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. 2015 Jul;83(7):2870-81.
doi: 10.1128/IAI.00106-15. Epub 2015 May 4.

Mutational Analysis of the Chlamydia muridarum Plasticity Zone

Krithika Rajaram  1 , Amanda M Giebel  1 , Evelyn Toh  2 , Shuai Hu  2 , Jasmine H Newman  3 , Sandra G Morrison  4 , Laszlo Kari  5 , Richard P Morrison  4 , David E Nelson  6
Affiliations

Mutational Analysis of the Chlamydia muridarum Plasticity Zone

Krithika Rajaram et al. Infect Immun. 2015 Jul.

Abstract

Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add, and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed--and some may produce functional proteins--but are dispensable for infection of the murine genital tract.

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Figures

FIG 1
FIG 1
Map of the C. muridarum PZ and diagram of PZ ORFs. The ORFs are color coded according to function: red, MACPF; green, PLD; yellow, cytotoxin; orange, purine nucleotide synthesis; gray, conserved hypothetical; blue, peptide ABC transporter; pink, dsbB. The locations of promoters identified by β-galactosidase assays in this study are shown by the bent arrows. The genomic locations and nucleotide changes of the nonsense mutations discussed in this study are indicated in the corresponding ORFs. The diagram is not drawn to scale.
FIG 2
FIG 2
Transcription of C. muridarum PZ ORFs initiates in the early to middle phase of the developmental cycle. Total RNA was isolated from C. muridarum-infected McCoy cells at 0, 6, 12, 18, 24, and 30 h p.i. (A) RT-PCR at 24 h p.i. indicated that all genes in the PZ are transcribed. Whether amplification was performed with (+) or without (−) reverse transcriptase is indicated. (B) The kinetics of transcription of select PZ ORFs was characterized by qRT-PCR. Transcript levels were normalized to standard curves of dilutions of C. muridarum chromosomes. The data represent the mean transcript levels with standard deviations (SD) from three independent experiments. euo, ompA, and omcB represent early, mid-late, and late genes, respectively.
FIG 3
FIG 3
C. muridarum PZ mutants exhibit mild growth defects. rIFU analysis was performed for C. muridarum (CM) and PZ mutants at various time intervals following infection of McCoy cells. The data shown are from experiments performed in parallel. The data represent the average (+SD) ratios of input to output IFU from three independent experiments performed in triplicate. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 by two-way ANOVA with Bonferroni post hoc test.
FIG 4
FIG 4
tc0437 and tc0439 mutants have reduced cytotoxicity. (A) HeLa cells were infected at an MOI of 250 in the presence of rifampin and were fixed 3 h p.i. Cell morphology and actin structure were visualized by staining with Alexa Fluor 488-phalloidin (green) and DAPI (blue). Overlays of fluorescence micrographs of cells that were mock infected or infected with C. muridarum (CM), C. trachomatis (CTL), tc0437, tc0438, or tc0439 are depicted. The images are representative of the results of three independent experiments. (B) LDH in supernatants of HeLa cells infected at various MOI in the presence of rifampin. Shown are C. muridarum (black), C. trachomatis (gray), tc0437 (hatched), tc0438 (white), and tc0439 (crosshatched). Triton X-100-treated cells were used as positive controls for LDH release. The data show the mean percentages (+SD) of the positive control from three independent experiments performed in triplicate. ****, P < 0.0001; **, P < 0.01 by one-way ANOVA with Dunnett's post hoc test.
FIG 5
FIG 5
PZ mutants are resistant to IFN-γ. The sensitivity to IFN-γ treatment of C. muridarum (CM), C. trachomatis (CTL), and various PZ mutants was assessed by IFU assay (A) and rIFU assay (B). The data are represented as the average (+SD) percentages of untreated (no IFN-γ) infected controls from three independent experiments performed in triplicate. ***, P < 0.001; **, P < 0.01 by one-way ANOVA with Dunnett's post hoc test.
FIG 6
FIG 6
Mouse genital tract infections with C. muridarum PZ mutants. Groups of mice were challenged intravaginally with 50,000 IFU of C. muridarum (CM) or various PZ mutants. The infection curve for C. muridarum-infected mice (n = 30) is reproduced in each panel for comparison. (A) Lower genital tract shedding of IFU by mice infected with C. muridarum differed significantly from that by mice infected with the tc0439 mutant (n = 8) at day 3 postinfection (P < 0.01), but not that by mice infected with tc0437 (n = 8) or tc0438 (n = 8). (B and C) IFU shedding from mice infected with CM did not vary significantly from that from mice infected with the t0440 (n = 12) (B) or guaA (n = 16) mutant (C). (D) Shedding by mice infected with the add (n = 8) mutant was significantly less than shedding by mice infected with C. muridarum at 3 days (P < 0.0001) and 7 days (P < 0.01) postinfection. IFU shedding from mice infected with the add (n = 8) mutant was also significantly reduced at 3 days (P < 0.0001), 7 days (P < 0.05), and 10 days (P < 0.001) postinfection. The data are presented as mean numbers of IFU (+SD) for the mice in each group. Statistical differences were analyzed by two-way ANOVA with Bonferroni post hoc test.
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