Kinetics of Genetic Variation of the Mycoplasma genitalium MG192 Gene in Experimentally Infected Chimpanzees

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is capable of causing chronic infections, though mechanisms for persistence remain unclear. Previous studies have found that variation of the MgPa operon occurs by recombination of repetitive chromosomal sequences (known as MgPars) into the MG191 and MG192 genes carried on this operon, which may lead to antigenic variation and immune evasion. In this study, we determined the kinetics of MG192 sequence variation during the course of experimental infection using archived specimens from two chimpanzees infected with M. genitalium strain G37. The highly variable region of MG192 was amplified by PCR from M. genitalium isolates obtained at various time points postinfection (p.i.). Sequence analysis revealed that MG192 sequence variation began at 5 weeks p.i. With the progression of infection, sequence changes accumulated throughout the MG192 variable region. The presence of MG192 variants at specific time points was confirmed by variant-specific PCR assays and sequence analysis of single-colony cloned M. genitalium organisms. MG192 nucleotide sequence variation correlated with estimated recombination events, predicted amino acid changes, and time of seroconversion, a finding consistent with immune selection of MG192 variants. In addition, we provided evidence that MG192 sequence variation occurred during the process of M. genitalium single-colony cloning. Such spontaneous variation suggests that some MG192 variation is independent of immune selection but may form the basis for subsequent immune selection.     

Persistence,Immune Response,and Antigenic Variation of Mycoplasma genitalium in an Experimentally Infected Pig-Tailed Macaque (Macaca nemestrina)

Mycoplasma genitalium is a sexually transmitted pathogen associated with several acute and chronic reproductive tract disease syndromes in men and women. To evaluate the suitability of a pig-tailed macaque model of M. genitalium infection, we inoculated a pilot animal with M. genitalium strain G37 in the uterine cervix and in salpingeal pockets generated by transplanting autologous Fallopian tube tissue subcutaneously. Viable organisms were recovered throughout the 8-week experiment in cervicovaginal specimens and up to 2 weeks postinfection in salpingeal pockets. Humoral and cervicovaginal antibodies reacting to MgpB were induced postinoculation and persisted throughout the infection. The immunodominance of the MgpB adhesin and the accumulation of mgpB sequence diversity previously observed in persistent human infections prompted us to evaluate sequence variation in this animal model. We found that after 8 weeks of infection, sequences within mgpB variable region B were replaced by novel sequences generated by reciprocal recombination with an archived variant sequence located elsewhere on the chromosome. In contrast, mgpB region B of the same inoculum propagated for 8 weeks in vitro remained unchanged. Notably, serum IgG reacted strongly with a recombinant protein spanning MgpB region B of the inoculum, while reactivity to a recombinant protein representing the week 8 variant was reduced, suggesting that antibodies were involved in the clearance of bacteria expressing the original infecting sequence. Together these results suggest that the pig-tailed macaque is a suitable model to study M. genitalium pathogenesis, antibody-mediated selection of antigenic variants in vivo, and immune escape.     

Fluoroquinolone and Macrolide Resistance-Associated Mutations in Mycoplasma genitalium

Mycoplasma genitalium is a significant sexually transmitted pathogen, causing up to 25% of cases of nongonococcal urethritis in men, and it is strongly associated with cervicitis and pelvic inflammatory disease in women. Currently, the usual first-line treatment is the macrolide antibiotic azithromycin, but an increasing incidence of treatment failure over the last 5 years suggests the emergence of antibiotic resistance. The mutations responsible for macrolide resistance have been found in the 23S rRNA gene in numerous M. genitalium populations. A second-line antibiotic, the fluoroquinolone moxifloxacin, was thought to be a reliable alternative when azithromycin began to fail, but recent studies have identified mutations that may confer fluoroquinolone resistance in the genes parC and gyrA. The aim of this study was to determine the prevalence of antibiotic resistance in M. genitalium in Sydney, Australia, by detecting relevant mutations in the 23S rRNA gene, parC, and gyrA. M. genitalium-positive DNA extracts of specimens, collected from patients attending sexual health clinics in Sydney, were tested by PCR amplification and DNA sequence alignment. The 186 specimens tested included 143 initial patient specimens and 43 second, or subsequent, specimens from 24 patients. We identified known macrolide resistance-associated mutations in the 23S rRNA gene in 43% of the initial patient samples and mutations potentially associated with fluoroquinolone resistance in parC or gyrA sequences in 15% of the initial patient samples. These findings support anecdotal clinical reports of azithromycin and moxifloxacin treatment failures in Sydney. Our results indicate that further surveillance is needed, and testing and treatment protocols for M. genitalium infections may need to be reviewed.     

Single-nucleotide polymorphisms in the rRNA operon and variable numbers of tandem repeats in the lipoprotein gene among Mycoplasma genitalium strains from clinical specimens

We characterized the single-nucleotide polymorphisms in the rRNA operon and variable numbers of tandem repeats in the lipoprotein gene MG309 among Mycoplasma genitalium strains from clinical specimens by PCR and sequencing. Analysis of 31 M. genitalium-infected patient specimens and 7 American Type Culture Collection strains identified six types of rRNA sequences and 11 different numbers of MG309 repeats. Examination of sequential specimens from 10 patients showed that these genotypes were stable for at least 5 weeks. These data suggest the potential usefulness of the rRNA genotypes and the MG309 repeats for genotyping of M. genitalium.     

Analysis Identifying Common and Distinct Sequences among Texas Clinical Strains of Mycoplasma genitalium

Mycoplasma genitalium is a human bacterial pathogen linked to urethritis and other sexually transmitted diseases. Here, we assessed the incidence of M. genitalium infection in patients attending a sexually transmitted disease clinic in San Antonio, TX, by use of diagnostic real-time PCR. Overall, 16.8% of women and 15.1% of men were found M. genitalium positive. Regions of the mgpB gene, which encodes the MgPa adhesin, were amplified from positive clinical specimens and evaluated for sequence variability, which demonstrated transmission of the pathogen between sexual partners. Follow-up analysis of a subset of patient specimens revealed reinfection by a different strain of M. genitalium, indicating the absence of protective immunity. Eighteen DNA sequence variants were obtained and compared with all other available clinical sequences. Detailed analysis revealed silent mutations of six amino acid residues within the encoded region of the MgPa adhesin in numerous clinical strains. In addition, missense mutations of limited numbers of amino acids were observed. Alignment of putative amino acid sequences revealed the simultaneous occurrence of several mutations and the existence of identical or similar protein variants in strains from different locations.Mycoplasma genitalium is associated with numerous human genitourinary tract maladies, including nonchlamydial, nongonococcal urethritis in men and cervicitis, endometritis, and pelvic inflammatory disease in women (1, 4, 5, 12, 19, 29, 30, 35, 39). Understanding the epidemiological aspects of infections and developing effective treatment require reliable typing of pathogenic microorganisms. Since M. genitalium is very difficult to culture from clinical specimens (13, 21), classical microbiological typing methods are not readily applicable. Furthermore, serological approaches are not widely used because of cross-reactivity with other mycoplasmas, especially with Mycoplasma pneumoniae (15, 26). Therefore, typing of M. genitalium strains relies on DNA sequence data. Recently, sequence variability of the rRNA operon and tandem repeats in the MG309 locus have been evaluated with clinical specimens (28). However, the majority of M. genitalium clinical sequence data available today is based upon the gene mgpB (locus MG191 of sequenced reference strain G37 [11]) encoding the M. genitalium adhesin MgPa (14, 17, 21). An M. genitalium-specific diagnostic PCR assay was designed to target the proximal unique region of this gene (using primers MgPa-1 and MgPa-3 [22] and designated here “MgPa-13”). The MgPa-13 region has exhibited sequence stability in persistently infected patients for up to 21 months (17). Despite this intrastrain stability, high levels of sequence variability between clinical isolates were observed (14, 17). Based on this sequence region, transmission of M. genitalium between sexual partners was shown, as was colonization of different anatomical sites of the same patient by identical strains (14).Here, we present analysis of MgPa-13 sequences generated from clinical specimens collected at the Project S.A.F.E. sexually transmitted disease (STD) clinic in San Antonio, TX (36), from recruited female participants and their male partners. Alignment of MgPa-13 sequences corroborated transmission between partners and colonization of different anatomical sites by the same strain. Comparison of newly recovered sequences with all currently available clinical data (14, 17, 21) revealed not only the presence of several common variants but also distinct sequences. Analyses of encoded amino acids uncovered mutations that determine common features among MgPa-13 region variants.     

Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis

Mycoplasma genitalium is a sexually transmitted organism commonly treated with azithromycin. However, macrolide resistance has been reported and is associated with point mutations in the 23S rRNA gene. To evaluate the prevalence of macrolide resistance in M. genitalium isolates from clinical specimens from France, we first used a previously reported high-resolution melting assay. Because susceptible and resistant M. genitalium isolates were hardly discriminated in M. genitalium-positive clinical specimens, we developed a new molecular assay for the rapid detection of macrolide resistance. An assay using real-time PCR based on fluorescence resonance energy transfer (FRET) coupled with melting curve analysis was designed. The assay was first validated on characterized macrolide-resistant M. genitalium isolates and then applied to 202 urogenital M. genitalium-positive specimens collected from 178 patients from France in 2011 and 2012. Resistant genotypes were confirmed by 23S rRNA gene sequencing. Among the 202 M. genitalium-positive specimens, 155 were amplified, demonstrating a sensitivity of 76.7%. A substitution in the 23S rRNA gene was found in 14.2% of the patient samples. Nine and six patients had M. genitalium isolates with a substitution at positions 2059 and 2058, respectively. In four cases, a mixed population of wild-type and mutated M. genitalium isolates was observed. The prevalence of M. genitalium macrolide resistance has been stable in France since its detection in 2006. Our FRET PCR assay is able to discriminate between wild-type and resistant genotypes directly from clinical specimens. This assay will allow clinicians to shorten the time to the initiation of effective disease treatment.     

The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 Genes Encode RecA Homologs That Promote Homologous DNA Strand Exchange

The P1, P40, and P90 proteins of Mycoplasma pneumoniae and the MgPa and P110 proteins of Mycoplasma genitalium are immunogenic adhesion proteins that display sequence variation. Consequently, these proteins are thought to play eminent roles in immune evasive strategies. For each of the five proteins, a similar underlying molecular mechanism for sequence variation was hypothesized, i.e., modification of the DNA sequences of their respective genes. This modification is thought to result from homologous recombination of parts of these genes with repeat elements (RepMp and MgPar elements in M. pneumoniae and M. genitalium, respectively) that are dispersed throughout the bacterial genome. Proteins that are potentially involved in homologous DNA recombination have been suggested to be implicated in recombination between these repeat elements and thereby in antigenic variation. To investigate this notion, we set out to study the function of the RecA homologs that are encoded by the M. pneumoniae MPN490 and M. genitalium MG339 genes. Both proteins, which are 79% identical on the amino acid level, were found to promote recombination between homologous DNA substrates in an ATP-dependent fashion. The recombinational activities of both proteins were Mg²⁺ and pH dependent and were strongly supported by the presence of single-stranded DNA binding protein, either from M. pneumoniae or from Escherichia coli. We conclude that the MPN490- and MG339-encoded proteins are RecA homologs that have the capacity to recombine homologous DNA substrates. Thus, they may play a central role in recombination between repetitive elements in both M. pneumoniae and M. genitalium.Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory infections, including atypical pneumonia. This bacterium causes up to 40% of all community-acquired pneumonias and approximately 18% of cases requiring hospitalization for children (for reviews, see references 1 and 35). The closest known relative of M. pneumoniae on the basis of sequence similarity is Mycoplasma genitalium, which is an etiological agent of various diseases of the human reproductive tract, such as urethritis (see reference 28 for a review).Although M. pneumoniae and M. genitalium represent the smallest self-replicating species regarding both cellular dimensions and genome size, it is interesting to note that a significant part of their genomes consists of repeated DNA elements. In M. pneumoniae strain M129 (6, 12), approximately 8% of the 816-kb genome is composed of variants of four different types of repetitive DNA elements (RepMP1, RepMP2/3, RepMP4, and RepMP5) (29, 34, 36), while in M. genitalium strain G-37^T, 4% of the 580-kb genome consists of MgPa repeats (or MgPar sequences) (10, 26, 27). Common features of the two types of repetitive elements are that (i) their representatives are similar but not identical in sequence and (ii) they are also contained in open reading frames (ORFs) encoding surface-exposed, antigenic proteins. Among these proteins is the M. pneumoniae P1 protein, which plays an essential role in bacterial adhesion to host cells (2). The ORF encoding the P1 protein, MPN141, contains both a RepMP4 element and a RepMP2/3 element. It has been hypothesized that homologous recombination between these RepMP elements with elements elsewhere in the genome could generate sequence changes within MPN141. These changes could subsequently lead to amino acid sequence variation of the antigenic P1 protein and thereby contribute to bacterial evasion of the host''s immune system. Strong evidence, albeit indirect, for recombination among RepMP sequences has come from the observation that all naturally occurring sequence variations within the MPN141 gene originate from RepMP2/3 and RepMP4 elements located at distant sites within the M. pneumoniae genome (32). In addition, several RepMP2/3 and RepMP4 elements outside of the MPN141 gene, as well as RepMP1 elements, appeared to have recombined in a number of strains (24, 32). In each of these cases, RepMP sequence information seemed to be copied from the donor site to the recipient site in a unidirectional fashion, which is indicative of a gene conversion-like mechanism of homologous DNA recombination (18, 32).In analogy, the M. genitalium MgPar elements are thought to provide a pool of sequence variation of the mgpB and mgpC genes (16). These genes encode the proteins MgPa and P110, respectively, which are antigenic proteins involved in host cell attachment (3, 7, 13). Interestingly, recombination between MgPar sequences in M. genitalium appeared to be mediated by reciprocal homologous DNA recombination events rather than by unidirectional gene conversion-like processes (15, 16).In order to elucidate the molecular mechanisms that underlie recombination between RepMP elements in M. pneumoniae, we previously initiated a study aimed at the identification of proteins that may be involved in DNA recombination in this bacterium (31). In the current study, we have focused on the putative enzymes from both M. pneumoniae and M. genitalium that may play central roles in homologous DNA recombination and DNA repair (for a review, see the work of Carvalho et al. [4]). These enzymes, which are encoded by the M. pneumoniae MPN490 ORF (12) and the M. genitalium MG339 ORF, show significant sequence similarity with RecA proteins from other organisms. Here we have shown that the proteins encoded by MPN490 and MG339 promote recombination between homologous DNA substrates and may therefore play a central role in recombination between RepMP and MgPar elements in M. pneumoniae and M. genitalium, respectively.     

Persistent Mycoplasma genitalium Infection of Human Endocervical Epithelial Cells Elicits Chronic Inflammatory Cytokine Secretion

Infection with Mycoplasma genitalium has been associated with male and female urogenital disease syndromes, including urethritis, cervicitis, pelvic inflammatory disease (PID), and tubal factor infertility. Basic investigations of mucosal cytotoxicity, microbial persistence, and host immune responses are imperative to understanding these inflammatory urogenital syndromes, particularly in females, considering the potential severity of upper tract infections. Here, we report that M. genitalium can establish long-term infection of human endocervical epithelial cells that results in chronic inflammatory cytokine secretion and increased responsiveness to secondary Toll-like receptor (TLR) stimulation. Using a novel quantitative PCR assay, M. genitalium was shown to replicate from 0 to 80 days postinoculation (p.i.), during which at most time points the median ratio of M. genitalium organisms to host cells was ≤10, indicating that low organism burdens are capable of eliciting chronic inflammation in endocervical epithelial cells. This observation is consistent with clinical findings in women. Persistently secreted cytokines predominately consisted of potent chemotactic and/or activating factors for phagocytes, including interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1β (MIP-1β). Despite persistent cytokine elaboration, no host cell cytotoxicity was observed except with superphysiologic loads of M. genitalium, suggesting that persistent infection occurs with minimal direct damage to the epithelium. However, it is hypothesized that chronic chemokine secretion with leukocyte trafficking to the epithelium could lead to significant inflammatory sequelae. Therefore, persistent M. genitalium infection could have important consequences for acquisition and/or pathogenesis of other sexually transmitted infections (STIs) and perhaps explain the positive associations between this organism and human immunodeficiency virus (HIV) shedding.     

Characterization of hypervariable region in hepatitis C virus envelope protein during acute and chronic infection

Summary. Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure.In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.     

Prévalence des infections à Chlamydia trachomatis, Neisseria gonorrhoeae et Mycoplasma genitalium chez des patients admis aux urgences

Study objective

To estimate the prevalence of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG) in patients under 31 years of age admitted to the emergency department of the University Hospital of Montpellier, for which a urinalysis was performed.

Patients and methods

CT, NG and MG specific real-time PCRs were performed in the urine samples from 301 patients between July 2010 and January 2011.

Results

CT DNA was detected in 11% of patients, NG DNA in 3.7% of patients and MG DNA in one patient. Seventy-five percent of male patients and only 13% of women were diagnosed with sexually transmitted infection (STI). No patient with leucocyturia below 10⁴/mL had a positive PCR result for one of the three bacteria. Of the patients with leucocyturia greater or equal to 10⁴/mL, CT was detected in 23.4% of men and 11% of women, NG in 19.2% of men and 1% of women, and MG in 2.1% of men.

Conclusion

The prevalence of NG and CT detection in our population was high while that of MG was low. The diagnosis was facilitated by the use of PCR on the urine sample although this sample is not recommended for the molecular detection of bacterial agents of STIs and may explain the low detection of MG. The study allowed diagnosing STIs in 14.3% of our patient population.     

Multidrug-resistant <Emphasis Type="Italic">Mycoplasma genitalium</Emphasis> infections in Europe

In Japan and Australia, multidrug-resistant Mycoplasma genitalium infections are reported with increasing frequency. Although macrolide-resistant M. genitalium strains are common in Europe and North America, fluoroquinolone-resistant strains are still exceptional. However, an increase of multidrug-resistant M. genitalium in Europe and America is to be expected. The aim of this paper is to increase awareness on the rising number of multidrug-resistant M. genitalium strains. Here, one of the first cases of infection with a genetically proven multidrug-resistant M. genitalium strain in Europe is described. The patient was a native Dutch 47-year-old male patient with urethritis. Mycoplasma genitalium was detected, but treatment failed with azithromycin, doxycycline and moxifloxacin. A urogenital sample was used to determine the sequence of the 23S rRNA, gyrA, gyrB and parC genes. The sample contained an A2059G single nucleotide polymorphism (SNP) in the 23S rRNA gene and an SNP in the parC gene, resulting in an amino acid change of Ser83 → Ile, explaining both azithromycin and moxifloxacin treatment failure. The SNPs associated with resistance were probably generated de novo, as a link with high-prevalence areas was not established. It is, thus, predictable that there is going to be an increase of multidrug-resistant M. genitalium strains in Europe. As treatment options for multidrug-resistant M. genitalium are limited, the treatment of M. genitalium infections needs to be carefully considered in order to limit the rapid increase of resistance to macrolides and fluoroquinolones.     

B-cell epitopes in hypervariable region 1 of hepatitis C virus obtained from patients with chronic persistent hepatitis

The hypervariable domain (HVR1) within the N-terminus of the E2 protein of hepatitis C virus (HCV) is known to be variable antigenically during the course of persistent infection. The aim of the study was to detect B-cell epitopes in HVR1 responsible for neutralizing HCV. The B-cell epitopes were analyzed using two series of synthetic peptides: 25 heptapeptides from the most common amino acids within 73 HVR1 sequences, and 216 heptapeptides, the sequences of which cover more than 65% of the 73 HVR1 sequences. Sera from three patients with chronic hepatitis C were tested for reactivity to the synthetic peptide sequences by enzyme-linked immunosorbent assay (ELISA). The post-interferon (IFN) serum of one patient who had a long-term response to treatment reacted specifically with 13 heptapeptides of 216 variable sequences of HVR1. Some of the amino acid sequences (amino acids 398, 399, 400, 404) of the heptapeptides were also found in those deduced from the nucleotide sequences of HCV genomes in the pre-IFN serum. The sera of the other two patients who did not respond to treatment did not react with the 13 heptapeptides. It is concluded that the B-cell epitopes in HVR1 may be relevant for eliminating viremia in the case of the patient who had a good response to treatment. These results suggest that the analysis of the B-cell epitopes recognized in HVR1 may be important in understanding the mechanism of persistent infection and progression of hepatitis. © 1996 Wiley-Liss, Inc.     

Stability of human cytomegalovirus genotypes in persistently infected renal transplant recipients

Although most genes are highly conserved among strains of human cytomegalovirus (HCMV), several are unusually variable. By analyzing the sequence of two variable genes (UL146 and UL74) amplified directly from whole blood DNA extracts, multiple HCMV strains were detected in blood samples from 5/11 virus-infected renal transplant patients. These five patients were seronegative prior to transplantation and their likely acquisition of the virus was via the donated organ; HCMV-positive immunocompetent donors may thus be capable of harboring and transmitting multiple virus genotypes. In sequential samples taken up to 140 days post transplant no mutations in either gene were detected from 9/10 patients, and in the remaining patient a single nucleotide change was detected in UL146, and none in UL74. All sequences grouped into previously defined genotypes, with the detection of multiple members of the UL74 group 5 genotype establishing this previously tentative genotype. Additionally, identical sequences were identified in viruses from different patients, including examples from geographically distinct regions. Thus, although UL146 and UL74 exhibit impressive variation among strains, their sequences are maintained stably within and between infected individuals, suggesting that sequence differences between genotypes may be driven by differing gene function.     

Molecular evidence of persistent echovirus 13 meningoencephalitis in a patient with relapsed lymphoma after an outbreak of meningitis in 2000

Enteroviral meningoencephalitis was diagnosed in a patient with an immunodeficiency syndrome acquired after treatment with rituximab for a relapsed primary B-cell lymphoma. A second meningoencephalitic episode was diagnosed 6 months later and was successfully treated with a combination of immunoglobulins and pleconaril. The infection was persistent since the enterovirus genome was detected in five sequential specimens of cerebrospinal fluid collected over 9 months. An echovirus 13 isolate was isolated in the first three samples. The viral sequence encoding the VP1 capsid protein of the three isolates was determined and was compared with that of four control viruses. The virus isolates recovered from the patient shared >99% nucleotide sequence similarity with one another. In a phylogenetic tree, they were directly related to a control virus obtained from a patient hospitalized in 2000 during an outbreak of enterovirus meningitis. The epidemiological origin of a chronic echovirus infection in a patient with immune deficiency suggests that the echovirus had been continuously circulating in the general population after the outbreak that had revealed its emergence.     

A triacylated lipoprotein from Mycoplasma genitalium activates NF-kappaB through Toll-like receptor 1 (TLR1) and TLR2

Mycoplasma genitalium is a sexually transmitted bacterial pathogen that causes nongonococcal chlamydia-negative urethritis, mucopurulent cervicitis, endometritis, pelvic inflammatory disease, and tubal factor infertility in humans. However, pathogenic agents that induce inflammatory responses have not been identified in M. genitalium. In this study, we examined the involvement of Toll-like receptors (TLRs) in activation of the immune response by a lipoprotein from M. genitalium and their active component responsible for NF-κB activation. The Triton X-114 detergent phase of M. genitalium was found to induce NF-κB through TLR2. The active component of the Triton X-114 detergent phase was a lipoprotein precursor, MG149. The activation of NF-κB by MG149 was inhibited by a dominant negative (DN) construct of TLR1 but not by a DN construct of TLR6. These results indicate that the activation of NF-κB by MG149 is dependent on TLR1 and TLR2. A synthetic lipopeptide derived from MG149 containing three acyl chains also induced NF-κB through TLR1 and TLR2. Thus, the results show that MG149, a triacylated lipoprotein from M. genitalium, activates NF-κB through TLR1 and TLR2.     

Mycoplasma genitalium Prevalence,Coinfection, and Macrolide Antibiotic Resistance Frequency in a Multicenter Clinical Study Cohort in the United States

The prevalence rates of Mycoplasma genitalium infections and coinfections with other sexually transmitted organisms and the frequency of a macrolide antibiotic resistance phenotype were determined in urogenital specimens collected from female and male subjects enrolled in a multicenter clinical study in the United States. Specimens from 946 subjects seeking care from seven geographically diverse clinical sites were tested for M. genitalium and for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. Sequencing was used to assess macrolide antibiotic resistance among M. genitalium-positive subjects. M. genitalium prevalence rates were 16.1% for females and 17.2% for males. Significant risk factors for M. genitalium infections were black race, younger age, non-Hispanic ethnicity, and female symptomatic status. Female M. genitalium infections were significantly more prevalent than C. trachomatis and N. gonorrhoeae infections, while the M. genitalium infection rate in males was significantly higher than the N. gonorrhoeae and T. vaginalis infection rates. The macrolide-resistant phenotype was found in 50.8% of females and 42% of males. These results show a high prevalence of M. genitalium single infections, a lower prevalence of coinfections with other sexually transmitted organisms, and high rates of macrolide antibiotic resistance in a diverse sample of subjects seeking care across a wide geographic area of the United States.     

Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli

Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion of gp45, indicating the highly conserved and immunodominant nature of these regions. Other gp90 segments, both from conserved and variable env sequences, displayed variable reactivities with the panel of equine sera. A panel of MAbs was also used in Western blot assays with the recombinant protein fragments for physical localization of previously identified MAb epitopes. The binding sites of two neutralizing MAbs were localized to a highly variable region of gp90, while nonneutralizing epitopes were localized to conserved and variable regions of the envelope glycoproteins. These results, in addition to localizing important antigenic sites on EIAV glycoproteins, indicate that previously defined conserved and variable env nucleotide sequences indeed encode protein sequences constituting conserved and variable immunogens during persistent infection by EIAV.     

Clinical Characteristics Associated with Mycoplasma genitalium among Female Sex Workers in Nairobi,Kenya

The prevalence of Mycoplasma genitalium is high in vulnerable populations of women in low-resource settings. However, the epidemiology of infection in these populations is not well established. To determine the prevalence of Mycoplasma genitalium and its association with cervical cytology and other correlates, we recruited 350 female sex workers (FSW) who were 18 to 50 years old in Nairobi, Kenya, for a cross-sectional study. A questionnaire was administered at baseline to obtain information on sociodemographics and sexual behaviors. Women underwent a pelvic exam, during which a physician collected cervical-exfoliation samples for conventional cytology and sexually transmitted infection (STI) testing. Samples were tested for M. genitalium and other STI organisms (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis) and the E6/E7 mRNA of human papillomavirus (HPV) by Aptima nucleic amplification assays. The prevalence of M. genitalium was 12.9%. FSW who engaged in sexual intercourse during menses were less likely to have M. genitalium infection than those who did not (odds ratio [OR], 0.3; 95% confidence interval [95% CI], 0.1, 0.9). M. genitalium was also less prevalent among FSW who had worked in prostitution for >5 years (6.2%) than among those who had worked for <3 years (17.6%) (OR, 0.3; 95% CI, 0.1, 0.8). FSW who reported more frequent condom use were more likely to be infected with M. genitalium than those who reported less frequent use (OR, 3.8; 95% CI, 1.2, 11.6). These correlates differ from those found in M. genitalium studies conducted with FSW from West Africa and China. Further longitudinal analyses assessing associations with persistent M. genitalium infection are needed.