Sergi Torres PuigInstitut for Biokemi og Molekylær Biologi · Biochemistry and Molecular Biology
11.75· Doctor of Philosophy
Research Items (17)
Adhesion of pathogenic bacteria to target cells is a prerequisite for colonization and further infection. The main adhesins of the emerging sexually transmitted pathogen Mycoplasma genitalium, P140 and P110, interact to form a Nap complex anchored to the cell membrane. Herein, we present the crystal structures of the extracellular region of the virulence factor P110 (916 residues) unliganded and in complex with sialic acid oligosaccharides. P110 interacts only with the neuraminic acid moiety of the oligosaccharides and experiments with human cells demonstrate that these interactions are essential for mycoplasma cytadherence. Additionally, structural information provides a deep insight of the P110 antigenic regions undergoing programmed variation to evade the host immune response. These results enlighten the interplay of M. genitalium with human target cells, offering new strategies to control mycoplasma infections.
In the human pathogen Mycoplasma genitalium, homologous recombination is under the control of σ20, an alternative sigma factor that boosts the generation of genetic and antigenic diversity in the population. Under laboratory growth conditions, σ20 activation is rare and the factors governing its intermittent activity are unknown. Two σ20-regulated genes, rrlA and rrlB, showed to be important for recombination of homologous DNA sequences in this bacterium. Herein, we demonstrate that rrlA and rrlB code for two small proteins that participate in a feed-forward loop essential for σ20 function. In addition, we identify novel genes regulated by σ20 and show that several non-coding regions, which function as a reservoir for the generation of antigenic diversity, are also activated by this alternative sigma factor. Finally, we reveal that M. genitalium cells can transfer DNA horizontally by a novel mechanism that requires RecA and is facilitated by σ20 overexpression. This DNA transfer system is arguably fundamental for persistence of M. genitalium within the host since it could facilitate a rapid dissemination of successful antigenic variants within the population. Overall, these findings impose a novel conception of genome evolution, genetic variation and survival of M. genitalium within the host.
Over the last two decades, replicative OriC plasmids have been successfully developed for several Mollicutes. The OriC region of these shuttle vectors contains DnaA binding sites similar to the Escherichia coli consensus sequence 5’-TTATCCACA-3’. Therefore, the presence of conserved DnaA boxes is a hallmark of mycoplasmal OriC sites, which actually facilitates the identification of the replication origin in the chromosome. So far, the identification of the Mycoplasma genitalium OriC region (Mge-OriC) has been elusive. The usual locations of the OriC, i.e. flanking the dnaA gene (MG_469) or between the parA (MG_470) and dnaN (MG_001) genes, are devoid of conserved DnaA boxes in this bacterium. In order to identify the Mge-OriC, we used a computational method aimed to predict stress-induced DNA destabilization sites (SIDD). Susceptibility to stress-induced destabilization is closely associated with several classes of DNA regulatory regions, including promoters, terminators or replication origins. Using the SIDD method, we identified a putative OriC region between the parA and dnaN genes. We found that plasmids containing the putative OriC region were capable to replicate when introduced into M. genitalium. Transformation efficiencies obtained with the Mge-OriC plasmid were similar to those obtained with transposons. When the replicative OriC plasmid was introduced into the reference strain G37, it rapidly integrated into the chromosome at a high frequency. The absence of sequences with less than two mismatches with respect to the DnaA box consensus within the identified Mge-OriC indicates that DnaA binding sites of M. genitalium are significantly divergent. Of note, the Mge-OriC contains four instances of a 10-bp long palindromic sequence that is only found seven additional times in the chromosome. Deletion of some of these sequences abrogated the self-replicating capacity of the OriC plasmid. This result suggests the existence of novel regulatory sequences controlling replication initiation in M. genitalium. Work is in progress to pinpoint the DnaA binding sites and to establish the participation of additional sequences in the replication of the M. genitalium chromosome.
The Mycoplasma genitalium MG428 protein shows homology to members of the sigma-70 family of sigma factors. Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function. Deletion of MG_428 or some of the up-regulated unknown genes led to severe recombination defects. Single cell analyses revealed that activation of the MG428-regulon is a rare event under laboratory growth conditions. A conserved sequence with sigma-70 promoter architecture (TTGTCA-N18/19-ATTWAT) was identified in the upstream region of all of the MG428-regulated genes or operons. Primer extension analyses demonstrated that transcription initiates immediately downstream of this sigma70-type promoter in a MG428-dependent manner. Furthermore, mutagenesis of the conserved -10 and -35 elements corroborated the requirement of these regions for promoter function. Therefore, a new mycoplasma promoter directs transcription of a unique recombination regulon. Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor. Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism. Since recombination is an important mechanism to generate antigenic variation, MG428 emerges as a novel factor contributing to M. genitalium virulence. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.