Chlamydia trachomatis Polymorphic Membrane Protein D Is a Virulence Factor Involved in Early Host-Cell Interactions

Abstract

Chlamydia trachomatis is an obligate intracellular mucosotropic pathogen of significant medical importance. It is the etiological agent of blinding trachoma and bacterial sexually transmitted diseases, infections that afflict hundreds of millions of people globally. The C. trachomatis polymorphic membrane protein D (PmpD) is a highly conserved autotransporter and the target of broadly cross-reactive neutralizing antibodies; however, its role in host-pathogen interactions is unknown. Here we employed a targeted reverse genetics approach to generate a pmpD null mutant that was used to define the role of PmpD in the pathogenesis of chlamydial infection. We show that pmpD is not an essential chlamydial gene and the pmpD null mutant has no detectable deficiency in cultured murine cells or in a murine mucosal infection model. Notably, however, the pmpD null mutant was significantly attenuated for macaque eyes and cultured human cells. A reduction in pmpD null infection of human endocervical cells was associated with a deficiency in chlamydial attachment to cells. Collectively, our results show that PmpD is a chlamydial virulence factor that functions in early host-cell interactions. This study is the first of its kind using reverse genetics to evaluate the contribution of a C. trachomatis gene to disease pathogenesis. Chlamydia trachomatis is an obligate intracellular mucosotropic bacterium that is the most common cause of preventable blindness (1, 2) and bacterial sexually transmitted infections worldwide (3-5). The study of these medically important pathogens has been severely limited in the past by the lack of genetic tools. However, newly developed genetic approaches enable us to ask definitive questions about the contribution of specific chlamydial genes to pathogenesis (6-8). Polymorphic membrane protein D (PmpD) is one of nine putative autotransporters (ATs) encoded in the C. trachomatis genome (9). ATs are members of the Gram-negative bacterial type V secretion system and are important virulence factors that function in host-cell interactions and immune evasion (10). PmpD exhibits classical AT processing resulting in a membrane translocator domain that facilitates the presentation of a passenger domain on the chlamydial surface (11). PmpD is highly conserved among all C. trachomatis strains and is the target of broadly neutralizing antibodies (12). Despite its conserved nature, surface localization, and immunological importance, little is known about the function of PmpD in the pathogenesis of C. trachomatis infection.Here, we made a C. trachomatis pmpD null mutant using a targeted reverse genetic approach (6). pmpD was not essential for C. trachomatis growth, and the pmpD null mutant showed no deficiency in cultured murine cell lines in vitro or in a mouse urogenital infection model. However, the pmpD null mutant was attenuated in cultured human endocervical and conjunctival cells and in a nonhuman primate model of C. trachomatis ocular infection. Our findings show that C. trachomatis PmpD is a virulence fact...

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“…140,141 An important addition around the same time was the use of chemical mutagens, such as ethyl methanesulfonate, to generate libraries of chlamydial mutants. [142][143][144] Unfortunately, these mutants tend to have multiple mutations, but nevertheless, it is possible to screen such libraries to identify clones with your gene of interest disrupted, and subsequently to evaluate such mutants in vitro. 144 By complementing the mutated gene with an intact gene on the plasmid shuttle vector, it is now possible to more definitively test the role of key virulence targets.…”

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