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.

Mycoplasma genitalium is a human pathogen adhering to host target epithelial cells and causing urethritis, cervicitis and pelvic inflammatory disease. Essential for infectivity is a transmembrane adhesion complex called Nap comprising proteins P110 and P140. Here we report the crystal structure of P140 both alone and in complex with the N-terminal domain of P110. By cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) we find closed and open Nap conformations, determined at 9.8 and 15 Å, respectively. Both crystal structures and the cryo-EM structure are found in a closed conformation, where the sialic acid binding site in P110 is occluded. By contrast, the cryo-ET structure shows an open conformation, where the binding site is accessible. Structural information, in combination with functional studies, suggests a mechanism for attachment and release of M. genitalium to and from the host cell receptor, in which Nap conformations alternate to sustain motility and guarantee infectivity.

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.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models’ natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.

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.