Characterization of Coxiella burnetii outbreak strains

Abstract

Q fever is a worldwide zoonotic infectious disease caused by the bacterium Coxiella burnetii. During 2007-2010, the largest Q fever outbreak was reported in The Netherlands, where more than 4000 human cases were registered showing a serious burden of the disease. During this outbreak, goats harboring predominantly the CbNL01 genotype strain were identified as the major source of disease in humans and drastic measures such as mass culling of infected goats were implemented to reduce the spread of the pathogen and control the disease. In order to minimize such complications in the future, it is crucial to have a thorough understanding of the disease causing pathogen and to develop effective Q fever vaccines. The causes of the large Dutch outbreak are not well-understood and one of the main reasons speculated were the hyper-virulent behavior of the circulating C. burnetii isolates. The research described in this thesis focuses on the characterization of C. burnetii outbreak strains isolated from infected goats, cattle, sheep and human clinical materials. Our studies were initiated to better understand the bacterial pathogenesis, virulence, evolution, adaptations in various environments, host immune responses and to identify pathogen related factors that have modulated the disease outbreak. We specifically aimed to identify the virulence factors and mechanisms that contributed to the increased zoonotic potential of the strain associated with the Dutch Q fever outbreak. The studies presented in this thesis majorly applied Pathogenomic approaches at the genome and transcriptome level to decipher host-pathogen interactions and to develop new tools to study C. burnetii infections. A transcriptome analysis of the outbreak C. burnetii strain of the CbNL01 genotype grown under in vivo and in vitro conditions resulted in the identification of distinct metabolic adaptations and virulence mechanisms of the bacterium. Detailed comparative analysis of complete genome sequences of C. burnetii strains showed a high similarity between strains of the same genotype. Genome sequences of the Dutch outbreak CbNL01 genotype strains were more divergent than the genome sequences of the less prevalent CbNL12 genotype strains and the NM reference strain. The analysis also showed that the high virulence of the outbreak strains was not associated with acquiring novel virulence-related genes arguing against the idea that the Dutch outbreak was due to emergence of hyper-virulent strains though horizontal gene transfer. Among the prominent genetic differences in the CbNL01 outbreak strains compared to CbNL12 and NM, were the presence of several point mutations and increased transposon mediated genome plasticity, which might have contributed to its epidemic potential. Point mutations, especially in a large number of membrane proteins, could also have contributed to the increased zoonotic potential of CbNL01 strains allowing this clone to escape the host immune responses in goats and humans. In addition, mutations in critical genes involved in virulence and evasion of the host immune system could be potentially involved in the increased virulence of the CbNL01 outbreak strains. On the contrary, studies on host immune responses in an in vivo (experimental infections in mice) and an in vitro (human PBMC’s stimulation) model did not show any difference associated with the strain genotype. However, differences in immune responses were found to be associated with the host-origin of the C. burnetii strains. Among different host-origin strains, strains derived from goats and humans generated significantly lower innate and adaptive immune responses than strains derived from cattle, whereas no differences in immune responses were observed when strains were grouped based upon their genotype. These observations support immune evasions as a major virulence strategy of goat and human strains in hosts and further suggest that bacteria originating from goats have a greater potential to cause outbreaks in humans. This indicates that for Q fever prevention purposes goats should be efficiently monitored for the presence of C. burnetii. Taken together, the results described in this thesis suggest that the virulence potential of C. burnetii strains is not only based on genetic differences, but also on other host-adaptation mechanisms such as transposition of genomic elements and/or differential regulation of gene expression. Finally, the results from this thesis provide a framework for future studies in the development of vaccines and diagnostic tools for Q fever.