Multidisciplinary investigation on “Candidatus Midichloria” bacteria aimed at disentangling the evolution of the genus and of its intramitochondrial tropism

The world around us is populated by an immense variety of eukaryotic and prokaryotic organisms characterized by a wide range of sizes and behaviours and engaged in endless interactions. In this diverse set of interactions, the endosymbiotic bacterium Midichloria mitochondrii stands out for its unique intracellular tropism, colonizing the mitochondria of its tick host oocytes. Many hypotheses have been made on the lifestyle and role of this bacterium: it has been proposed that Midichloria mitochondrii preys mitochondria in a Bdellovibrio-like lifestyle, but it has also been suggested that this bacterium could be a mutualist of ticks and that the interaction with mitochondria is a key feature in the beneficial effect on the host. However, neither of these hypotheses can be accepted or refused through the available evidence, due to technical shortcomings: in vitro cultures are not available for Midichloria cells alone, nor tick oocytes infected by this symbiont. Furthermore, the entire Midichloria genus is understudied: the intramitochondrial tropism has been proven just for two Midichloria strains in, and there is little information on the distribution of this bacterium worldwide. In addition, no details on the interaction between Midichloria bacteria and other microorganisms have ever been provided, and from the in depth analysis of such interactions much information could derive and lead to uncover features and metabolic capabilities of Midichloria organisms. Finally, only one genome has ever been sequenced, thus not allowing a variety of analyses that could point to the roles played by Midichloria bacteria in their hosts. In this study we provide new data on the Midichloria diversity and distribution in African Countries, where the information was the scarcest, and analysed the coinfections in the ticks sampled in this area. We also provide qualitative, quantitative and genomic data on a Midichloria-Francisella coinfection in Hyalomma marginatum ticks sampled in Europe and Middle-East, leading to intriguing hypotheses on the interactions among the two bacterial symbionts in this tick species. Furthermore, we present additional ultrastructural observations on Midichloria mitochondrii, allowing us to perform mathematical modelling and test the Bdellovibrio-like hypothesis on mitochondrial colonization, and to propose a new one where Midichloria mitochondrii moves within mitochondrial networks without damaging the host mitochondria. Finally, we expand our knowledge on this genus by adding tropism information on three more Midichloria strains harboured by different tick species, revealing new strains capable of mitochondrial colonization and a strain unable to invade mitochondria. Thanks to these ultrastructural data coupled with the corresponding genomic sequences, we performed several comparative genomics analyses, allowing us to indicate genes potentially involved in the features and evolution of the intramitochondrial tropism in the Midichloria genus.