered physiological features that enhance our view of oligotrophic bacterioplankton ecology.
Cellular response of insect cells to virus infection
(University of Hawaii at Manoa, 2014-05) Yang, Baojun
RNA interference (RNAi) is the dsRNA-triggered gene regulatory mechanism that is evolutionally conserved in most eukaryotic cells. It has been widely used as a powerful tool for functional genomics in various organisms. In flies, mosquitoes or other insect cells, gene functional analysis by RNAi is usually performed through introduced dsRNAs that are synthesized by in vitro transcription. RNAi serves as an important innate immunity against viruses in plants and invertebrates. It has recently been shown that Aedes albopictus mosquito C6/36 cells, commonly used for arbovirus propagation, possess an impaired RNAi pathway. In this study, we developed in vitro Dicer assay using extracts prepared from mosquito cells. Our results confirmed the inability of C6/36 cells to process dsRNAs into siRNAs, which is consistent with the loss-of-function of Dcr-2 due to a frameshift mutation. However, such a defect could not be complemented by introduction of Drosophila Dicer-2. To evaluate the RNAi-based antiviral mechanism in C6/36 cells, we analyzed the replication of a mutant Nodamura virus (NoV) genomic RNA1 of which viral RNAi suppressor B2 is not expressed (NoVR1ΔB2) and cannot accumulate to a detectable level in RNAi-competent cells. In C6/36 cells, the defective RNAi gives rise to complete restoration of NoVR1ΔB2 replication, suggesting that RNAi is the primary antiviral immunity in mosquito cells. At present, dsRNA, as the trigger of the antiviral RNAi pathway in invertebrate and plants, is the major efficiency limitation factor in RNAi. In this study, a plasmid-based system was developed to express dsRNA intracellular from a DNA cassette containing two convergent T7 promoters in Drosophila S2 cells. Efficient knockdown of a transiently expressed reporter gene or an endogenous gene can be achieved by dsRNA expressed from the system. A random cDNA library was constructed in the dsRNA expression cassette and initial screening led to identification of two host factors that are involved in antiviral response in Drosophila S2 cells. The plasmid-based dsRNA expression system provides an alternative tool for functional genomics in Drosophila and other insect cells.
Identification and characterization of F17: a novel subviral agent that depends on dengue virus
(University of Hawaii at Manoa, 2014-08) Kakinami, Cherie Kuulei
Dengue is the most important arboviral disease of humans. It is estimated that over 40% of the world's population are at risk of dengue. In recent years, a resurgence of dengue virus has been seen with increased incidences and a wider geographical distribution. Dengue is spread to humans through the bite of an infected female Aedes mosquito. There is currently no vaccine or specific drug treatment for dengue. In this study, we identified and partially characterized a subviral agent of dengue virus, which we designated as F17. This is the first report of a subviral agent of dengue virus. F17's genome is positive-sense and single-stranded RNA and may encode a capsid protein but the viral particle is unenveloped. F17 was found to replicate efficiently in C6/36 Ae. albopictus and ATC-10 Ae. aegypti cell lines and like many subviral agents, it was found to interfere with its helper virus' replication. In C6/36 cells, the presence of F17 had an enhancing effect and an increase in dengue virus replication was observed. Interestingly, in human U937 DC-SIGN cells, F17 was found to have an inhibitory effect on dengue virus replication. Furthermore, in Vero cells, F17 inhibits dengue virus plaque formation. This is the first report of a subviral agent of dengue virus and one of a few reported outside of plants. Although arthropod-borne diseases are a major contributor to the burden caused by infectious diseases worldwide, little remains known about the virus-vector-host interactions. The transmission of dengue virus to humans is dependent on a mosquito vector and vector control remains the primary means of preventing vector-borne diseases. F17 was found to increase dengue virus replication in humans and this may have implications in the transmission of dengue virus. The relationship between dengue virus and its subviral agent may also provide insights into the pathogenesis of dengue.
Functional analysis of dengue virus nonstructural protein 1
(University of Hawaii at Manoa, 2014-08) Li, Yujia
Dengue virus NS1 is a glycoprotein that plays different roles in different stages of virus life cycle. It exists in multiple forms: intracellular membraneassociated form which is co-localized with dsRNA and essential in viral replication; cell surface-associated form which is associated with lipid raft at the plasma membrane and mediated signaling and complement activation pathways; secreted form which eventually released into the blood stream. In the first part of this study, we investigate the interaction between intracellular form NS1 and dsRNA by EMSA. Our results show that intracellular form NS1 binds dsRNA in vitro and has multiple dsRBDs. Results of the first part implicate a novel role of intracellular form NS1 in viral replication. In the second part of this study, we investigate the binding partner of secreted form NS1. We use affinity pull-down assay to isolate NS1 complex from the supernatant of transfected AD293 cells, and analyze the components by mass spectrometry. We find that ApoA-I, the major structural protein of HDL in serum, is co-immunoprecipitated with NS1. In addition, NS1 is also associated with ApoA-I that is produced and secreted from human HepG2 cells. Delipidation of ApoA-I disrupts its interaction with NS1. Formation of NS1-ApoA-I complex modulates VCAM-1 expression in HMEC-1 cells. Results of the second part indicate a novel role of secreted form NS1 in modulating endothelial cell functions, which may contribute to dengue pathogenesis. In the third part of this study, we investigate the factor that presents in human serum leading to an enhanced DV infectivity in various types of cells. Through co-immunoprecipitation, we reveal that ApoA-I is associated with DV particles and is able to promote DV infection. We further find that siRNA knockdown of SR-BI, the cell receptor of ApoA-I, abolishes the activity of ApoA-I in enhancement of DV infection. FACS analysis of cell surface dengue antigen after virus absorption further confirms that ApoA-I enhanced DV infection via promoting initial attachment of virus to cells. Results of the third part illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.
Role of hetF and patU3 in the regulation of heterocyst development in Anabaena sp. Strain pcc 7120
(University of Hawaii at Manoa, 2014-08) Tom, Sasa K.
A central paradigm in developmental biology concerns the differentiation of cells despite the fundamental sameness of the genetic complement shared by all cells in the organism. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is an ideal model system for the study of development. In response to nitrogen deprivation, Anabaena differentiates nitrogen-fixing heterocyst cells in a periodic pattern. Anabaena research has practical applications in human health, sustainable agriculture, and biofuel production. This study aimed to characterize novel interactions to refine the current understanding of Anabaena development. Investigations were performed to elucidate the hetF-dependent activation of differentiation. A novel genetic regulatory network involving hetF, a CHF class protease, and the negative regulator patU3 and other developmental genes was identified. A component of the hetZ-patU5-patU3 gene cluster, PatU3 was shown to directly interact with HetZ, another activator of differentiation. Genetic epistasis analysis determined that PatU3 suppressed positive regulation by HetZ and HetR. These negative feedback loops explain the elevated HetR-GFP concentrations in hetF-dependent strains despite the paradoxical absence of heterocysts. The HetF-dependent pathway may act as a control point prior to commitment to the heterocyst cell fate. Lateral inhibition by PatS and HetN, which both contain the same RGSGR pentapeptide sequence, involves regulation by HetR. The unique domains present in the structure of HetR may relate to its activity as a transcriptional activator. In this study, genetic and cytological approaches were used to identify residues in HetR necessary for interaction with PatS, HetN and RGSGR. A related investigation demonstrated that the RGSGR-pentapeptide derived from HetN directs pattern formation by direct cytoplasmic exchange. Lastly, protein phosphorylation plays a prominent role in varied biological processes. The PP2C-type protein phosphatase All1758 was characterized in this study. The corresponding all1758 gene is controlled by the developmental genes ntcA and hetR. All1758 affects later stages of differentiation and may represent a critical link between cell growth, cell division and morphogenesis by controlling putative sigma factor regulators (antisigma factors and antisigma factor agonists) and cell division genes (including FtsZ and MinCE). Taken together, these findings support additional regulatory mechanisms necessary for proper Anabaena development.
The HetR regulon of Anabaena sp. strain Pcc 7120
(University of Hawaii at Manoa, 2014-08) Videau, Patrick Jean-Adrien
The process of cellular differentiation relies on the action of transcriptional regulators to enact the developmental fate of the cell. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a model system for studying pattern formation and cellular differentiation. When combined nitrogen is limiting, Anabaena forms a periodic pattern of nitrogen-fixing heterocyst cells separated by 10-20 photosynthetic vegetative cells. The pattern of cells that can differentiate is defined by the interaction of the master regulator of differentiation, HetR, and the inhibitors of differentiation, PatS and HetN. The crystal structure of HetR bound to DNA determined the amino acids that interact with DNA and mutations in these amino acids yielded non-functional hetR alleles. In this work, a 17 bp inverted repeat in the promoter of the hepA gene was bound by HetR in vitro and found to be necessary for heterocyst-specific transcription in vivo. A search of the genome identified 166 potential hepA-like HetR sites and investigation of a subset of these sites found that HetR could act as either a transcriptional activator or repressor. This study also identified a HetR-binding site in the promoter region of the trpE gene, which encodes an anthranilate synthase involved in tryptophan biosynthesis. Mutation trpE yielded a strain that differentiated heterocysts in the presence of nitrate, a nitrogen source that normally represses differentiation. Analysis of the intracellular concentration of the Kreb's cycle intermediate 2-oxoglutarate (2-OG) showed a roughly 2.5 fold increase within one hour after the transition from growth on ammonia to nitrate. This spike in 2-OG is characteristic of a nitrogen starvation response and suggests a role for amino acid metabolism in the perception of nitrogen starvation. The final study utilized an FMN-dependent fluorphore, EcFbFP, to determine the transcriptional profiles of hetR, patS, and hetN in mature heterocysts. The transcription of hetR and hetN persisted in mature heterocysts while patS expression ceased. This is consistent with roles for PatS in pattern formation, HetN in pattern maintenance, and HetR in all stages of development. This work expands the direct HetR regulon and suggest a role for amino acid metabolism in the induction of differentiation.
Assessing the Persistence and Multiplication of Fecal Indicator Bacteria in Hawai‘i Soil Environment
(Water Resources Research Center, University of Hawaii at Manoa, 2000-12) Byappananhalli, Muruleedhara N.
Traditional fecal indicator bacteria such as fecal coliform, E.coli and enterococci have been shown to be unreliable indicators of the hygienic quality of recreational waters under tropical conditions. One of the major reasons for considering these bacteria as ineffective indicators of water quality in warm, tropical regions is that they are consistently found in natural environments (plants, soil, water) in the absence of any significant contamination of these environments. Since preliminary studies conducted in Hawaii had indicated soil as the major environmental source of elevated concentrations of these bacteria in environmental waters, the aim of this study was to focus on the soil environment to specifically address two assumptions made by regulatory agencies in using fecal bacteria as indicators of water quality: first, there should not be an environmental source of these indicator bacteria unrelated to sewage or fecal matter contamination, and second, the indicator bacteria do not multiply in the environment. To determine the validity of these two assumptions under tropical conditions in Hawaii and possibly other tropical locations, various experiments were conducted. The major findings are as follows. 1) Analysis of soil samples collected from various locations representing major soil groups on the island of Oahu showed that fecal indicator bacteria are naturally found in most of the soil environments, indicating that the fecal bacteria have adapted to the soil conditions to become part of soil biota. 2) Evidence was obtained to show that the soil contains adequate nutrients to sustain the populations of these bacteria. 3) Growth and multiplication of fecal indicator bacteria in natural soil was dependent on available nutrients (particularly carbon), moisture and competing microorganisms. In conclusion, tropical soil conditions are suboptimal for the multiplication of fecal indicator bacteria. Consequently, these bacteria in natural soil conditions will probably grow and multiply sporadically when conditions are relatively optimal. Although concentrations of fecal indicator bacteria in soil represent only a small fraction of the microbiota, their counts are significant enough in numbers not only to impact the quality of recreational waters but also to nullify two of the assumptions used in the application of recreational water quality standards. Thus, there is a need for an alternate and more reliable indicator of water quality in Hawaii and other tropical locations.

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