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Characterization of putative virulence-associated genes of Burkholderia pseudomallei

  • Ziel dieser Arbeit war die funktionelle Charakterisierung von zwei putativen Virulenzgenen von Burkholderia pseudomallei. Es wurde ein klassischer LysR-Typ Transkriptionsregulator (BPSL0117) mittels Tn5 Mutagenese als wichtiger Virulenzfaktor gefunden und mit verschiedenen in vitro, in vivo und weiterer molekularer Methoden wie gelfreie Proteomics untersucht. Daneben wurde ein hypothetisches Protein (BPSS1528 = bapA) mit unbekannter Funktion aus einen Typ-III-Sekretionssystem gezielt deletiert und funktionell beschrieben. Diese Mutante wies letztlich keine eingeschränkte Virulenz im Vergleich zum Wildtypstamm aus.
  • The Gram-negative rod Burkholderia pseudomallei (Bp) is the causative agent of the disease melioidosis, an often fatal infection of humans and animals. The bacteria can be isolated from soil and water in tropical and sub-tropical areas around the world. Moreover, Bp is a facultative intracellular bacterium that can survive in phagocytic cells. Bp possesses a wide array of virulence genes including numerous secretion systems such as three type 3 secretion system clusters (T3SS) and six type VI secretion clusters (T6SS). Furthermore, the genome of Bp consists of 88 paralogous LysR-type transcriptional regulator (LTTR) genes including BPSL0117 gene that may regulate other virulence factors. In the first part of this study, we characterised a role of the putative LTTR BPSL0117 of Bp for the virulence of this pathogen. For this purpose, a Bp ΔBPSL0117 transposon mutant that was already available was successfully complemented by using pUC18T-mini-Tn7T-FRT-Zeo vector. The Bp ΔBPSL0117 transposon mutant exhibited impaired biofilm formation, motility, exoproteases activity, and intracellular growth. Bp ΔBPSL0117 also showed more sensitivity to beta-lactamse antibiotics. Besides in vitro phenotypes, Bp ΔBPSL0117 presented reduced bacterial load in organs and highly attenuated virulence in the BALB/c mouse model of melioidosis. All observed phenotypes were reverted to wild type level in the complemented mutant strain. Next, a protein expression profile analysis should help to examine BPSL0117 dependent protein expression. Using stable isotope labeling by amino acids in cell culture (SILAC), we were able to detect 1608 cytoplasmic proteins. Beside others, we found that BPSL0117 regulates the expression of T3SS3 and T6SS1 associated proteins and enzymes involved in metabolic pathways. In silico analysis, predicted BPSL0117 structure showed high similarity with full-length crystal structures of LTTR proteins like CbnR and CrgA. Collectively, BPSL0117 works as both a positive and a negative regulator. Finally, due to the high attenuation in mice of the Bp ΔBPSL0117 transposon mutant, we examined whether this strain might serve as a promising live vaccine candidate. BALB/c mice were intranasally immunized with a high dose of Bp ΔBPSL0117 and then challenged with Bp E8 wild type via either the intranasal, intraperitoneal or intravenous route. Vaccinated mice showed increased titers of specific IgG anti-Bp antibodies and a delayed time to death compared to unvaccinated mice. Thus, Bp ΔBPSL0117 could partially protect mice against melioidosis but was less protective efficiency compared to other experimental vaccine strategies against Bp. In the second part of this study, the focus was to unravel function for the genes bapA, bapB and bapC, which are encoded within the T3SS cluster 3 of Bp and have rarely been addressed in other studies so far. In a first set of experiments, it was shown that the expression levels of bapB and bapC genes increased when Bp was cultivated in vitro under high salt concentrations. While the expression levels of all bap genes increased when Bp was cultured in vitro under low pH. To further evaluate the role of the bapA gene, an in-frame deletion mutant was constructed. The Bp ΔbapA#1 mutant strain derived from Bp K96243 wild type exhibited increased biofilm formation compared to wild type bacteria. Interestingly, this mutant was also able to replicate more efficiently inside macrophages and showed increased virulence in BALB/c mice. However, these phenotypes were not observed in the independently derived mutants Bp ΔbapA#2 from parental Bp K96243 and Bp ΔbapA#3 from parental Bp 1026b since it was not possible to successfully complement the Bp ΔbapA#1 mutant strain. Thus, the bap genes seem to play a role for the adaptations to environmental stress, but at least the bapA gene is not directly involved in the pathogenic potential of Bp in experimental murine infection.

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Author: Duong Tuan Linh
Title Additional (German):Charakterisierung von putativen virulenzassoziierten Genen von Burkholderia pseudomallei
Title Additional (English):Characterization of putative virulence-associated genes of Burkholderia pseudomallei
Advisor:Prof. Dr. Jan Buer, Prof. Dr. Ivo Steinmetz
Document Type:Doctoral Thesis
Date of Publication (online):2014/12/19
Granting Institution:Ernst-Moritz-Arndt-Universität, Universitätsmedizin (bis 31.05.2018)
Date of final exam:2014/11/21
Release Date:2014/12/19
Tag:Effektorprotein; LysR-Type Regulator; Typ-III-Sekretionssystem
Burkholderia pseudomallei; attenuation; type-III-secretion-system; virulence
GND Keyword:Transkriptionsfaktoren, Virulenz, Attenuierung, Metabolismus, Pathogenität
Faculties:Universitätsmedizin / Friedrich-Loeffler-Institut für Medizinische Mikrobiologie
DDC class:600 Technik, Medizin, angewandte Wissenschaften / 610 Medizin und Gesundheit