BackgroundBacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems.ResultsHere we show that the trypanosomatid Angomonas deanei, which harbors a β-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei.ConclusionsAfter previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0820-z) contains supplementary material, which is available to authorized users.
b Linezolid-dependent growth was recently reported in Staphylococcus epidermidis clinical strains carrying mutations associated with linezolid resistance. To investigate this unexpected behavior at the molecular level, we isolated active ribosomes from one of the linezolid-dependent strains and we compared them with ribosomes isolated from a wild-type strain. Both strains were grown in the absence and presence of linezolid. Detailed biochemical and structural analyses revealed essential differences in the function and structure of isolated ribosomes which were assembled in the presence of linezolid. The catalytic activity of peptidyltransferase was found to be significantly higher in the ribosomes derived from the linezolid-dependent strain. Interestingly, the same ribosomes exhibited an abnormal ribosomal subunit dissociation profile on a sucrose gradient in the absence of linezolid, but the profile was restored after treatment of the ribosomes with an excess of the antibiotic. Our study suggests that linezolid most likely modified the ribosomal assembly procedure, leading to a new functional ribosomal population active only in the presence of linezolid. Therefore, the higher growth rate of the partially linezolid-dependent strains could be attributed to the functional and structural adaptations of ribosomes to linezolid. O xazolidinone antibiotics inhibit protein synthesis by binding to the peptidyltransferase center (PTC) of the ribosome and inhibiting the growth of bacteria (1). Although it has been suggested that they are involved in the initiation of translation, many reports have been contradictory, mainly because the ratio of drug to ribosome that was used was extremely high (2). Moreover, the inhibitory effect of oxazolidinones on peptide bond formation has not been demonstrated so far, despite structural data suggesting the binding of linezolid in the peptidyltransferase center (3-5). In contrast, linezolid perturbs translational accuracy in vivo, even at concentrations lower than the MIC (6).Linezolid (LZD) was the first FDA-approved oxazolidinone used to treat serious infections due to Gram-positive bacteria. Although LZD was completely synthetic (7), resistance readily emerged and was attributed mainly to mutations in 23S rRNA and ribosomal proteins L3 and L4. Mutations in 23S rRNA implicated in linezolid resistance include not only bases near the binding site, like G2061, C2452, A2503, U2504, and G2505, but also bases that are located more distantly from the binding site, such as A2062, G2447, A2453, C2499, U2500, and G2576 (reviewed in reference 8). Additional mechanisms include acquisition of the cfr gene, which encodes a methyltransferase which modifies A2503 in 23S rRNA, and a mutation in the RlmN gene, which naturally modifies A2503 (8-14). Recently, four nosocomial Staphylococcus epidermidis isolates belonging to the same pulsed-field gel electrophoresis type were described to exhibit partial linezolid dependence, an adaptation reported for only a few antibiotics and bacterial species in the ...
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