Identification of Pseudomonas aeruginosa Genes Important for Production of Mixed Species Biofilm with Burkholderia cenocepacia

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2015-12
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Vo, Hung
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[Honolulu] : [University of Hawaii at Manoa], [December 2015]
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Pseudomonas aeruginosa (Pa) is globally prevalent and dangerous opportunistic bacterial pathogen, which has challenged Cystic Fibrosis (CF) patients in the United States. Pa causes serious chronic infections by forming biofilm in the CF patient’s lungs and respiratory tracts, which help the bacteria’s resistance to host defense immunity and antibiotics. The co-infection Pa and Burkholderia cenocepacia (Bc) further endangers the CF patients because Pa and Bc can form a more productive biofilm with increased virulence in mixed species biofilm (MSB) compared to Pa acting alone in a single species biofilm (SSB). Therefore, it is imperative to determine how these two bacterial pathogens form biofilm together, specifically, which genes of Pa and Bc are important for MSB. Identifying these genes could lead to discoveries in new methods for prevention and treatment of the infection. Moreover, these studies help us to further understand the bacterial communication network and interactions in MSB, which is currently not well characterized. Biofilms grow vertically and expand outward following attachment of the bacteria to a surface and subsequent cell-cell adhesion, proliferation, maturation and dispersion (Seminara et al., 2012). Bacterial cells in the biofilm exhibit a spatially different expression to adapt to the host-specific environment (Davey et al., 2000). Our previous transcriptome microarray study of different spatial regions (surface, middle, and bottom) of vertically sectioned biofilms using Laser Capture Microdissection (LCM) was performed and determined 49 Pa genes that have different spatial expression in MSB in comparison to SSB (unpublished data) (Table 1 and Figure 1). My hypothesis is that the reason these Pa genes are altered is that they are important for MSB with Bc. In this study, Pa mutants of these 49 genes were screened for biofilm formation ability alone (SSB) and in the presence of Bc (MSB) using a crystal violet assay. The mutants of these genes, which can form SSB but cannot form wild-type levels of MSB, are important for MSB. To validate microarray data, non-disruptive gfp transcriptional gene fusions were constructed and tested in a drip flow reactor to visualize the gene expression in mature SSB and MSB. From the 49 genes exhibiting spatially different expression in SSB and MSB, 8 genes (Group 1, Table 1) implied importance for MSB because its mutants could form biofilm in SSB but exhibited reduced biofilm formation in MSB compared to wildtype Pa (PA01) and Bc (LO6). This thesis focuses on three of the eight Pa genes from Group 1 (important for MSB), PA1497, PA2422 and PA5150. The gene PA2422, a predicted secreted protein, is strongly expressed at the bottom of MSB while expressing lowly in surface and middle in SSB. The gene PA5150, a predicted short chain dehydrogenase/reductases (SDRs), showed low and discontinuous expression in surface and middle of SSB but high and continuous expression in surface of MSB. The gene PA1497 exhibited low expression in SSB but high expression in surface and middle of MSB. Moreover, the gene PA1497 was initially demonstrated to be a probable urea transporter and related to urea uptake for nutrient or urea stress reduction in MSB. To investigate their roles in virulence, colonization and biofilm formation in vivo, survival curves and competitive indexes of the mutants of each Pa gene in single and mixed infection with LO6 were studied in vivo by the D. melanogaster feeding model. The results indicated that these genes were not related to virulence since its mutants still killed flies similar to wild-type. Interestingly, genes PA2422 and PA5150 indicated importance for mixed aggregation as well as mixed biofilm formation in vivo since their mutants could not compete and aggregate to its complements to form MSB with LO6 in the fly crop. The gene PA1497 showed importance for MSB in vitro but not for MSB in D. melanogaster feeding model since its mutant could compete and aggregate to its complement to form MSB in the fly crop. These findings further contribute to the information of the physiology of bacteria interaction in MSB, which is important to develop new methods for treatment and prevention of co-infection with Pa and Bc for CF patients.
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M.S. University of Hawaii at Manoa 2015.
Includes bibliographical references.
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Molecular Biosciences & Bioengineering
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