Browsing by Author "Schweizer, Herbert P., advisor"
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Item Open Access Counter-selection markers for allele replacement in Burkholderia pseudomallei(Colorado State University. Libraries, 2008) López Peláez, Carolina María, author; Schweizer, Herbert P., advisorBurkholderia pseudomallei is a gram negative bacillus that lives in the soil of tropical regions around the planet and causes melioidosis in humans, a disease endemic in regions of Southeast Asia and Northern Australia. The United States government has classified B. pseudomallei, and its relative Burkholderia mallei, as potential bioterrorism agents. The increased interest in these complex pathogens initiated a quest to better understand the biology of these bacteria at the molecular level. Completed genome sequences of diverse strains have provided a wealth of information that opened new venues for further study. Many genetic tools have been successfully adapted for use in Burkholderia species, but others are yet to be discovered. The ability to introduce unmarked single nucleotide changes or other genetic modifications into the B. pseudomallei genome, by way of the host's natural homologous recombination pathways, has been hampered by the lack of a suitable counter-selection marker that works efficiently in different wild-type strains. Counter-selection markers allow for the positive selection of strains that have lost the marker and other unwanted sequences around them. This dissertation describes the search for a system that allows isolation of unmarked mutations and single nucleotide changes in the B. pseudomallei genome. Two different systems were proven effective and provide alternative options for isolation of allelic mutants of genes of interest. The first method uses a mutated allele of the B. pseudomallei pheS gene. This gene encodes for a subunit of phenylalanine tRNA synthase. A specific PheS mutant protein exhibits relaxed substrate specificity, allowing for incorporation of a toxic chlorinated phenylalanine analog into proteins resulting in death of cells expressing the mutant protein. Counter-selection based on the mutant pheS gene of B. pseudomallei allowed for the creation of amrRAB-oprA deletion mutants of different B. pseudomallei strains. The AmrAB-OprA efflux pump is responsible for intrinsic resistance to aminoglycosides and macrolides in B. pseudomallei. Consequently, efflux pump mutants became sensitive to selected aminoglycosides. Also, as a proof of concept experiment, a clean unmarked purM mutant was created. purM mutants are thiamine and adenine auxotrophs and have been shown to result in a strong attenuation of virulence in a mouse model of melioidosis. A second system based on the I-SceI homing endonuclease of Saccharomyces cerevisiae was also developed. Expression of the endonuclease in cells containing chromosomal I-SceI recognition sites integrated in their chromosomes in place of counter-selection markers via homologous recombination, leads to the selection of isolates that have lost the sites and thus unwanted sequences containing them. This is because I-SceI creates double-strand breaks and promotes recombination between nearby homologous sequences. As a proof of concept experiment this system was also used to create a B. pseudomallei purM mutant. Furthermore, by creating a temperature sensitive fabD mutant due to a point mutation in the fabD gene proved that I-SceI could be used to create point mutations. FabD is an essential enzyme of the bacterial fatty acid biosynthesis pathway. In summary, this report describes the first counter-selection markers that work in wild-type B. pseudomallei strains. Availability of the markers will allow the routine generation of mutants required for studies of the biology and pathogenesis of this understudied pathogen and the related B. mallei.Item Open Access Drug efflux systems and antibiotic resistance in Burkholderia pseudomallei(Colorado State University. Libraries, 2009) Trunck, Lily, author; Schweizer, Herbert P., advisorBurkholderia pseudomallei, the etiologic agent of melioidosis, is intrinsically resistant to most antibiotics. A predicted 10 RND efflux transporters are encoded by the B. pseudomallei genome; 3 have been characterized (AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC) as major contributors to the intrinsic aminoglycoside, macrolide, chloramphenicol, and trimethoprim resistance of this organism. AmrAB-OprA is constitutively expressed in most strains and confers resistance to aminoglycosides. Gentamicin susceptible clinical isolates have been identified and work in this dissertation demonstrates that such susceptibility occurs as a result of either insufficient expression or deletion of amrAB-oprA. The mechanisms regulating expression in these strains are unclear, but are not related to mutations in this operon's putative repressor (AmrR) or mutations in the regulatory regions of amrAB-oprA. Expression analysis of seven B. pseudomallei RND efflux pumps (amrB, bpeB, bpeF, bpeH, BPSL0309, BPSL1267, and BPSL1567) in 60 clinical and environmental B. pseudomallei isolates from Thailand demonstrated (i) efflux pump expression is prevalent in both clinical and environmental strains (ii) bpeH is expressed at a higher level in clinical isolates as compared to environmental isolates and (iii) efflux pump expression correlates with resistance/susceptibility to several antibiotics. These data suggest possible substrates for uncharacterized efflux pumps or, alternatively, co-regulation of resistance determinants. Since a deficit in efflux resulted in susceptibility to otherwise clinically useful drugs and that expression of efflux pumps was prevalent in B. pseudomallei, we suggest that efflux pump inhibitors would broaden the spectrum of antibiotics useful for treatment of melioidosis. To facilitate discovery and characterization of such compounds, we have developed a panel of efflux deficient B. thailandensis strains that express the B. pseudomallei efflux pumps, amrAB-oprA and bpeAB-oprB, which can be handled under BSL2 conditions. When expressed in the surrogate background, these pumps have the same substrate profile and can be inhibited by clinically insignificant efflux pump inhibitors to a degree similar to that observed in the native background. In summary, we have assessed the contribution of efflux to antibiotic resistance in B. pseudomallei and described a tool for discovery and characterization of efflux pump inhibitors for pumps expressed in this organism.Item Open Access Folate pathway inhibitor resistance mechanisms in Burkholderia pseudomallei(Colorado State University. Libraries, 2013) Podnecky, Nicole L., author; Schweizer, Herbert P., advisor; Dow, Steven W., committee member; Slayden, Richard A., committee member; Stargell, Laurie A., committee memberAntimicrobials are invaluable tools used to facilitate the treatment of infectious diseases. Their use has saved millions of lives since their introduction in the early 1900's. Unfortunately, due to the increased incidence and dispersal of antimicrobial resistance determinants, many of these drugs are no longer efficacious. This greatly limits the options available for treatment of serious bacterial infections, including melioidosis, which is caused by Burkholderia pseudomallei, a Gram-negative saprophyte. This organism is intrinsically resistant to many antimicrobials. Additionally, there have been reports of B. pseudomallei isolates resistant to several of the antimicrobials currently used for treatment, including the trimethoprim and sulfamethoxazole combination, co-trimoxazole. The overarching goal of this project was to identify and characterize mechanisms of trimethoprim and sulfamethoxazole resistance in clinical and environmental isolates, as well as in laboratory induced mutants. Prior to these studies, very little work has been done to identify and characterize the mechanisms by which B. pseudomallei strains are or could become resistant to folate-pathway inhibitors, specifically trimethoprim and sulfamethoxazole. During the initial phases of these studies, we determined the antimicrobial susceptibilities of a large collection of clinical and environmental isolates from Thailand and Australia (n = 65). A high frequency of naturally-occurring resistance to trimethoprim alone (40%) was observed. However these strains were susceptible to sulfamethoxazole and to the co-trimoxazole combination. Trimethoprim resistance in a subset of these strains was due to increased expression of an efflux pump belonging to the resistance nodulation and cellular division (RND) superfamily, BpeEF-OprC, in the presence of trimethoprim. This efflux pump had been previously shown to efflux trimethoprim, chloramphenicol and tetracyclines when expressed in surrogate bacterial strains. The molecular mechanism of increased BpeEF-OprC expression in these isolates remains unknown. Similarly, decreased susceptibility in laboratory mutants selected on trimethoprim were due to mutations leading to amino acid substitutions in BpeT, which caused overexpression of BpeEF-OprC, or FolA, the trimethoprim drug target. This is the first description of mutations to FolA conferring trimethoprim resistance in B. pseudomallei, though similar mutations have been observed in B. cenocepacia and Escherichia coli. A similar study to select for sulfamethoxazole resistance, instead suggested that B. pseudomallei may be able to tolerate high concentrations of the drug. Studies to characterize laboratory induced mutants selected on co-trimoxazole led to the identification of two novel resistance determinants. Mutations to BpeS, a newly named LysR-type regulator with high similarity to the cognate BpeEF-OprC efflux pump regulator, BpeT, cause increased BpeEF-OprC expression in these strains. Additionally mutations to Ptr1, an annotated pteridine reductase, partially contributed to the decreased co-trimoxazole susceptibility. However, it is unclear what function Ptr1 has in the folate synthesis pathway, as deletion of this gene also caused slight decreases in antimicrobial susceptibility. Finally, in a collection of co-trimoxazole resistant clinical isolates from Thailand, high-level expression of the BpeEF-OprC was found in the resistant isolates. A mutation to BpeS was also observed in two of the clinical isolates that had BpeT-independent BpeEF-OprC overexpression. Co-trimoxazole resistant isolates were each resistant to both trimethoprim and sulfamethoxazole individually. However, deletion of the bpeEF-oprC efflux pump structural genes in all isolates resistant to co-trimoxazole or isolates resistant to trimethoprim alone (except those with a mutant FolA) resulted in antimicrobial susceptibility to trimethoprim, co-trimoxazole and sulfamethoxazole. These data suggest that sulfamethoxazole is also a substrate of the BpeEF-OprC efflux pump and this RND pump is the major resistance determinant contributing to clinically relevant folate pathway inhibitor resistance in B. pseudomallei. To summarize, we have identified and described several resistance determinants in B. pseudomallei causing decreased susceptibilities to trimethoprim, sulfamethoxazole and/or co-trimoxazole; these include drug target and metabolic pathway modifications and overexpression of the BpeEF-OprC efflux pump. Further characterization of these mechanisms and the development of specific detection assays could allow for rapid determination of antimicrobial resistance and provide useful information for the development of novel antimicrobials against B. pseudomallei.Item Open Access Interactions between plants and an opportunistic human pathogen, Pseudomonas aeruginosa(Colorado State University. Libraries, 2008) Weir, Tiffany L., author; Schweizer, Herbert P., advisor; Vivanco, Jorge M., advisorPseudomonas aeruginosa is an opportunistic human pathogen that can be found living in soil, water, or saprophytically on plant tissues. It is important to understand the pathology of this organism under variable conditions because of its ability to survive in diverse environments, its role in human disease, and its use as a model organism in studies on biofilm formation, quorum-sensing, and pathogenicity. To this end, a number of unconventional model systems, including plants, nematodes, and fruit flies, have been developed to study the pathology of P. aeruginosa. In the present study, the interactions between P. aeruginosa and plants, with respect to pathogenicity, quorum-sensing, and microbial ecology are further explored. To examine what factors are important in the pathogenicity of P. aeruginosa in a plant system, compatible and incompatible cultivars of Nicotiana tabacum were infiltrated with the pathogen. Bacterial growth in planta was monitored and P. aeruginosa PAO1 gene expression was examined 24 hours after infiltration into the hosts. The data suggests that, in addition to known virulence factors, the acquisition of micronutrients such as sulfate and inorganic phosphate are also important in disease development. The results of this study also suggest that type III secretion systems may be important in P. aeruginosa's ability to infect plants, and that differences in host response, i.e. salicylic acid signaling, are determining factors in host compatibility. Another aspect of this study was to utilize the natural interactions between plant roots and soil-borne bacteria to identify root exudates that interfere with bacterial quorum sensing (QS), particularly in P. aeruginosa. Quorum sensing in P. aeruginosa controls the expression of several secreted factors that are important in virulence of the pathogen, and preventing infections by inhibition of quorum sensing is a current therapeutic target. Unfortunately, while many of the exudates appeared to have some affect on QS in general, none had strong activity against P. aeruginosa QS systems. However, one class of chemicals, triterpene saponins, was shown to be active in a lux-based QS reporter. Finally, preliminary data suggesting that root exudates can influence competitive outcomes between two soil-borne bacterial species are also presented. The interactions between bacteria are typically studied in nutrient rich medium under defined laboratory conditions. Under these conditions, P. aeruginosa outcompetes Agrobacterium tumefaciens, two bacteria that potentially compete for the same niche in the soil. However, when Arabidopsis thaliana is factored into this equation, growth of A. tumefaciens is favored. Furthermore, the negative effects of P. aeruginosa on the growth of A. thaliana were reduced.Item Open Access Sub-cellular localization of the PenA β-lactamase in Burkholderia pseudomallei(Colorado State University. Libraries, 2013) Randall, Linnell B., author; Schweizer, Herbert P., advisor; Borlee, Brad R., committee member; Argueso, Juan Lucas, committee memberBurkholderia pseudomallei, a Gram-negative soil bacterium found in tropical regions, is the etiologic agent of melioidosis. B. pseudomallei is intrinsically resistant to many antibiotics, and melioidosis treatment involves prolonged antibiotic therapy. PenA, a chromosomal beta-lactamase in B. pseudomallei, confers resistance to many beta-lactams. Point mutations in penA leading to PenA amino acid changes can cause resistance to ceftazidime and amoxicillin-clavulanate, which can result in treatment failure. Typically beta-lactamase enzymes are found in the periplasm of Gram-negative bacteria. Previous studies have shown that PenA is secreted via the twin arginine translocase (Tat) system, but failed to demonstrate periplasmic localization. The purpose of this study was to determine the sub-cellular localization of PenA in B. pseudomallei. Using alkaline phosphatase as a periplasmic marker, we optimized a method for extracting periplasmic proteins from B. pseudomallei. Through subcellular fractionations, immunoblotting, and colorimetric enzyme assays, we have shown that PenA does not localize to the periplasm. Rather, it is present in a detergent-soluble fraction of the cellular membranes. Further experiments including site-directed mutagenesis, metabolic labeling with 14C-palmitate, globomycin treatment, and mass spectrometry indicate that PenA is likely a lipoprotein with post-translational lipid modification of the cysteine residue at position 23. This work implicates PenA as the first example of a beta-lactamase that is a Tat-secreted lipoprotein, and provides a better physiological understanding of an important antibiotic resistance mechanism in B. pseudomallei.