Analysis of the Mycoplasma genitalium MgpB Adhesin to Predict Membrane Topology, Investigate Antibody Accessibility, Characterize Amino Acid Diversity, and Identify Functional and Immunogenic Epitopes

Abstract

Mycoplasma genitalium is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that M. genitalium may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized mgpB heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to functionality, variability, and immunogenicity during persistent M. genitalium infection.

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium

“…It had been established that P140 and P110 proteins are reciprocally stabilized and are the major surface antigens surrounding the terminal organelle (Hu et al ., ; Burgos et al ., , Svenstrup et al ., ; Iverson‐Cabral et al ., ) which suggested that the two proteins could be forming a membrane complex in M. genitalium . Solubilization of the membrane proteins with detergents and the attachment of an affinity tag at the C‐terminal end of P110, predicted to be inside the cell, allowed the purification of a complex containing equimolar ratios of P140 and P110 (Fig.…”

“…The smaller α and α' lobes most likely correspond each to a single P110 protein and the bigger β and β' lobes correspond each to a single P140 protein. The C2 symmetry of the NAPs extracellular region reflects the organization of the P140 and P110 subunits with a large N‐terminal domain forming a globular region followed by a stalk, which corresponds to a smaller domain that reaches the cell membrane and crosses it with the single transmembrane helix predicted from the sequences of both proteins (Iverson‐Cabral et al ., ) (Supporting Information Fig. S4).…”

“…In fact, functional and phylogenetic studies show that components of the terminal organelle are orthologous and closely related in M. genitalium and M. pneumoniae , both belonging to the pneumoniae cluster of mycoplasmas (Miyata and Hamaguchi, ). The mg191 and mg192 gene products, known as P140 and P110 proteins in M. genitalium (Burgos et al ., ), are surface‐exposed and localize mainly at the terminal organelle (Hu et al ., ; Iverson‐Cabral et al ., ). The ortholog of P140 in M. pneumoniae is the P1 adhesin, and the ortholog of P110 is the polypeptide of the MPN142 gene product, post‐translationally cleaved into the P90 and P40 proteins (Layh‐Schmitt and Herrmann, ).…”

Virulence and molecular adaptation of human urogenital mycoplasmas: a review

“…Adhesins P140 and P110 stimulate humoral and cervicovaginal antibody production during M. genitalium infection. Both mgpB and mgpC produce sequence variation through recA mediated homologous recombination with MgPar donor sequences leading to antigenic diversity in these adhesins [43][44][45][46]. M. genitalium attaches to mucin, a component of the host's epithelial mucus membrane via ligands of the bacterial surface-exposed enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”

“…The constant variation in surface lipoproteins results in a membrane that is rather plastic, a feature that is key to the survival of the species of Mycoplasma. As discussed previously, Mycoplasma can vary their surface antigens by changing the size of the immunogenic lipoproteins, physically masking the lipoproteins or switching on and off the expression of the antigen [44,60]. Whichever mechanism is used, the result is that the host humoral immune system is evaded allowing the Mycoplasma species to persist.…”