Amoebophilus asiaticus mechanisms for host cell interaction

Free-living amoebae are abundant in terrestrial habitats and are among the most important predators of microorganisms, thereby having a major impact on microbial community composition. During evolution some bacteria have learned to evade phagocytosis by amoebae, to use them as hosts, and to establish a stable and symbiotic association with these protozoa. In total, five groups of symbionts of Acanthamoeba sp., belonging to three different phyla, are known to date. All these Acanthamoeba symbionts are obligate intracellular, i.e. they can not be cultivated in cell-free media. Due to their only recent discovery and their intracellular life style our knowledge about these elusive bacteria is still scarce. We have determined and analyzed the genome of `Candidatus Amoebophilus asiaticus` (hereafter: A. asiaticus) strain 5a2, a virtually uncharacterized obligate amoeba symbiont, belonging to the phylum Bacteroidetes. Compared to other bacteria, the A. asiaticus genome shows massive proliferation of IS elements (24% of all predicted of all open frames), indicating rapid genome evolution allowing novel insights into the adaptation of Bacteroidetes to an intracellular life style. Genome analyses revealed that central metabolic pathways are reduced or absent. The A. asiaticus genome encodes a hitherto unmatched arsenal of proteins with eukaryotic domains including ankyrin-repeats, TPR/SEL1 repeats, and leucine-rich repeats - most likely used for host cell interaction. Among prokaryotic genomes, the A. asiaticus genome contains the highest number of proteins targeting the host ubiquitin system so far, including F-and U-box proteins and putative ubiquitin-specific proteases; indicating an important role of ubiquitin interference for host cell interaction. The main aim of this study is to identify important factors controlling this symbiotic system by sequencing the mRNA pool of the symbiont and its amoeba host. This allow us to identify and characterize mechanisms important for host cell interaction by A. asiaticus, as well as important proteins from the Acanthamoeba host influencing this symbiosis.

Free-living amoebae are abundant protozoa and are among the most important predators of microorganisms. During evolution some bacteria have learned to evade predation by amoebae, to use them as hosts, and to establish a stable and symbiotic association with these protozoa. Due to their only recent discovery and their obligate intracellular life style our knowledge about these elusive bacteria is still scarce. In this project we performed RNA sequencing of the obligate intracellular symbiont Amoebophilus (A.) asiaticus and its Acanthamoeba host in order to identify genes which are important for this symbiosis. We could show that proteins harbouring eukaryotic domains are highly expressed by the symbiont, suggesting an important role in host cell interaction. We also identified a novel putative secretion system in A. asiaticus which is also highly expressed, particularly during the infective parts of its life cycle. The A. asiaticus genome harbors an among bacteria extremely high number of mobile genetic elements (mostly transposases). A high number of these transposases were found to be highly expressed, although we could not identify signs for recent transpositional activity of transposases. Interestingly, we found evidence that the transposases influence the transcription of downstream genes. We also characterized three transporters in A. asiaticus which import ATP or the cofactor S-adenosylmethionine from the amoeba host and thus compensate for its inability to synthesize ATP, and the cofactor S-adenosylmethionine de novo.