Oscar Q. Pich

Mycoplasma genitalium P140 and P110 Cytadhesins Are Reciprocally Stabilized and Required for Cell Adhesion and Terminal-Organelle Development

Mycoplasma genitalium is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in M. genitalium cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis. 17(Suppl. 1):S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of M. genitalium.

Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence

Isolation and characterization of transposon‐generated Mycoplasma genitalium gliding‐deficient mutants has implicated mg200 and mg386 genes in gliding motility. The proposed role of these genes was confirmed by restoration of the gliding phenotype in deficient mutants through gene complementation with their respective mg386 or mg200 wild‐type copies. mg200 and mg386 are the first reported gliding‐associated mycoplasma genes not directly involved in cytadherence. Orthologues of MG200 and MG386 proteins are also found in the slow gliding mycoplasmas, Mycoplasma pneumoniae and Mycoplasma gallisepticum, suggesting the existence of a unique set of proteins involved in slow gliding motility. MG200 and MG386 proteins share common features, such as the presence of enriched in aromatic and glycine residues boxes and an acidic and proline‐rich domain, suggesting that these motifs could play a significant role in gliding motility.

Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature

Motility is often a virulence factor of pathogenic bacteria. Although recent works have identified genes involved in gliding motility of mycoplasmas, little is known about the mechanisms governing the cell gliding behaviour. Here, we report that Mycoplasma genitalium MG217 is a novel protein involved in the gliding apparatus of this organism and it is, at least, one of the genes that are directing cells to move in narrow circles when they glide. In the absence of MG_217 gene, cells are still able to glide but they mainly move drawing erratic or wide circular paths. This change in the gliding behaviour correlates with a rearrangement in the terminal organelle disposition, suggesting that the terminal organelle operates as a guide to steer the mycoplasma cell in a specific direction. Immunogold labelling reveals that MG217 protein is located intracellular at the distal end of the terminal organelle, between the cell membrane and the terminal button. Such location is consistent with the idea that MG217 could act as a modulator of the terminal organelle curvature, allowing cells to move in specific directions.

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

The terminal organelle is a differentiated structure that plays a key role in mycoplasma cytadherence and locomotion. For this reason, the analysis of Mycoplasma genitalium mutants displaying anomalous terminal organelles could improve our knowledge regarding the structural elements required for proper locomotion. In this study, we isolated several M. genitalium mutants having transposon insertions within the mg218 or mg317 genes, which encode the orthologues of Mycoplasma pneumoniae HMW2 and HMW3 cytoskeletal proteins, respectively. As expected, mg218(-) and mg317(-) mutants exhibit a reduced gliding motility, although their ability to attach to solid surfaces was not completely abolished. Interestingly, most of the mg218(-) mutants expressed N-terminal MG218 derivatives and showed the presence of short terminal organelles retaining many of the functions displayed by this structure in the wild-type strain, suggesting that the N-terminal region of this protein is an essential element in the architecture of the terminal organelle. Separately, the analysis of mg317(-) mutants indicates that MG317 protein is involved in the formation of the terminal button and contributes to anchoring the electron-dense core to the cell membrane. The results presented here clearly show that MG218 and MG317 proteins are implicated in the maintenance of gliding motility and cytadherence in M. genitalium.

A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium

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.

P110 and P140 Cytadherence-Related Proteins Are Negative Effectors of Terminal Organelle Duplication in Mycoplasma genitalium

Background The terminal organelle is a complex structure involved in many aspects of the biology of mycoplasmas such as cell adherence, motility or cell division. Mycoplasma genitalium cells display a single terminal organelle and duplicate this structure prior to cytokinesis in a coordinated manner with the cell division process. Despite the significance of the terminal organelle in mycoplasma virulence, little is known about the mechanisms governing its duplication. Methodology/Principal Findings In this study we describe the isolation of a mutant, named T192, with a transposon insertion close to the 3′ end of the mg192 gene encoding for P110 adhesin. This mutant shows a truncated P110, low levels of P140 and P110 adhesins, a large number of non-motile cells and a high frequency of new terminal organelle formation. Further analyses revealed that the high rates of new terminal organelle formation in T192 cells are a direct consequence of the reduced levels of P110 and P140 rather than to the expression of a truncated P110. Consistently, the phenotype of the T192 mutant was successfully complemented by the reintroduction of the mg192 WT allele which restored the levels of P110 and P140 to those of the WT strain. Quantification of DAPI-stained DNA also showed that the increase in the number of terminal organelles in T192 cells is not accompanied by a higher DNA content, indicating that terminal organelle duplication does not trigger DNA replication in mycoplasmas. Conclusions/Significance Our results demonstrate the existence of a mechanism regulating terminal organelle duplication in M. genitalium and strongly suggest the implication of P110 and P140 adhesins in this mechanism.

MultitaskProtDB-II: an update of a database of multitasking/moonlighting proteins

Abstract Multitasking, or moonlighting, is the capability of some proteins to execute two or more biological functions. MultitaskProtDB-II is a database of multifunctional proteins that has been updated. In the previous version, the information contained was: NCBI and UniProt accession numbers, canonical and additional biological functions, organism, monomeric/oligomeric states, PDB codes and bibliographic references. In the present update, the number of entries has been increased from 288 to 694 moonlighting proteins. MultitaskProtDB-II is continually being curated and updated. The new database also contains the following information: GO descriptors for the canonical and moonlighting functions, three-dimensional structure (for those proteins lacking PDB structure, a model was made using Itasser and Phyre), the involvement of the proteins in human diseases (78% of human moonlighting proteins) and whether the protein is a target of a current drug (48% of human moonlighting proteins). These numbers highlight the importance of these proteins for the analysis and explanation of human diseases and target-directed drug design. Moreover, 25% of the proteins of the database are involved in virulence of pathogenic microorganisms, largely in the mechanism of adhesion to the host. This highlights their importance for the mechanism of microorganism infection and vaccine design. MultitaskProtDB-II is available at http://wallace.uab.es/multitaskII.

Mycoplasma genitalium adhesin P110 binds sialic-acid human receptors

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.How the Mycoplasma genitalium cytadhesins P140 and P110 promote host cell invasion remains poorly understood. Here, combining structural analysis with functional assays, Aparicio et al. identify the P110 domain that binds to sialylated receptors essential for mycoplasma cytadherence.

Activation of σ20-dependent recombination and horizontal gene transfer in Mycoplasma genitalium

Abstract 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 over-expression. 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.