Altered developmental expression of polymorphic membrane proteins in penicillin-stressed Chlamydia trachomatis

Abstract

Late Chlamydia trachomatis inclusions express each member of the surface-exposed polymorphic membrane protein family (Pmp subtypes A through I) with a reproducible distribution of fully-on, fully-off and intermediate phenotypes. This observation is consistent with observed variable Pmp antibody profiles in C. trachomatis-infected patients and has led to the hypothesis that the pmp gene family forms the basis of a phase variation-like mechanism of antigenic variation. Here we investigate and compare the developmental expression of each of the nine pmp genes under conditions of optimal in vitro growth with that under conditions that promote prolonged survival of chlamydiae when exposed to penicillin-induced stress. We demonstrate that the pmp gene family includes distinct transcriptional units that are differentially expressed along development and differentially responsive to stress. In particular, our results indicate that expression of pmpA, pmpD and pmpI is uniquely unaffected by stress, suggesting that the PmpA, PmpD and PmpI proteins play a critical role in the pathogenesis of C. trachomatis.

Figure 1. pmpABC, pmpEF and pmpGH are single transcriptional units

(A) Organization of the pmpABC, pmpFE and pmpGH putative operons with gene sizes and intergenic distances (not drawn to scale). Total RNAs from C. trachomatis cultures grown under normal conditions (B) or with 200U penicillin (C) were obtained at different times post-infection as indicated and amplified by RT-PCR with specific primers flanking the intergenic region of pmpA-pmpB, pmpB-pmpC, pmpF-pmpE, and pmpG-pmpH, respectively. C+, positive control C. trachomatis genomic DNA. C-, negative control cDNA from uninfected HeLa cells; RT/−RT, with/without reverse transcriptase. Molecular sizes were obtained by alignment with a 50 bp ladder (Fermentas).

Figure 2. Transcription of specific pmp genes of C. trachomatis is differentially regulated along development and upon exposure to penicillin

Transcript levels were measured by real time RT-qPCR in C. trachomatis grown under normal conditions (white bars) and with penicillin (black bars) at 2, 6, 12, 18, 24, 32, 48 and 72 hpi. Transcript levels of the tufA gene encoding EF-Tu are used for comparison.

Figure 3. Aberrant inclusions are present in normal cultures

Infected HeLa cells were fixed at 42 hpi and double stained with anti-EF-Tu antibody and antibodies specific for PmpB, PmpC or PmpH as indicated, and visualized at 400x magnification. Single channel and merged images (m) are shown. White arrowheads indicate aberrant inclusions.

Figure 4. Production of Pmps is altered in stressed inclusions

C. trachomatis-infected HeLa cells were fixed at 24 and 48 hpi, and double-stained with EF-Tu-specific antibody and Pmp-specific antibody as indicated. Merged images (m) and magnified insets thereof are shown in the two right-most columns. For each Pmp, inclusions grown under normal culture conditions or penicillin-induced stress conditions (pen) are shown. Only results obtained for PmpA (A), PmpB (B), PmpD (C) and PmpI (D) are shown as they are representative of all other Pmps. Staining patterns at 24 and 48 hpi obtained for PmpC, PmpE, PmpF, PmpG and PmpH (not shown) were respectively similar to those obtained for PmpB, PmpD, PmpA, PmpD and PmpD. (E) IF staining quantified at 24 and 48 hpi under normal (white) and penicillin-stressed (black) conditions. Statistically significant differences (P<0.01) are indicated with *.

Figure 4. Production of Pmps is altered in stressed inclusions

C. trachomatis-infected HeLa cells were fixed at 24 and 48 hpi, and double-stained with EF-Tu-specific antibody and Pmp-specific antibody as indicated. Merged images (m) and magnified insets thereof are shown in the two right-most columns. For each Pmp, inclusions grown under normal culture conditions or penicillin-induced stress conditions (pen) are shown. Only results obtained for PmpA (A), PmpB (B), PmpD (C) and PmpI (D) are shown as they are representative of all other Pmps. Staining patterns at 24 and 48 hpi obtained for PmpC, PmpE, PmpF, PmpG and PmpH (not shown) were respectively similar to those obtained for PmpB, PmpD, PmpA, PmpD and PmpD. (E) IF staining quantified at 24 and 48 hpi under normal (white) and penicillin-stressed (black) conditions. Statistically significant differences (P<0.01) are indicated with *.

Figure 4. Production of Pmps is altered in stressed inclusions

C. trachomatis-infected HeLa cells were fixed at 24 and 48 hpi, and double-stained with EF-Tu-specific antibody and Pmp-specific antibody as indicated. Merged images (m) and magnified insets thereof are shown in the two right-most columns. For each Pmp, inclusions grown under normal culture conditions or penicillin-induced stress conditions (pen) are shown. Only results obtained for PmpA (A), PmpB (B), PmpD (C) and PmpI (D) are shown as they are representative of all other Pmps. Staining patterns at 24 and 48 hpi obtained for PmpC, PmpE, PmpF, PmpG and PmpH (not shown) were respectively similar to those obtained for PmpB, PmpD, PmpA, PmpD and PmpD. (E) IF staining quantified at 24 and 48 hpi under normal (white) and penicillin-stressed (black) conditions. Statistically significant differences (P<0.01) are indicated with *.

Figure 4. Production of Pmps is altered in stressed inclusions

C. trachomatis-infected HeLa cells were fixed at 24 and 48 hpi, and double-stained with EF-Tu-specific antibody and Pmp-specific antibody as indicated. Merged images (m) and magnified insets thereof are shown in the two right-most columns. For each Pmp, inclusions grown under normal culture conditions or penicillin-induced stress conditions (pen) are shown. Only results obtained for PmpA (A), PmpB (B), PmpD (C) and PmpI (D) are shown as they are representative of all other Pmps. Staining patterns at 24 and 48 hpi obtained for PmpC, PmpE, PmpF, PmpG and PmpH (not shown) were respectively similar to those obtained for PmpB, PmpD, PmpA, PmpD and PmpD. (E) IF staining quantified at 24 and 48 hpi under normal (white) and penicillin-stressed (black) conditions. Statistically significant differences (P<0.01) are indicated with *.

Figure 4. Production of Pmps is altered in stressed inclusions

C. trachomatis-infected HeLa cells were fixed at 24 and 48 hpi, and double-stained with EF-Tu-specific antibody and Pmp-specific antibody as indicated. Merged images (m) and magnified insets thereof are shown in the two right-most columns. For each Pmp, inclusions grown under normal culture conditions or penicillin-induced stress conditions (pen) are shown. Only results obtained for PmpA (A), PmpB (B), PmpD (C) and PmpI (D) are shown as they are representative of all other Pmps. Staining patterns at 24 and 48 hpi obtained for PmpC, PmpE, PmpF, PmpG and PmpH (not shown) were respectively similar to those obtained for PmpB, PmpD, PmpA, PmpD and PmpD. (E) IF staining quantified at 24 and 48 hpi under normal (white) and penicillin-stressed (black) conditions. Statistically significant differences (P<0.01) are indicated with *.