J. Microbiol. Biotechnol. (2015), 25(10), 1621–1628 http://dx.doi.org/10.4014/jmb.1503.03072 Research Article Review jmb

Identification of Plasmid-Free muridarum Organisms Using a Pgp3 Detection-Based Immunofluorescence Assay Chaoqun Chen1,2, Guangming Zhong1,2*, Lin Ren1, Chunxue Lu1, Zhongyu Li1, and Yimou Wu1*

1Departments of Medical Microbiology and Immunology, Hunan Province Key Laboratory for Special Pathogens Prevention and Control, University of South China School of Medicine, Hengyang, Hunan 421001, P.R. China 2Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA

Received: March 20, 2015 Revised: May 26, 2015 Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in Accepted: June 3, 2015 chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms

First published online represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor June 9, 2015 immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole

*Corresponding authors organisms plus DNA staining was used to screen C. muridarum organisms selected with G.Z. novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then Phone: +210-567-1169; used to sequence the genomes of plasmid-free C. muridarum candidates and the parental Fax: +210-567-0293; E-mail: [email protected] C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum Y.W. organisms by using a combination of novobiocin treatment and screening plaque-purified Phone: +86-13707340050; Fax: +86-734-8282907; clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR E-mail: [email protected] analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pISSN 1017-7825, eISSN 1738-8872 pathogenic mechanisms. Copyright© 2015 by The Korean Society for Microbiology Keywords: Chlamydia muridarum, plasmid depletion, Pgp3 detection, immunofluorescence and Biotechnology

Introduction organisms cause no known human diseases, they have been and are still extensively used to study the mechanisms of , consisting of 19 different serovars, C. trachomatis pathogenesis and immunity in mouse models can cause a broad spectrum of diseases, including trachoma [11, 19] . and tubal factor infertility, and the even disseminated Most C. trachomatis clinical isolates carry a highly sexually transmitted infection. Recently, several studies conserved plasmid comprising eight predicted coding have associated chlamydial infection with cervical and sequences (CDSs; the gene products are designated as ovarian cancer development [4] . However, the pathogenic Pgp1-8) [8, 9], and naturally occurring plasmid-deficient mechanisms of C. trachomatis-induced diseases remain clinical isolates are very rare. The plasmid may play a unknown and there is still no licensed anti-C. trachomatis significant role in C. trachomatis pathogenesis since plasmid- vaccine. The mouse chlamydial pathogen Chlamydia free trachoma serovar A organisms no longer caused muridarum (also known as mouse pneumonitis, MoPn) is pathology in primate ocular tissues [7]. This finding was closely related to C. trachomatis. Although C. muridarum consistent with an earlier observation that a C. muridarum

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strain after depletion of the plasmid failed to induce Reagents, Antibodies, and Constructs hydrosalpinx in mice [12]. It is now known that the Chemicals were obtained from Sigma-Aldrich. Secondary Cy2- chlamydial plasmid not only encodes eight Pgps but also conjugated antibodies against rabbit IgG, and Cy3-conjugated regulates expression of more than 20 chlamydial genomic against mouse IgG were obtained from Jackson ImmunoResearch genes [2]. Plasmid-free chlamydial organisms generally Laboratories, Inc. The AccuPrime Pfx SuperMix and 1 kb plus DNA ladder were from Invitrogen; mAb MC22 against chlamydial displayed a reduced expression of glycogen synthesis major outer membrane protein (MOMP) were from Zhong’s Lab genes and lacked glycogen accumulation in the inclusions. [22]; and HRP-conjugated goat anti-mouse IgG were from Santa Recently, researchers developed a chlamydial plasmid Cruz Biotech. shuttle vector-based transformation system that allowed Antiserum against Pgp3 was raised by immunization of a the full complementation of the plasmid-free organisms mouse with GST-Pgp3 fusion protein. The pgp3 gene (TCA04) with an engineered plasmid [21]. This technology has from the C. muridarum plasmid was used to build the fusion offered an opportunity for chlamydiologists to genetically construct. Purification of GST-Pgp3 was performed as described manipulate chlamydial organisms and define the molecular previously [3]. The mouse anti-GlgA (TC0181) polyclonal antiserum basis of plasmid-dependent pathogenesis [6, 17]. However, was raised with a GST-GlgA fusion protein. this genetic manipulation depends on the availability of plasmid-free organisms. Thus, the generation and Chlamydial Plaque-Forming Assay identification of plasmid-free chlamydial organisms from A plaque-forming assay was adapted from a previously described protocol [13, 14]. Briefly, the survived chlamydial different serovars/clinic isolates have become important. organisms harvested from novobiocin-treated cultures were Herein, we report a convenient immunofluorescence assay serially diluted and inoculated onto confluent monolayers of for identifying plasmid-free chlamydial organisms using McCoy cells in 6-well tissue culture plates. The infection was C. muridarum selected with novobiocin as a model system. facilitated by centrifugation at 483 RCF for 1 h at 37°C. The inoculum was then removed and replaced with DMEM containing Materials and Methods 1 µg/ml of cycloheximide and 10% FBS. Four hours later, the medium was replaced with 2 ml of agarose overlay medium Chlamydial Organisms and Infection (1× EMEM, 3.5 g/l glucose, 2 mM L-glutamine, 1 mM sodium Chlamydia muridarum Nigg strain was propagated in HeLa cells pyruvate, 10% FBS, 10 µg/ml gentamicin, 2 µg/ml cycloheximide, (human cervical carcinoma epithelial cells) or McCoy cells (mouse and a final concentration of 0.55% of agarose) followed by fibroblast cells). Briefly, cells were grown in 24-well plates with or dispensing 4 ml of pre-warmed DMEM supplemented with 10% without coverslips or 6-well plates containing DMEM supplemented FBS on top of the agarose layer. The cultures were then incubated with 10% FBS overnight at 37°C in an incubator supplied with 5% for 3-5 days to allow plaques to form. Individual plaques (well

CO2. The following day, the cell monolayers were pretreated with separated) were picked up into 100 µl of SPG buffer for making DEAE-dextran and chlamydial stocks were diluted in serum-free lysates by vortexing with glass beads and stored at -80°C. A DMEM for inoculation. The chlamydial organisms were allowed portion of each plaque-derived lysate was used to infect fresh to attach to the monolayers for 30 min at 37°C before being monolayers of HeLa cells grown in 96-well microplates. The 96- centrifuged at 483 RCF (relative centrifugal force) for 1 h at 37°C. well plate cultures were processed for tricolor immunofluorescence Following centrifugation, the cell culture supernatant was screening for lack of the plasmid-encoded Pgp3 antigen. removed and DMEM with 10% FBS and 2 µg/ml of cycloheximide was added to the infected cells, which were incubated in 5% CO2 Indirect Immunofluorescence Assay for 24-30 h. Elementary bodies (EBs) were purified by density HeLa cells grown in 96- or 24-well plates with or without gradient centrifugation using MD-76 (MD-76%: 0.1 g diatrizoate chlamydial infection were washed once with PBS, and fixed with sodium and 0.66 g diatrizoate meglumine; Mallinckrodt Inc.). 4% paraformaldehyde dissolved in PBS for 30 min at room In some experiments, novobiocin was added to the culture temperature (RT), followed by permeabilization with 2% saponin medium at final concentrations of 50, 62.5, 100, 125, and 150 µg/ml for an additional 30 min. After being washed three times with PBS at 4 h post-infection. After incubation for another 40 h, the and blocked with 4% BSA in PBS for 1 h, the cell samples were novobiocin-treated cultures were harvested by trypsinization and stained with primary antibodies diluted in 2% BSA in PBS the cell pellets were lysed by vortexing with glass beads in a overnight at 4°C. A mouse anti-Pgp3 or anti-GlgA antibody was sucrose-phosphate-glutamate (SPG) buffer (218 mM sucrose, used to detect Pgp3 or GlgA, whereas a rabbit anti-chlamydial

3.76 mM KH2PO4, 7.1 mM K2HPO4, and 4.9 mM glutamate (pH organism antibody, raised by immunization with purified 7.2)). The lysates were serially diluted for titrating live organisms C. muridarum EBs, was used to monitor chlamydial organisms. on HeLa cell monolayers in 24-well plates or for plaque forming After intensive washing, the binding of primary antibodies was on McCoy cell monolayers in 6-well plates. visualized by a Cy3-conjugated goat anti-mouse (red) or a Cy2-

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conjugated goat anti-rabbit (green) IgG, and Hoechst 33258 (blue) Western Blot Assay was used to visualize nuclear DNA. The immunofluorescence HeLa cells with or without C. muridarum infection were images were acquired using an Olympus IX-80 fluorescence solubilized in 2% SDS sample buffer and loaded onto 15% SDS- microscope equipped with multiple filter sets and Simple PCI polyacrylamide gel wells. After electrophoresis, the proteins were imaging software as described previously [3]. wet transferred onto nitrocellulose membrane. Following transfer, For titrating inclusion forming units (IFUs), the inclusions were the membrane was incubated overnight at 4°C with mouse counted under a fluorescence microscope, where five random polyclonal antiserum against Pgp3 diluted 1:2,000 in 5% skim fields were counted per coverslip. The total number of IFUs was milk, prior to being incubated with the appropriate HRP- calculated based on the number of IFUs per field, number of fields conjugated goat anti-mouse IgG secondary antibody for 1 h at RT per coverslip, and dilution factors. An average was taken from the before detection using an ECL kit (Santa Cruz Biotech). After that, serially diluted samples. the membrane was incubated and agitated in stripping buffer (25 mM glycine, 1% SDS, pH 2.0) for 30 min at RT, and then Iodine Staining washed with 0.05% PBST for 3× 10 min. It was re-blocked with HeLa cells cultured overnight on glass coverslips in 24-well milk and subjected to the western blot procedure. The primary plates were infected with plasmid-free or wild-type C. muridarum antibody was mAb MC22. as described above. After 28h incubation, the culture medium was removed and the monolayer was rinsed several times with Preparation of Chlamydial DNA for Sequencing PBS and fixed with ice-cold methanol for 10 min at RT. The cell Genomic DNA was prepared as previously described [1] with samples were stained with 5% iodine stain solution (5% some modifications. A plasmid-free clone, CMUT3, and the potassium iodide and 5% iodine in 50% ethanol) for 40 min at RT. parent C. muridarum Nigg strain wild type were selected for The coverslips were mounted in 50% glycerol containing 5% experimentation. Briefly, plasmid-free or wild-type C. muridarum potassium iodide and 5% iodine. Stained cells were immediately was large-scale propagated in HeLa cells. At 30 h post-infection, photographed and the images were acquired by using an infected monolayers were detached with PBS containing 0.05% Olympus CH-30 microscope equipped with a Canon EOS Rebel trypsin/0.02% EDTA, suspended in DMEM with 10% FBS and T3i Digital SLR Camera. centrifuged at 500 RCF for 10 min at 4°C. The pellet was combined and resuspended in ice-cold PBS and ruptured by sonication to PCR release the chlamydial organisms from the host cells. Low-speed The chlamydial organisms were lysed with 0.1% SDS and the spun supernatant (10 min at 500 RCF and 4°C) was subjected to lysates were used as PCR templates after dilution at 1:1,000. The high-speed centrifugation (30 min at 14,000 rpm and 4°C) to pellet PCR conditions were as follows: initial denaturation at 94°C for the chlamydial organisms. The final pellet was resuspended in 5 min, followed by 30 cycles of denaturation (94°C) for 30 sec, PBS, homogenized by sonication, and incubated with DNaseI annealing (50°C) for 45 sec, and elongation (72°C) for 2 min. We (0.03 IU/ml) (Invitrogen) and RNase A (100 µg/ml) (Sigma) in a separated PCR products in a 1% (w/v) agarose gel and visualized water bath at 37°C for 1 h. This suspension was layered onto 35% them by staining with ethidium bromide. The gene-specific MD-76 in Hepes/NaCl and centrifuged at 16,000 rpm for 1 h at primers used are listed in Table 1. 4°C in a Beckman SW32Ti rotor. The resulting pellet was

Table 1. PCR primers used in the current manuscript. Forward primer Reverse primer TC0199 CGCGGATCCATGACTATTCTTTCTAATGATGG TTTTCCTTTTGCGGCCGCTTAAGAAATCGAAGCACGTCTC TC0602N CGCGGATCCATGAGTCATAAAAGTTTTTCCGT TTTTCCTTTTGCGGCCGCTTATGAGCAAACAAAAATAGGAC TCA01 CGCGGATCCATGAACTCAAAATTTTACCACAG TTTTCCTTTTGCGGCCGCTCACAATATAGTTGGTATATTTG TCA02 TTGCGTTTAGGTTATATCACGAGGAA TTATATTAGGGCCATCTTCTTTGAGGC TCA03 CGCGGATCCATGAAAACTAATTCTGAAATAGAA TTTTCCTTTTGCGGCCGCTTACCATACTTTTTTAATAGCAGA TCA04 CCGGAATTCATGACAGAACCTCTTACAGAT TTTTCCTTTTGCGGCCGCTTAAGTGTTTTTTTGAGGTATCAC TCA05 CGCGGATCCATGCAAAACAAAAGAAAACTGAGA TTTTCCTTTTGCGGCCGCCTATTCTGCCTTAGAAAACATGT TCA06 TTGGAGTACACTTTGCAAACCTTAGTTT TCACAGTGTCCTCTGGGAAAAGTC TCA07 CGCGGATCCGTGAATAAACTGGAAAAGGAAG TTTTCCTTTTGCGGCCGCTCAGCTCTTTTGCTTATAGACT TC0199: 648 bp TC0602N: M1~S245, 735 bp TCA01: 918 bp; TCA02: 1,023 bp; TCA03: 1,356 bp; TCA04: 723 bp, TCA05: 309 bp; TCA06: 807 bp; TCA07: 741 bp;

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homogenized and resuspended in cold PBS. The suspension was completed by the UTHSCSA Greehey Children’s Cancer Research overlaid onto a discontinuous-density gradients consisting of Institute Genome Sequencing Facility TX, USA. Briefly, purified 40%, 44%, and 52% MD-76 and centrifuged at 17,000 rpm for 1.5 h DNA was fragmented by a Covaris S220 Ultra Sonicator and a at 4°C. After centrifugation, the EB fraction (located at the 44-52% DNA sequencing (DNA-Seq) library was prepared by using the interface) was collected, diluted in PBS, and then centrifuged at TruSeq DNA Sample Preparation kit (Illumina, Inc.) according to 14,000 rpm for 30 min at 4°C. The resulting pellets were washed the manufacturer’s protocol. DNA-Seq libraries were sequenced with PBS to remove residual MD-76 and resuspended in PBS. with a 50 bp single-end sequencing module on the Illumina Purified EBs were lysed with proteinase K (100 µg/ml) (Life HiSeq2000 platform. After demultiplexing with CASAVA, Technologies) at 50°C overnight. The DNA was extracted twice sequence reads and associated qualities were exported in FASTQ with 25:24:1 phenol:chloroform:isoamyl alcohol and once with format. Reads in FASTQ format were mapped to the C. muridarum chloroform and precipitated with alcohol. The genomic DNA was strain Nigg reference (NC_002620.2) and pMoPn then purified using a QIAamp DNA mini-kit according to the plasmid (NC_02182.1) [15]. Sequencing reads and associated manufacturer’s protocol. Prior to sequencing, several assays were coverage were visualized using the IGV browser [18]. performed to assess the purity and quality of DNA recovered by this method. The quality of DNA was verified by agarose gel Results electrophoresis, and the DNA was quantified by nanodrop method, and 2 µg of highly pure DNA for use was finally stored at 4°C. Generation and Identification of Plasmid-Free C. muridarum Organisms Genome Sequencing and Sequence Analysis To generate plasmid-free C. muridarum organisms, we Next-generation sequencing of purified genomic DNA was

Fig. 1. Plaque-forming assay and screening for C. muridarum organisms lacking plasmid. (A) Live organisms freshly harvested from novobiocin-treated cultures were serially diluted and plated on McCoy cell monolayers grown on 6- well plates. After the 6-well plate cultures were incubated for 3 to 5 days in soft agar medium, visible plaques (well separated from each other) were picked up and a portion of each plaque-derived lysate (made with glass beads) was used to infect fresh monolayers of HeLa cells grown in 96-well cell plates. The 96-well plate cultures were processed for tricolor immunofluorescence screening for lack of the plasmid-encoded Pgp3 antigen. (B) Pgp3 was detected with a mouse anti-Pgp3 antibody that was visualized with a goat anti-mouse IgG conjugated with Cy3 (red), whereas the chlamydial organisms were visualized with a rabbit anti-C. muridarum organism antibody plus a Cy2-conjugated goat anti-rabbit IgG (green). The DNA was visualized with Hoechst dye (blue). Among the 153 plaques screened, five plaques were found to contain 100% (CMUT2, 3, & 15, panels c, d, & f) or >50% (CMUT1 & 9, panels b & e) of C. muridarum organisms lacking the plasmid-encoded Pgp3. The organisms from these five plaques were re-plaqued, and the second-round plaques that contained 100% plasmid-free organisms were identified as shown in the bottom row images (panels h-l). The organisms harvested from the second round of plaques were re-plaqued for a third time to ensure monoclonality of the plasmid-free organisms (data not shown).

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first titrated novobiocin for its ability to inhibit C. muridarum replication. We found that novobiocin at 62.5 µg/ml achieved 99.5% killing of C. muridarum organisms. This was the dose chosen for the subsequent selection experiment. As described in Fig. 1A, after the novobiocin selection, the remaining live organisms were harvested from novobiocin-treated cultures and serially diluted for plating on McCoy cell monolayers grown on 6-well plates supplied with soft agar medium to allow plaque formation. The well-separated visible plaques were each individually picked up for making lysates with glass beads. A portion of each lysate was used to infect fresh monolayers of HeLa cells grown in 96-well cell culture microplates, while the remaining lysates were stored at -80°C. After culturing for 30 h, the 96-well microplate cultures were processed for tricolor immunofluorescence screening for lack of the plasmid-encoded Pgp3 antigen (Fig. 1B). Among the 153 plaques screened, five plaques were found to contain 100% (CMUT2, 3, & 15, panels c, d, & f) or >50% (CMUT1 & 9, panels b & e) of C. muridarum organisms lacking the plasmid-encoded Pgp3. The organisms from these five plaques were re-plaqued and the second-round plaques that contained 100% plasmid-free organisms were plaqued for the third time to ensure monoclonality of the plasmid- free organisms (data not shown).

In Vitro Characterization of Plasmid-Free C. muridarum Organisms As shown in Fig. 2, the five plasmid-free clones along with the wild-type C. muridarum organisms were detected with PCR for DNA sequences of both genomic open reading frames (ORFs) TC0199 (coding for a hypothetical protein homologous to CT813) and TC0602N (N-terminal portion of C. muridarum-specific helicase) and plasmid CDSs TCA01 (coding for Pgp7), TCA02 (Pgp8), TCA03 (Pgp1), TCA04 (Pgp3), TCA05 (Pgp4), TCA06 (Pgp5), and TCA07 (Pgp6). The PCR primers for amplifying the Fig. 2. PCR detection of plasmid genes. corresponding DNA sequences are listed in Table 1. The five plasmid-free (panels b-f) and wild-type C. muridarum (a) Although all expected PCR products were detected in the organisms were subjected to PCR detection of DNA sequences of wild-type C. muridarum organisms, only the genomic ORF genomic open reading frames (ORFs) TC0199 (coding for a PCR products were detected in the plasmid-free C. muridarum hypothetical protein homologous to CT813) and TC0602N (N- organisms. These observations demonstrated that the terminal portion of C. muridarum-specific helicase) and plasmid CDSs plasmid-free C. muridarum organisms indeed lacked all TCA01 (coding for Pgp7), TCA02 (Pgp8), TCA03 (Pgp1), TCA04 plasmid CDSs. We further characterized plasmid-free (Pgp3), TCA05 (Pgp4), TCA06 (Pgp5), and TCA07 (Pgp6) as shown on top of the gel images. All expected PCR products were detected in the C. muridarum in cell cultures by detecting Pgp3, GlgA, or wild-type C. muridarum organisms whereas only the genomic ORF glycogen (Fig. 3). The wild-type but not plasmid-free PCR products were detected in the plasmid-free C. muridarum C. muridarum organisms expressed both Pgp3 and GlgA. organisms (data are representative of two independent experiments). Glycogen accumulation was only detected in the wild-type All PCR primers are listed in Table 1.

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Fig. 3. In vitro characterization of plasmid-free C. muridarum. HeLa cells infected with C. muridarum wild type (panels a, g, and m) or deficient in plasmid (b-f, h-l, n-r) were subjected to immunofluorescence detection of Pgp3 (a-f) and GlgA (g-l) or iodine detection of glycogen (m-r). Mouse antibodies plus a Cy3-conjugated goat anti-mouse IgG were used to detect either Pgp3 or GlgA (red for both), and a rabbit antibody plus a Cy2-conjugated goat anti-rabbit IgG for detecting C. muridarum organisms (green). Hoechst dye was used to detect DNA (blue). The wild-type but not plasmid-free C. muridarum organisms expressed both Pgp3 and GlgA. Representative inclusions stained positive (arrows, panel a) or negative (arrowheads, n-r) with iodine are marked. Glycogen accumulation was detected in wild-type but not plasmid-free C. muridarum cultures. Data are representative of three independent experiments.

but not plasmid-free C. muridarum-infected cultures. On a C. muridarum-infected HeLa cell lysates. As a control, the western blot (Fig. 4), Pgp3 was detected only in C. muridarum anti-MOMP antibody detected MOMP in the wild-type and wild-type-infected HeLa cell lysates, not in plasmid-free plasmid-free C. muridarum-infected HeLa cell samples and ensured that all lanes with C. muridarum organism-containing samples had equivalent amounts of the organisms loaded. These results together have independently confirmed that plasmid-free C. muridarum candidates lacked the plasmid.

Genomic Characterization of Plasmid-Free C. muridarum Clone CMUT3 Compared with the genomic information of the parental C. muridarum Nigg strain (GenBank Accession No. CP009608.1.), CMUT3 (GenBank Accession No. CP006974.1) lacked the plasmid. Interestingly, a single mutation was detected in CMUT3 within TC0412: an additional T at 472839 that led to a premature stop at amino acid 47. Significantly, we determined that this mutation was also present in the parental C. muridarum Nigg strain, indicating that the Fig. 4. Western blot detection of Pgp3 in fractions of plasmid- mutation in CMUT3 was present prior to novobiocin free or wild-type C. muridarum-infected HeLa cells. treatment. The gene TC0412 is predicted to encode a protein Normal HeLa cell lysates, or plasmid-free or wild-type C. muridarum of unknown function that is 365 amino acids in length. (Nigg)-infected HeLa cell lysates were resolved on a SDS polyacrylamide gel and the resolved protein bands were transferred onto nitrocellulose membrane for western blot detection with anti-Pgp3 antibody. Pgp3 Discussion was detected only in wild-type C. muridarum infected-HeLa cell lysates. The same membrane was stripped and reprobed with anti- Chlamydial plasmids are small, highly conserved, MOMP. Data are representative of two independent experiments. nonconjugative, and nonintegrative DNA molecules that

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are nearly ubiquitous in many chlamydial [16]. can induce hydrosalpinx in mice, it is necessary to use the Studies in both C. trachomatis [7] and C. muridarum [12] C. muridarum infection of mouse urogenital tract model to have revealed an important role for the chlamydial plasmid define plasmid-dependent virulence factors. Thus, the in the expression of key virulence properties. Meanwhile, generation and identification of plasmid-free C. muridarum the need to identify plasmid-free chlamydial organisms has organisms represent our first step towards understanding become even greater owing to the recent advancement in the molecular mechanisms of chlamydial pathogenesis. The transforming chlamydial organisms [6, 20]. This is because availability of plasmid-free CMUT series of C. muridarum the current transformation method is dependent on organisms will provide chlamydial researchers with delivering a chlamydial plasmid-based shuttle vector back additional experimental tools in using the murine model into plasmid-free chlamydial organisms. The traditional system for investigating chlamydial pathogenesis. In vitro approach for identification of plasmid-free chlamydial characterization of the newly generated CMUT organisms organisms was based on glycogen staining [10], which is revealed that all five plasmid-free C. muridarum clones inherently insensitive and inconsistent. Various groups lacked plasmid genes and failed to accumulate GlgA have successfully used PCR-based screening for lack of protein or synthesize glycogen. These in vitro characteristics plasmid genes to identify plasmid-free chlamydial plaques are typical features of plasmid-free organisms [13]. More selected with novobiocin [7, 13]. In the current study, we importantly, whole-genome sequencing characterization of have demonstrated that a convenient immunofluorescence the plasmid-free C. muridarum organism and the parental assay can be used for identifying plasmid-free chlamydial Nigg strain revealed no additional mutations other than organisms. This tricolor immunofluorescence assay for loss of the plasmid in plasmid-free C. muridarum clone simultaneously detecting the plasmid-encoded Pgp3 (red) CMUT3. Efforts are under way to use the plasmidless and whole organisms (green) plus DNA staining (blue) CMUT organisms for dissecting the mechanisms of the was used to screen cells infected with survived chlamydial chlamydial plasmid-dependent pathogenicity. clones from novobiocin-treated cultures. In a single screening assay carried out in a 96-well microplate, five Acknowledgments clones with the organisms labeled green but without Pgp3- labeling were identified. These clones lacked plasmid This work was supported in part by the Hundred Talents genes with undetectable levels of GlgA protein and Program of Hunan Province to Zhong Guangming, and glycogen. These observations have both revealed a rapid/ National Natural Science Foundation of China (81072417), reliable immunofluorescence assay for identifying plasmid- and program of Hunan Province Key Laboratory for free chlamydial organisms and validated the phenotype of Special Pathogens Prevention and Control (2014-5). plasmid-free chlamydial organisms. We would like to thank Lai Zhao from UTHSCSA Pgp3 is a highly conserved plasmid protein that is both Greehey Children’s Cancer Research Institute Genome abundant and immunogenic [8, 9]. It has been shown that Sequencing Facility and Turner Conrad from UTHSCSA Pgp3 is both associated with the chlamydial outer membrane for their help with the genome sequence analysis. and secreted into the host cell cytosol. Thus, the Pgp3 detection signal is both strong and widely distributed across References the entire infected cells. The roles of plasmid in pathogenesis of C. trachomatis ocular infection and C. muridarum 1.Borges V, Ferreira R, Nunes A, Sousa-Uva M, Abreu M, urogenital tract infection have been demonstrated using Borrego MJ, et al. 2013. Effect of long-term laboratory plasmid-free organisms [7, 12]. However, not all chlamydial propagation on Chlamydia trachomatis genome dynamics. organisms can be attenuated by depleting the plasmid. For Infect. Genet. Evol. 17: 23-32. example, the C. caviae GPIC organisms retained virulence 2. Carlson JH, Whitmire WM, Crane DD, Wicke L, Virtaneva K, Sturdevant DE, et al. 2008. The Chlamydia trachomatis even after losing the plasmid [5]. Clearly, chlamydial plasmid is a transcriptional regulator of chromosomal genes pathogenic determinants can be coded by both the plasmid and a virulence factor. Infect. Immun. 76: 2273-2283. and the genome. 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