- Research article
- Open Access
A serine/threonine phosphatase encoded by MG_207 of Mycoplasma genitalium is critical for its virulence
- Mario A Martinez1, 2,
- Kishore Das†1,
- Sankaralingam Saikolappan1,
- Luis A Materon2 and
- Subramanian Dhandayuthapani1Email author
© Martinez et al.; licensee BioMed Central Ltd. 2013
Received: 30 October 2012
Accepted: 19 February 2013
Published: 21 February 2013
Bacterial signal transduction systems like two component system (TCS) and Serine/Threonine kinase (STK) and Serine/Threonine phosphatase (STP) play important roles in the virulence and pathogenesis of bacterial pathogens. Mycoplasma genitalium, a mollicute that causes the urogenital diseases urethritis and cervicitis in men and women, respectively, is a pathogen which lacks TCS but possesses STK/STP. In this study, we investigated the biochemical and virulence properties of an STP protein encoded by the gene MG_207 of this species.
We overexpressed MG207 in Escherichia coli overexpression system as a recombinant His10MG207 protein and purified it with affinity chromatography. This recombinant protein readily hydrolyzed the substrate p-nitrophenyl phosphate (pNPP) in a dose-dependent manner. Additional studies using synthetic peptides as substrates revealed that the recombinant protein was able to hydrolyze the threonine phosphate. Further, a transposon insertion mutant strain of M. genitalium (TIM207) that lacks the protein MG207 showed differentially phosphorylated proteins when compared to the wild type G37 strain. Mass spectrometry revealed that some of the key proteins differentially phosphorylated in TIM207 strain were putative cytoskeletal protein encoded by the gene MG_328 and pyruvate dehydrogenase E1 α chain encoded by the gene MG_274. In addition, TIM207 was noticed to be less cytotoxic to HeLa cells and this correlated with the production of less hydrogen peroxide by this strain. This strain was also less efficient in inducing the differentiation of THP-1 cell line as compared to wild type M. genitalium.
The results of the study suggest that MG207 is an important signaling protein of M. genitalium and its presence may be crucial for the virulence of this species.
Bacteria adapt to changing environments by regulating their gene expression through signal transduction systems. Two kinds of signal transduction systems exist in bacteria; the two component system (TCS) and serine/threonine kinases (STK) and phosphatases (STP) system [1–4]. Although both systems transduce signals by phosphorylation events, they have distinct ways of doing this. While TCS uses a sensor histidine kinase and a regulator protein to transduce the signals, the STK /STP regulate gene expression by protein-protein interaction [3, 4]. However, it should be noted that not all kinases and phosphatases associated with serine or threonine residues in prokaryotes are STK/STP. The STK/STP has special signature motifs [5, 6] and is restricted to selected species of bacteria. It was once thought that bacteria have only TCS but not STK/STP. However, evidence for the occurrence of STK/STP in bacteria continues to accumulate . Also, it has been reported that bacterial TCS and STK/STP systems cross talk with each other .
In addition to their role in the physiology, STK/STP plays a significant role in the virulence of some pathogenic bacteria, including bacteria relevant to public health such as Yersinia and Mycobacteria [4, 8]. For instance, YpkA, an STK of Yersinia pseudotuberculosis, is critical for the disruption of host cytoskeleton during infection [9, 10]. In Mycobacterium tuberculosis, lack of STK PknG and PknH has been reported to show reduced viability of this bacterium and increased bacterial load, respectively, in mouse models [11, 12]. The significance of STK in the pathogenesis of Staphylococcus aureus[13, 14], Streptococcus pneumoniae, S. pyogenes, Pseudomonas aeruginosa, S. agalactiae[18, 19], Mycoplasma pneumoniae and Salmonella enterica has also been reported. With respect to STP, relatively few studies have been undertaken in understanding their role in bacterial virulence and most of them focus on Pneumococcus . An STP (SP-STP) of S. pyogenes is required for the production of hemolysin and to cause apoptosis in the host cells [16, 22, 23]. Its homologue, STP1, in group B Streptococcus sp is also associated with the production of hemolysin and lack of this STP leads to less efficient systemic infection by this bacterium . Very recently, an STP (PhpP) of S. pneumoniae is found to have a role in the adherence of this species . Besides, an STP of Listeria monocytogenes is reported to be essential for the growth of this bacterium in murine model .
Mycoplasma genitalium is a bacterium that lacks a cell wall and is one of the smallest self-replicating organisms with a genome size of 580 kb . It is the etiological agent for the diseases non-gonococcal urethritis and cervicitis in men and women, respectively [28, 29]. In women, it is also implicated in diseases like endometritis, pelvic inflammatory syndrome and tubal infertility [30–32]. Additionally, M. genitailum coinfection in HIV patients has been reported to have increased shedding of HIV in urogenital mucosal regions of the female . Although it was initially thought that M. genitalium primarily attaches with epithelial cells of the host to cause the disease, evidences indicate that it invades epithelial cells and is localized on the periphery of the nucleus of the infected cells [34, 35]. The intracellular M. genitailum is reported to persist within the infected cells for a long time [34, 36]. It should be noted that intracellular survival and persistence of this bacterium may require signal transduction mediated adaptation, as do other bacteria in similar circumstances [37–39]. Strikingly, however, M. genitalium and its close relative M. pneumoniae are lacking the classical bacterial TCS [27, 40, 41], although a few mycoplasmas like M. penetrans and M. iowae do have TCS (NCBI data base). Besides, both species have only a limited number of regulators controlling gene expression at the transcription level [27, 40], and this has been attributed to their small genomes due to reductive evolution. Nevertheless, these species have genes encoding STK and STP [27, 40, 41]. In fact, the STK of M. pneumoniae has been demonstrated to have an effect on the adherence of this species , although no such effect was noticed with an STP of this species (PrpC) .
Our long term objective is to determine the roles of STK and STP in M. genitalium pathogenesis and signal transduction. NCBI database of M. genitalium genome sequence  reveals that this bacterium possesses a gene encoding STK (MG_109) and three genes encoding STP (MG_108, MG_207 and MG_246). We initiated our studies first with MG_207 because we had a mutant strain for this gene readily available from a transposon mutant library . Here, we show that MG207 is an alkaline phosphatase and it dephosphorylates threonine phosphate. We also report that M. genitalium lacking in MG207 (TIM207 strain) shows differentially phosphorylated proteins in two-dimensional gels. In addition, we provide evidence that TIM207 has reduced virulence as compared to wild type M. genitalium.
Results and discussion
MG_207 encodes a functional phosphatase
To determine if the purified His10MG207 protein was functional, we assayed the phosphatase activity of this protein using the substrate p-nitrophenyl phosphate (pNPP). The His10MG207 readily hydrolyzed pNPP in a dose dependent manner (Figure 1B) in the alkaline pH of 8.0. To rule out the possibility that the observed phosphatase activity of His10MG207 was not due to E. coli host derived phosphatase, we used similarly overexpressed and purified His10Ohr protein of M. genitalium as a control. Reactions with this protein (His10Ohr) or reactions with heat inactivated His10MG207 or reactions with His10MG207 but without Mg2+ in the reaction mixture showed no color formation with pNPP (data not shown), indicating that the overexpressed protein was a functional phosphatase dependent upon Mg2+ ion for its activity. To further assess whether His10MG207 is a serine/threonine phosphatase, malachite green based phosphatase assay was performed using synthetic serine (RRApSSVA) or threonine (KRpTIRR) phosphopeptide. No activity was noticed with either peptide in the presence of Ni2+, a cation supplied with the assay kit (data not shown). However, substitution of Ni2+ with Mg2+ in the reaction mixture released the phosphate from threonine peptide (Figure 1C), but this failed to release the phosphate from serine peptide. We presume that the absence of activity with the serine phosphate peptide may be due to the requirement of appropriate conditions. Alternatively, it is possible that the serine phosphate in this particular peptide is un-accessible for the enzyme. However, the fact that MG207 requires a metal (Mg2+) for its activity with pNPP or with threonine peptide suggests that it is a metal dependent phosphatase.
This observation is consistent with reports of other STPs like Stp of L. monocytogenes, PhpP of S. pneumoniae, PrpC of M. pneumoniae and Stp1 of S. agalactiae, all of which required divalent metal cofactor Mn2+ for their activity. In bacteria, STP belongs to two families, phosphoprotein phosphatases (PPP) and metal dependent phosphatases (PPM). The major difference between these two groups appears to be their specificity for substrates. While PPM specifically hydrolyzes serine or threonine phosphates, the PPP hydrolyzes, in addition to serine and threonine phosphates, histidine and tyrosine phosphates . Although PP2C phosphatase, a member of the PPM family, has some catalytic similarities with PPP, this does not show any amino acid similarity with PPP . Further, it appears that MG207 is only a closely related protein to PP2C phosphatase, because the cluster of orthologous groups (COGs) classification has placed this protein in a different group of bacterial phosphatase.
TIM207 strain and its confirmation
Further, to determine whether the transposon insertion indeed disrupted the expression of MG207 protein, we analyzed the proteins of G37 and TIM207 strain in immunoblot with anti-MG207 antiserum. This antiserum detected the MG207 protein only in the wild type G37 strain and not in the TIM207 strain (Figure 2B), indicating that the disruption of the gene affected the expression of the protein. We do not expect that Tn4001 insertion in this strain (TIM207) will have any polar effects on its downstream genes, because the transcription of the downstream genes is predicted to be in the opposite orientation (Figure 2C). This situation implies that complementation of the TIM207 with a functional allele to assess the function of MG207 is of limited significance. Moreover, the only way by which the M. genitalium mutant strain can be complemented is through the use of a transposon which can insert a copy of the functional allele of the mutated gene in an unknown location of the chromosome. It is very likely that the unknown location may be a functional gene and this will affect the interpretation of the complimented phenotype. Therefore, we have used a M. genitalium strain called TIM262, which bears the same transposon as in TIM207, inserted in the gene MG_262, as a control strain in some experiments. The gene MG_262 is predicted to code for a 3´-5´ exonuclease (NCBI database).
TIM207 strain exhibits differentially phosphorylated proteins
The predominant difference was noticed to be at the high molecular weight (HMW) areas which are shown in large circles (Figure 3A and C). As can be seen, the gels from G37 showed relatively dense and larger stained areas as compared to gels from the TIM207 strain, suggesting that some HMW proteins are less phosphorylated in TIM207 strain. However, these dense areas have shown no corresponding protein spots in Sypro Ruby stained gels, thus indicating that these areas do not represent real proteins but represent some artifacts. Therefore, we focused only on well separated and differentially phosphorylated proteins. These included two proteins (shown in circles 1 and 2) which showed relatively dense staining in the gels of G37 strain but were weaker in the gels of TIM207 strain, and three proteins (shown in circles 3, 4 and 5) that showed stronger staining in the gels of TIM207 strains but were weaker in the gels of G37.
Phosphorylated proteins identified by Mass spectrometry
Pyruvate dehydrogenase E1 subunit α
Pyruvate dehydrogenase E1 subunit α
Putative cytoskeletal protein
Conserved hypothetical protein
Ability of TIM207 strain to adhere and invade eukaryotic cells
Nevertheless, the partial culture flask non-adherence phenotype that we observed with the TIM207 strain is different from that of the completely culture flask non-adhering phenotype of M. genitalium strain reported earlier . Feldner et al.  reported that adherence of mycoplasma to culture flasks are based on electrostatic forces rather than adhesion mediated. It is likely, therefore, that change in phosphorylation of some surface proteins, due to the absence of MG207, leads to change in membrane potential which ultimately affects the electrostatic force. In the case of M. pneumoniae, it is the STK, but not STP (PrpC), mutant which failed to adhere with culture flasks [20, 42]. Consistent with this negative adherence to culture flasks, this STK mutant strain (MPN248 mutant) exhibits reduced levels of adherence related proteins, including P1, in SDS-PAGE. However, recent studies have demonstrated that deletion of STP in strains of S. pyogenes (M1SF370)  and S. pneumoniae (D39) leads to reduced adherence to pharyngeal cells. It appears, therefore, that disruption of both STK and STP can lead to adherence negative phenotype but it varies from species to species. However, the mechanism behind partial adherence of TIM207 to cultures flask remains elusive and it requires further study.
TIM207 strain is less cytotoxic to HeLa cells
TIM207 strain fails to differentiate THP-1 cells
We presume that phosphorylation of some proteins associated with the differentiation of THP-1 cells is severely affected in this mutant which leads to reduced differentiation of THP-1 cells as compared to wild type. It is unknown at present whether or not the surface proteins like pyruvate dehydrogenase E1 α chain and MG328, which showed altered phosphorylation in this study, have any role in this process but such a possibility does exist. Nevertheless, since differentiation of monocytes is related to modulation of immune responses, the reduced ability of TIM207 strain to differentiate these cells may suggest that this mutant will have only limited ability to alter the immune system to its favor. This hypothesis is supported by the fact that an msrA mutant (ΔMG_408) of M. genitalium, which differentiates THP-1 cells only moderately, could induce only limited amounts of proinflammatory cytokines IL-1β and TNF-α as compared to wild type M. genitalium that has the full ability to differentiate THP-1 cells . It is our future goal to investigate whether absence of MG207 protein in M. genitalium has any relationship with induction of immune response in the host cells
In this study, we have shown that the product encoded by MG_207 in M. genitalium is a phosphatase and its absence may affect the phosphorylation of some proteins. We have also provided evidence that absence of MG207 leads to reduced virulence of this bacterium by affecting its ability to cause cytotoxicity and to differentiate monocytic cells. However, the partial adherence phenotype to culture flasks that we observed with TIM207 appears to be significant and what causes this transient phenotype remains a question. Similarly, the factors that led TIM207 to cause reduced cytotoxicity and reduced induction of differentiation of THP-1 cells also remain indefinable at this point. Whether the differentially phosphorylated proteins like MG274, MG328 and MG281 play any role in these processes needs additional investigation.
Bacterial strains and their culture
Escherichia coli strains were cultured in LB broth at 37°C with ampicillin 100 μg/ml.
M. genitalium wild type strain (G37) was grown in 100 ml of SP-4 medium at 37°C for 72 h in 150 cm2 tissue culture flasks (Corning, NY). M. genitalium transposon mutant strains TIM207 and TIM262, (kindly provided by Dr. John Glass, J. Craig Venter Institute, Rockville, MD) were also grown similarly in SP-4 medium with 4 μg/ml tetracycline or 50 μg/ml gentamicin.
Adherent M. genitalium from culture flasks was washed three times with PBS (pH 7.2) and scraped with cell scrapers (39 cm handle/3 cm blade; Corning, NY). The suspension was centrifuged at 20,000xg for 20 min at 4°C in Sorvall RC 5B centrifuge. The pellets were resuspended in PBS and passed through 18G needles and then through 23G needles to disperse bacterial clumps. The suspensions, diluted to OD600=1.0 (which is equivalent to 1 X107 CFU/ml) with PBS, were used to infect cell lines with different multiplicity of infection (MOI).
Cell lines and their culture
Human cell lines THP-1 (TIB-202) and HeLa (CCL-2) were purchased from American Type Culture Collection (ATCC, Manassas, VA). THP-1 and HeLa cells were cultured in RPMI and Dulbecco’s modified Eagle’s medium (DMEM), respectively, with 10% FBS at 37°C in a humid chamber with 5% CO2.
Plasmids from E. coli were isolated using QIAprep Spin kit (Qiagen). Genomic DNA from mycoplasma was isolated using DNA isolation kit (Invitrogen). Primers for amplification of MG_207 gene and subsequent site directed mutagenesis were synthesized at the DNA core facility, The University of Texas Health Science Center at San Antonio (UTHSCSA). The whole gene encoding MG207 was amplified by PCR using primers MG_207EX1 (5´-ACGCATATGCAAAACAAACTGATTAAGGTT-3´) and MG_207EX2 (5´-CAGTCGGATCCGTTAACTAACTTTTGAAGCTTG-3´) and M. genitalium genomic DNA as template. This fragment was cloned into pCR 2.1 to result in pMG207. The gene MG_207 has a TGA codon for tryptophan residue, which will be recognized as stop codon by E. coli, and this needed modification into TGG to express the gene in E. coli. To do this modification (point mutation), we used QuikChange Site-Directed Mutagenesis Kit (Stratagene) and primers MG_207M1 (5´-CAAAATGCTACTTTTTGGGTGGCAGGTAACAAC-3´) and MG_207M2 (5´-GTTGTTACCTGCCACCCAAAAAGTAGCATTTTG-3´). Plasmid pMG207 served as the template for point mutation. Subsequent to point mutation, the newly synthesized plasmid DNA (pMG207A) was transformed into E. coli, plasmid isolated and the sequence of the insert region was verified to confirm the point mutation. The coding region of MG_207 from pMG207A was digested with NdeI and BamHI and the fragment cloned into similarly cut pET16b expression vector. This plasmid (pMG207EX) was transformed into E. coli BL21 (DE3) strain to overexpress His10MG207 protein.
To reconfirm the insertion of transposon Tn4001 in MG_207, we performed Southern hybridization. Briefly, chromosomal DNA from M. genitalium G37 and TIM207 was cut with SpeI and separated in 1% agarose gels. The separated DNA fragments were transferred to Zeta probe membranes (Bio-Rad) by Southern blotting and crosslinked with UV. Prehybridization of the membranes was performed in a solution containing 50% formamide, 0.12 M Na2HPO4, 0.25 M NaCl, and 7% (wt/vol) sodium dodecyl sulfate (SDS) for 4 h. Hybridization of the membranes was done in the same solution with [α-32P]dCTP labeled probe DNA of MG_207 or gentamicin gene for overnight at 42°C. The membranes were washed at 42°C (each wash for 15 min with solutions A (2X SSC with 0.1% SDS), B (0.5X SSC with 0.1% SDS) and C (0.1X SSC with 0.1% SDS) for three times. Afterwards, the membranes were exposed to X-ray films for autoradiography.
Overexpression of MG207 in E. coli
Overexpression and purification of recombinant MG207 protein using pET16b were performed as detailed before [55, 56]. Briefly, E. coli strain BL21 (DE3) harboring the pMG207EX was induced with 0.5 mM IPTG at 37°C to overexpress the protein. The overexpressed protein was purified with Ni-NTA affinity column chromatography (Qiagen). The E. coli extracts and purified protein were separated on 12% SDS-PAGE to assess the expression and purification. The purified recombinant protein was designated as His10MG207. All purification and desalting procedures were performed with buffers based on Tris–HCl pH8.0 and use of phosphate buffer was avoided.
To determine if the overexpressed and purified His10MG207 was functional, we performed phosphatase assay with p-nitrophenyl phosphate (pNPP) as substrate (Sigma-Aldrich, St. Louis, MO). The assay was conducted in 96 well plates and the assay mixture (120 μl) contained 1 mM pNPP in 20 mM Tris–HCl pH 8.0, 5 mM MgCl2 and His10MG207 protein. Control reactions had no protein or heat inactivated His10MG207. Each reaction was done in triplicate wells. The reaction mixtures were incubated at 37°C for 1 h and the yellow color, developed due to the hydrolysis of pNPP, was read at 405 nm using a Spectramax plate reader (Molecular Devices, Sunnyvale, CA).
To determine the specificity of His10MG207 towards serine or threonine residue, we used Alkaline/Acid Phosphatase assay kit (Millipore, Temecula, CA). This uses synthetic peptides for serine phosphate (RRApSSVA) and threonine phosphate (KRpTIRR) as substrates for the enzyme assay. The reactions were done as described by the manufacturer in 96 well plates, except that the reaction mixture had MgCl2 instead of NiCl2. Amount of phosphate released was calculated using phosphate reference standards supplied with the kit.
SDS-PAGE and immunoblot
Premade SDS-PAGE gels (NuPAGE 12% Bis-Tris gel, Invitrogen, Carlsbad, CA) were used to separate proteins from E. coli and M. genitalium for coomassie staining of proteins and for Western blot. In these gels 50 μg of total protein was loaded per well. Protein concentration was determined by BCA method (Pierce). Western blots were probed with anti-MG207 rabbit antiserum (1:500 dilutions) to detect MG207 protein of M. genitalium strains. This rabbit antiserum was generated against purified His10MG207 protein using a commercial source (Alpha Diagnostic International Inc., San Antonio).
Two-dimensional gel analysis of proteins
Two-dimensional (2-D) gel analyses of total proteins of M. genitalium G37 and TIM207 strains were performed by Kendrick Lab Inc., (Madison, WI). Fifty μg of total proteins were separated by isoelectric focusing [IEF] in glass tubes with an inner diameter of 2.0 mm. The IEF gel contained 2% pH 4–6 ampholines (Servalytes, Serva, Heidelberg, Germany) and 2% pH 5–8 ampholines (GE Healthcare). After IEF, gels were equilibrated for 10 min in buffer “0” (10% glycerol, 50 mm dithiothreitol, 2.3% SDS and 0.0625 M Tris, pH 6.8). Thereafter, each tube gel was sealed to the top of a stacking gel that was overlaid above 10% SDS-PAGE acrylamide gels (slab gels, 0.75 mm thick) and gels were run for about 4 h at 15 mA/gel. The gels were then fixed twice in 50% methanol 10% acetic acid solution and stained with Pro-Q Diamond for phosphoproteins. Images of the gels were acquired by scanning the gels with Bio-Rad Molecular Imager FX ProPlus scanner. After destaining, the gels were stained with Sypro Ruby (Molecular Probes) and again scanned with Bio-Rad Molecular Imager FX ProPlus scanner to obtain the images of total proteins. The following proteins (Sigma Chemical Co., St. Louis, MO) were used as molecular weight standards: myosin (22,000), phosphorylase A (94,000), catalase (60,000), actin (43,000), carbonic anhydrase (29,000) and lysozyme (14,000).
Mass spectrometry analyses were conducted in our core facility at UTHSCA. Pro-Q Diamond-stained gel spots were manually excised and digested in situ with trypsin (Promega, modified) in 40 mM NH4HCO3 overnight at 37°C. The digests were analyzed by capillary HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) using a Thermo Fisher LTQ linear ion trap mass spectrometer fitted with a New Objective PicoView 550 nanospray interface. On-line HPLC separation was accomplished with an Eksigent NanoLC micro HPLC: column, PicoFrit™ (New Objective; 75 μm i.d.) packed to 11 cm with Vydac 218MSB5 (5 μm, 300 Å) using a scan strategy in which a survey scan was acquired followed by data-dependent collision-induced dissociation (CID) of the seven most intense ions in the survey scan above a set threshold. The uninterpreted CID spectra were searched by means of Mascot (Matrix Science) against the Swiss-Prot database [2011_03 (525,997 sequences; 185,874,894 residues)] as follows: enzyme, trypsin, one missed cleavage allowed; precursor and fragment ion mass tolerances, ± 1.5 Da and ± 0.8 Da, respectively; variable modifications, methionine oxidation and phosphorylation of serine, threonine and tyrosine. Cross correlation of the Mascot results with X! Tandem and determination of probabilities for peptide assignments and protein identities were accomplished by Scaffold™ (Proteome Software).
Attachment of mycoplasmas to the HeLa cells: HeLa cells (2.5 × 105) were grown on square cover slides in 6 well tissue culture plates (Corning, NY). M. genitalium strains were labeled with Fluorescein isothiocyanate isomer I (FITC: Sigma-Aldrich, St. Louis, MO) as described before  and infected with an MOI of 1:25 for 1 h at 37°C. The cell monolayer was then washed three times with PBS and images captured using at 488 nm in an inverted laser microscope (Olympus FV1000) with 20 X objective (NA 0.75).
Cytotoxicity of M. genitalium strains was assessed by infecting HeLa cell line as reported earlier . Briefly, HeLa cells (2.5 × 105) were grown on coverslips in 6 well plates for 24 h and then infected with wild type G37 and mutant (TIM207 and TIM262) M. genitalium strains with MOI=50 for 2–3 h. Heat killed M. genitalium (HKG37) was used as control. Cytotoxic effect was determined by evaluating the integrity of the infected cells using differential interference contrast  at 488 nm in an inverted laser scanning confocal microscope (Olympus FV1000) with 20X objective.
Determination of H2O2 in M. genitalium strains
Production of H2O2 by mycoplasma strains was measured by colorimetric ferrous ion oxidation in xylenol orange [FOX] method [58, 59]. Protein samples from strains of M. genitalium were used as the source for H2O2. Protein content of samples was determined using Pierce BCA Protein Assay Kit (Pierce). Equal amount of protein samples (each 25 μl) and cold FOX reagent (250 μl) were mixed and incubated for 30 min at room temperature. After incubation, absorbance was measured at 560 nm. The amount of hydrogen peroxide in each sample was determined using a standard curve generated with known amounts of H2O2. The results were expressed as μmoles H2O2/per μg protein.
Differentiation of monocytic THP-1 cells by M. genitalium strains
THP-1 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cells (0.5X105) were plated on 4 chamber 1.5 German cover glass slides (Nunc, Rochester, NY). The cells were then infected with (MOI 1:5) M. genitalium (G37 or TIM207 or TIM262 or HKG37) for 1 h. After incubation, the chambers were washed with PBS to remove non-adherent cells. Cells adhering to the cover slips were examined under FV1000 laser scanning inverted confocal microscope (Olympus, Japan) with 20X objective. Images were acquired and labeled cells in each image was counted using the NIH analyze particle plug-in of Image J software.
The data were analyzed by paired t- test using graphpad prism software.
This study was partly supported by NIH grant AI08346. We thank Dr. John Glass, J. Craig Venter Institute, Baltimore, MD, for the TIM207 and TIM262 strain of M. genitalium. Mass spectrometry analyses were conducted in the UTHSCSA Institutional Mass Spectrometry Laboratory. Confocal microscopic analyses were performed at the Optical Imaging Core Facility at UTHSCSA- Regional Academic Health Center at Edinburg, Texas. We thank Drs. Robert Edwards and Robert Gilkerson, Department of Biology, University of Texas Pan American for kindly reading the manuscript and correcting the language.
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