Plasmid-Cured Chlamydia caviae Activates TLR2-Dependent Signaling and Retains Virulence in the Guinea Pig Model of Genital Tract Infection

Loss of the conserved “cryptic” plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains

Effect of 2DG treatment on inclusion formation and transcription by <i>C. caviae</i>.

<p>(A) GPIC inclusions in L929 cells 40 hours post infection (A–C). Inclusions formed by GPIC in cells treated with 10 mM 2DG are smaller (B) and may contain aberrant forms (C). Cells were fixed with methanol then stained with an anti-LPS monoclonal antibody and anti-mouse Alexa488 (secondary) to detect chlamydiae and cytoplasm was counterstained with Evans Blue. All inclusions were imaged at 400x magnification. (D) GPIC differentially regulates candidate PRCL transcription in response to 2DG treatment. Total RNA was isolated 24 hours after infection from infected cells treated with 10 mM 2DG and transcripts were quantified as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone-0030747-g002" target="_blank">Fig. 2D</a>. (E) Reduced PRCL transcription is plasmid-independent in <i>C. caviae</i>. L929 cells infected with CC13 were treated with 2DG as for ‘C’ and transcription of plasmid-insensitive loci 24 hours after infection was examined as before. The transcriptional differences are presented as fold change in expression for each gene in a single representative experiment although each experiment was performed independently at least twice and samples were assayed in triplicate. <i>p</i> values for transcripts indicated with * were <0.05.</p

Cellular infiltrates are similar early during infection in estradiol-treated guinea pigs infected with GPIC or CC13.

<p>Groups of 5 estradiol-treated female guinea pigs infected with GPIC or CC13 were sacrificed on day 9. Pathology scores for inflammatory cells in the (A) uterine horns, and (B) oviducts were similar for GPIC- and CC13-infected animals. Boxes extend from the 25-75 percentiles and whiskers indicate the 5^th–95^th percentiles. (C, E) Representative oviduct histologic sections at 40X and 200X magnification from a guinea pig infected with GPIC. (D, F) Representative oviduct histologic sections at 40X and 200X magnification from a guinea pig infected with CC13. Data are from one experiment with 5 guinea pigs per group.</p

Transcriptional profiling of CC13 via microarray screening and quantitative RT-PCR reveals partial conservation of PRCL but a plasmid-independent glucose response in <i>C. caviae.</i>

<p>(A) CC13 replicates normally during synchronous infection of L929 cells. GPIC and CC13 were inoculated at an MOI ∼1 into L929 cells growing in 24 well dishes, then samples were harvested at intervals and titrated to determine IFU/well. (B) pCpGP1 transcripts are detected 24 hours after infection. Total RNA isolated from L929 cells 24 hours after infection with GPIC was treated with reverse transcriptase to generate cDNA and assayed via RT-PCR. Purified genomic GPIC DNA was used as positive control for the reactions while RNA processed for cDNA without reverse transcriptase was used as the negative control. (C) Quantitative RT-PCR confirms partial conservation of candidate PRCL in <i>C. caviae</i>. Total RNA was isolated from infected cells 24 hours after infection and PRCL transcripts were measured by quantitative RT-PCR. CCA00078 (<i>mip</i>) was included as a non-plasmid responsive control because we did not detect a difference in its transcription by microarray screen.</p

<i>C. caviae</i> CC13 is plasmid-deficient but displays no alteration in plaque size or efficiency.

<p>(A) Equal amounts of template were added to PCR reactions with primers directed against the pgp1 coding sequence expressed by pCpGP1 or the <i>C. caviae</i> genome and amplified products were analyzed on 2% agarose gels. (B) Genomic DNA was isolated from GPIC or CC13, digested with <i>Sph</i>I and separated on a 1% agarose gel. The DNA was transferred to a nylon membrane and probed for the presence of pCpGP1 DNA using a ∼1.6 Kb probe directed against a region spanning pGP4-6. Plasmid pCpGP1 was isolated from GPIC and digested with <i>Sph</i>1 to yield a single, linear fragment of ∼7.9 kb which was used as a positive control for hybridization. (C) Plaques formed by CC13 do not differ from those formed by GPIC. <i>C. caviae</i>-infected L929 monolayers were incubated for 5 days at 37 C, 5% CO2, then the media overlay was removed and the plaques visualized using crystal violet.</p

The impact of plasmid-curing on <i>Chlamydia sp</i>. gene expression, regulation and virulence.

<p>This diagram outlines the gene expression and regulation and virulence phenotypes of plasmid-cured <i>Chlamydia sp.</i>, and indicates the phylogenetic relationships of the plasmid carried by these species or strains and other genera. Data regarding phenotypic and transcriptional responses were obtained in the course of this or previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-OConnell1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-OConnell2" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Russell1" target="_blank">[5]</a>. NT indicates where phenotype was not examined. The dendrogram was constructed via CLUSTAL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Larkin1" target="_blank">[41]</a> and Neighbor-joining analysis was performed using plasmid sequences from <i>C. trachomatis</i> D/UW-3/Cx (pCTDLC1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Sturdevant1" target="_blank">[42]</a>, L2/434/Bu (pL2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Thomson1" target="_blank">[43]</a>, <i>C. muridarum</i> (pMoPn) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Read1" target="_blank">[44]</a>, <i>C. caviae</i> (pCpGP1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Read2" target="_blank">[45]</a>, <i>C. psittaci</i> pCpA1 (Lusher,M.E., Gregory,J., Storey,C.C. and Richmond,S.J., unpublished; Genebank: NC_002117, <i>C. felis</i> (pCfe1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Azuma1" target="_blank">[46]</a> and the non-human pathogens <i>C. pneumoniae</i> LPCoLN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Mitchell1" target="_blank">[31]</a> and N16 (pCpnE1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Thomas2" target="_blank">[47]</a> to determine branching order. The outcome of this analysis did not differ from the more detailed analysis published by Mitchell et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Mitchell1" target="_blank">[31]</a>.</p

Chlamydial burden and levels of IL-8 do not differ between GPIC and CC13 infected guinea pigs during genital tract infection.

<p>Groups of 5 untreated (A, B) or estradiol-treated (C, D) female guinea pigs were intravaginally infected with either GPIC or CC13 and sacrificed on day 30. (A) Monitoring of endocervical swabs during infection of untreated guinea pigs revealed that infectious bacteria were not detectable after day 9 for either strain. (B) Vaginal sponges collected through day 10 revealed similar levels of IL-8 in GPIC and CC13-infected guinea pigs. (C) The course of GPIC infection in estradiol-treated guinea pigs was prolonged compared to CC13. (D) IL-8 levels monitored during the first 10 days of infection were not different in estradiol-treated guinea pigs infected with GPIC and CC13. Bars represent the mean ± SD of five guinea pigs per group and each experiment was performed once.</p

PCR primer sets used in this study.

a<p>Chromosomal loci are identified by their annotated designations, <i>C. caviae</i> GPIC GenBank accession AE015925. Plasmid ORFs of pGPGP1 are indicated according to GenBank accession AE015926 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030747#pone.0030747-Read2" target="_blank">[45]</a>.</p><p>*Plasmid primers used to detect presence of pCpGP1 by PCR or DNA hybridization only.</p

CC13 signals via TLR2 and induces cytokine production at levels similar to wild-type GPIC.

<p>(A) IL-8 was measured in the supernatants of HEK 293 cells transfected with control plasmid, TLR2 or TLR4 and infected with GPIC or CC13 for 24 hrs. At both an MOI of 1 and 3, cytokine production did not differ between HEK-TLR2 cells infected with GPIC or CC13. Neither strain induced IL-8 levels above media for cells transfected with control plasmid or TLR4. The pattern of IL-8 production after stimulation with LPS (TLR4 agonist), TNF (NF-κB agonist), or Pam₃Cys (TLR2 agonist) indicated that cytokine production was specific for the transfected TLR. (B) GPIC and CC13 express a potent, plasmid-independent TLR2 stimulating activity. X-ray inactivated chlamydial suspensions at various MOIs were incubated with HEK-TLR2 cells transfected with an NF-κB reporter plasmid. After 24 h of incubation NF-κB-induced secreted alkaline phosphatase activity was assayed using QUANTI-Blue. Bars represent the mean ± SE for three independent experiments. (C) Murine BMDDCs infected with GPIC or CC13 for 24 h did not differentially produce IL-6, (D) IL-1β, or (E) TNF-α. Bars represent the mean ± SD for duplicate wells. The data presented are from a single representative experiment that was performed at least twice.</p