Date Published: June 18, 2018
Publisher: Public Library of Science
Author(s): Wayne Paes, Adam Dowle, Jamie Coldwell, Andrew Leech, Tim Ganderton, Andrzej Brzozowski, Eric Cascales.
Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen, and the leading cause of infectious blindness worldwide. We have recently shown that immunization with the highly conserved antigenic passenger domain of recombinant Ct polymorphic membrane protein D (rPmpD) is protective in the mouse model of Ct genital tract infection, and previously, that ocular anti-rPmpD antibodies are elicited following vaccination. However, the mechanisms governing the assembly and structure-function relationship of PmpD are unknown. Here, we provide a biophysical analysis of this immunogenic 65 kDa passenger domain fragment of PmpD. Using differential cysteine labeling coupled with LC-MS/MS analysis, we show that widespread intra- and intermolecular disulphide interactions play important roles in the preservation of native monomeric secondary structure and the formation of higher-order oligomers. While it has been proposed that FxxN and GGA(I, L,V) repeat motifs in the Pmp21 ortholog in Chlamydia pneumoniae mediate self-interaction, no such role has previously been identified for cysteine residues in chlamydial Pmps. Further characterisation reveals that oligomeric proteoforms and rPmpD monomers adopt β–sheet folds, consistent with previously described Gram-negative bacterial type V secretion systems (T5SSs). We also highlight adhesin-like properties of rPmpD, showing that both soluble rPmpD and anti-rPmpD serum from immunized mice abrogate binding of rPmpD-coated beads to mammalian cells in a dose-dependent fashion. Hence, our study provides further evidence that chlamydial Pmps may function as adhesins, while elucidating yet another important mechanism of self-association of bacterial T5SS virulence factors that may be unique to the Chlamydiaceae.
Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans, and the etiological agent of blinding trachoma and sexually transmitted diseases . Ocular Ct serovars (A-C) primarily infect human conjunctival epithelial cells, with repeated infections leading to corneal opacity and subsequent blindness. Collectively, serovars (D-K) and lymphogranuloma venereum (LGV) strains (L1-L3) are the most common sexually transmitted bacterial pathogens worldwide, responsible for ~131 million new cases each year . Urogenital infections are asymptomatic in 70–90% of women, with untreated ascending infections the main cause of substantial morbidity and post-infection sequelae such as pelvic inflammatory disease, ectopic pregnancies and infertility . Thus, efficacious vaccine design remains a high public health priority.
Ct PmpD is a highly immunogenic chlamydial antigen following natural infection in humans with a range of serovars [26, 27]. In line with the degree and frequency of immunogenicity observed during the course of natural infections, we have recently shown that immunization with rPmpD elicits robust humoral and cell-mediated immunity that confer protection in a mouse model of genital Ct infection, and further confirmed the presence of ocular anti-rPmpD antibodies elicited following intramuscular immunization [8, 17]. Furthermore, other bacterial T5SSs have been included in licensed prophylactic acellular vaccines that include the Bordetella pertussis pertactin type Va autotransporter , and the more recently developed quadravalent 4CMenB vaccine (Bexsero), which contains the Neisseria meningitidis adhesin A (NadA) protein, a type Vc autotransporter . In our study, we have utilized both biochemical and biophysical analytical techniques to further characterize rPmpD, a highly promising chlamydial vaccine antigen, importantly elucidating a previously undescribed mechanism of bacterial T5SS assembly involving an extensively disulphide-linked network of cysteine residues.
In summary, the data gathered in this study provide advancement in our understanding of chlamydial polymorphic membrane protein secondary structure and intermolecular assembly in vitro. We have shown that in addition to non-covalent interactions, extensive disulphide-mediated covalent interactions play a role in formation of higher-order structures, which represent a previously undescribed mechanism of T5SS self-association that differs markedly from other bacterial species and may be unique to the Chlamydiaceae. Subsequent work aims to assess whether modification of these disulphide interactions influences protein function in vitro and in vivo, in order to gain a more comprehensive molecular understanding of chlamydial membrane protein assembly. PmpD also displays an adhesin-like function in vitro, and future studies will aim to identify putative host cell ligands, as knowledge of Ct binding to human epithelial cells still remains rudimentary.