Identification of Colonization Factors in $\textit{Burkholderia}$ by Applying Genome-Wide Mutant Profiling

Abstract

In the 1980s, the first members of the Burkholderia cepacia complex (Bcc), which were previously known only as pathogens of onions, were isolated from cystic fibrosis (CF) patients. Infections with members of the Bcc are difficult to treat and are associated with high mortality rates in CF patients. Bcc infections are often severe and can cause the “cepacia syndrome”, a necrotizing pneumonia that results in the rapid decline of lung function. Treatment with antibiotics usually fails to eradicate Bcc infections due to their high metabolic adaptability and intrinsic antibiotic resistance. We employed transposon sequencing (Tn-seq) to investigate colonisation by and survival of the clinical Bcc isolate B. cenocepacia H111 in different infection models, with the aim to identify novel drug targets. Tn-seq is a combination of transposon insertional mutagenesis with massive parallel sequencing that allows the identification of those genes that provide a fitness benefit in a particular growth condition. In this thesis, we employed Tn-seq to investigate genetic factors responsible for the anoxic survival of the obligate aerobe B. cenocepacia H111 in vitro (Chapter 2). Of a total of 71 unique fitness determinants required for the anoxic survival of B. cenocepacia H111, five candidate genes were experimentally validated by mutation and phenotypic characterization. Among these was a homologue of the Crp-Fnr family transcriptional regulator Anr known to be involved in the induction of arginine fermentation and nitrate reduction under anaerobic conditions. We showed that upon deletion of both anr copies in B. cenocepacia H111, anoxic survival ability is completely abolished. Furthermore, we identified a two-component system (RoxS/RoxR), a bd type terminal oxidase (CydPABX) and the sigma factor FliA and showed that they are important for anoxic survival of B. cenocepacia H111. In addition, we found that mutants, in which roxS, cydA or both homologs of anr have been inactivated, showed reduced pathogenicity in a Galleria mellonella infection model. Finally, we showed that the fliA mutant is non-flagellated and impaired in its swimming and swarming abilities. In Chapter 3, the role of terminal oxidases for the survival of B. cenocepacia H111 in various conditions was investigated with the aim to provide a basis for the development of respective inhibitors for the treatment of Burkholderia infections. To this end, a bioinformatic analysis was performed to identify six loci coding for terminal oxidases: Three heme-copper oxidases (bo3, caa3 and aa3) and three bd-type oxidases (bd-I and two cyanide insensitive oxidases cio-1 and cio-2). Reporter fusions for each terminal oxidase were constructed and expression levels under various conditions were determined. We found that the aa3 terminal oxidase (cta) was constitutively expressed and that in oxygen-limited environments the bd-I oxidase (cyd) was induced. Interestingly, the cyanide-insensitive terminal oxidase (cio-1) confers resistance to exogenously added cyanide or cyanide produced by Pseudomonas aeruginosa PA14, which could provide a fitness benefit in the CF lung in the case of polymicrobial infections. We also showed that the Anr and RoxS/RoxR regulators are involved in the regulation of cyd and cio-1. In Chapter 4, the Tn-seq approach was used to identify genetic factors required for the colonization of two models systems, the in vivo G. mellonella infection model and an ex vivo porcine lung model (EVPL). A total of 698 and 117 fitness determinants were identified, respectively, of which 62 were shared in both models. The results highlight the importance of using multiple model organisms to identify colonization factors, as our data showed that some factors can be model-specific while others are common. Most interestingly, a novel genomic region (I35_RS03700-I35RS03770) was identified that is absent from most Burkholderia strains and we showed that it is involved in lipopolysaccharide biosynthesis/modification and is important for G. mellonella colonization while detrimental for the colonization of the pig lung.