Recombinant Human Peptidoglycan Recognition Proteins Reveal Antichlamydial Activity

Abstract

Peptidoglycan recognition proteins (PGLYRPs) are innate immune components that recognize the peptidoglycan and lipopolysaccharides of bacteria and exhibit antibacterial activity. Recently, the obligate intracellular parasite Chlamydia trachomatis was shown to have peptidoglycan. However, the antichlamydial activity of PGLYRPs has not yet been demonstrated. The aim of our study was to test whether PGLYRPs exhibit antibacterial activity against C. trachomatis Thus, we cloned the regions containing the human Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4 genes for subsequent expression in human cell lines. We obtained stable HeLa cell lines that secrete recombinant human PGLYRPs into culture medium. We also generated purified recombinant PGLYRP1, -2, and -4 and confirmed their activities against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. Furthermore, we examined the activities of recombinant PGLYRPs against C. trachomatis and determined their MICs. We also observed a decrease in the infectious ability of chlamydial elementary bodies in the next generation after a single exposure to PGLYRPs. Finally, we demonstrated that PGLYRPs attach to C. trachomatis elementary bodies and activate the expression of the chlamydial two-component stress response system. Thus, PGLYRPs inhibit the development of chlamydial infection.

FIG 1

Western blot analysis of recombinant human PGLYRPs from HeLa cells transfected with pEGFP-N1-PGLYRP vectors. Lanes: a, PGLYRP1; b, PGLYRP2; c, PGLYRP3; d, PGLYRP4; M, molecular mass markers (corresponding values [in kilodaltons] are shown on the left).

FIG 2

Coomassie G-250-stained Laemmli SDS-PAGE analysis of recombinant PGLYRPs isolated from culture medium supernatants of Expi293F cells transfected with pcDNA-IGG-PGLYRP vectors. Lanes: a to c, purified PGLYRP1, -2, and -4, respectively; m, molecular mass markers (corresponding values [in kilodaltons] are shown on the right).

FIG 3

Western blot analysis of the attachment of recombinant PGLYRP1 to C. trachomatis. Lanes: a, purified PGLYRP1 in HBSS; b, supernatant of a centrifuged mixture of PGLYRP1 with chlamydial EBs in HBSS; c, sediment of a centrifuged mixture of PGLYRP1 with chlamydial EBs in HBSS; d, sediment of centrifuged chlamydial EBs in HBSS. The upper unspecific bands (U.B.) correspond to nonspecific staining of chlamydial proteins. Lane M, molecular mass markers (corresponding values [in kilodaltons] are shown on the right).

FIG 4

Changes in parental and progeny C. trachomatis titers after treatment with purified separated PGLYRPs added to HeLa cells. The parental titers were calculated at 72 h postinfection (A). To normalize the data from parental titers, samples were diluted to achieve equivalent MOIs (horizontal line) to analyze the production of infectious progeny (B). We used a blank control sample as a control.

FIG 5

Analysis of the activation of chlamydial two-component stress response system gene expression after treatment with purified recombinant PGLYRPs at 1, 24, 48, 60, and 72 h postinoculation. The quantitative PCR data were normalized, and the fold change in the mRNA level was determined as 2^−ΔΔCT. Data are expressed as the mRNA fold change ± the standard error. A blank control sample was used as a control. Differences from the untransfected control were considered significant if the P value was <0.05 (*).

FIG 6

Analysis of the activation of chlamydial two-component stress response system-related gene expression after treatment with PGLYRPs at 15, 30, 60, 90, 120, 180, and 300 min postinoculation. The quantitative PCR data were normalized, and the fold change in the mRNA level was determined as 2.1^−ΔΔCT. A blank control sample was used as a control. Data are expressed as fold changes in mRNA levels ± the standard errors.