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.

FIG 1

pmpD^C1618T is a null mutant. (A) Summary of the principal forms of WT PmpD and sequencing information showing the location of the pmpD^C1618T stop codon. (B) Western blot analysis of whole EB proteins with a PmpD-specific antibody showed strong reactivity with the WT and no reactivity with pmpD^C1618T. Anti-HSP60 antibody was used as a loading control.

FIG 2

The pmpD null mutant exhibits unique morphological and ultrastructural phenotypes in vitro. (A) Phase microscopy of McCoy cells at 36 h postinfection revealed an atypical organism distribution associated with the pmpD null mutant but not the WT strain. (B) Confocal immunofluorescence microscopy at 36 h postinfection showed no immunostaining of the pmpD null mutant with a PmpD-specific antibody. Immunostaining of the MOMP and DAPI-stained DNA confirmed the atypical organism distribution in pmpD null mutant inclusions. (C) Transmission electron microscopy at 24 h postinfection showed a reduced association of pmpD null mutant RBs with the inclusion membrane compared to that seen in the WT strain (see Table S1 in the supplemental material).

FIG 3

No detectable role for C. trachomatis PmpD in murine infection models. (A) Enumeration of inclusions in murine fibroblasts (McCoy cells) and primary murine oviduct epithelial (Bm12.4) cells revealed no (McCoy cells) or very little difference (Bm12.4 cells, 1.22-fold) in the ability of the pmpD null mutant to infect cells in vitro compared to that of the WT. (B) Recoverable IFU harvested from McCoy and Bm12.4 cells at 36 h postinfection also showed no differences in the ability of the pmpD null mutant to grow in these cell lines compared to that of the WT. (C) The recoverable numbers of IFU and the duration of chlamydial shedding following urogenital infection of C3H/HeJ mice were similar for the pmpD null mutant (n = 10) and the WT strain (n = 10). No statistically significant difference was found at any time point (two-tailed t test). (D) H&E staining of C3H/HeJ upper genital tract tissues (ovaries, oviducts, and uterine horns) infected with the pmpD null mutant or the WT strain showed no histopathological differences up to 6 weeks postinfection. Average clinical disease scores with standard deviations are shown (n = 5). (E) H&E staining of oviducts (OD) is shown for pmpD null mutant-, WT-, and mock-infected animals.

FIG 4

The pmpD null mutant is attenuated in a nonhuman primate infection model. Six cynomolgus macaques were ocularly infected with the pmpD null mutant and six were infected with the WT strain to evaluate infection, the duration of shedding, and clinical pathology. (A) Ocular infection was monitored by swabbing the conjunctiva and culturing recoverable IFU on HeLa cell monolayers. Averages and standard deviations are shown on a log₁₀ scale. Detailed chlamydial shedding data are shown in Table S3 in the supplemental material. (B) Disease was scored on the basis of hyperemia and follicle formation on the upper conjunctival surfaces (score 0 = no disease, score 12 = maximum disease). Averages and standard deviations are shown. Macaques were monitored for shedding and disease for 100 days postinfection. *, statistically significant difference (P < 0.05, two-tailed Mann-Whitney U test, n = 6).

FIG 5

The pmpD null mutant is deficient in attachment to human cells. (A) Infection of human endocervical (A2EN) and conjunctival (HCjE) cells with the pmpD null mutant yielded significantly fewer inclusions than the number obtained by infection with the WT (P < 0.05, two-tailed Mann-Whitney U test, n = 3). pmpD null mutant inclusions are shown as a percentage of the number for the WT control for each cell line. (B) A decrease in the number of recoverable IFU concomitant with decreased numbers of inclusions was detected in both human cell lines following infection with the pmpD null mutant and the WT strain (P < 0.05, two-tailed Mann-Whitney U test, n = 3). The amount of pmpD null mutant recoverable IFU is shown as a percentage of the number of recoverable IFU for the WT control for each cell line. (C) A2EN cells were consecutively infected with the same inoculum zero, one, two, or three times before the titer of the nonattached EBs in the inoculum in McCoy cells was redetermined. The number of viable EBs remaining in the inoculum after A2EN cell infections, determined by redetermining the titer in McCoy cells, is shown (results are averages and standard deviations). The numbers of unattached pmpD null mutant IFU remaining in the inocula after one, two, and three infections of A2EN cells were statistically significantly higher (P < 0.05, two-tailed Mann-Whitney U test, n = 6).