Search for the presence of six Mycoplasma species in peripheral blood mononuclear cells of subjects seropositive and seronegative for human immunodeficiency virus.

The prevalence of Mycoplasma fermentans, Mycoplasma pirum, Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma penetrans was investigated by using specific PCR assays with peripheral blood mononuclear cells from subjects infected or not infected with the human immunodeficiency virus (HIV). Only M. fermentans was detected in 5.8% of 154 HIV-seropositive and 11.1% of 90 HIV-seronegative subjects.     

Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival strategies of minimalist prokaryotes

Despite their very small genomes mycoplasmas are successful pathogens of man and a wide range of animal hosts. Because of the lack of effective therapeutics and vaccines, mycoplasma diseases continue to be a significant problem for public health as well as livestock production with major socio-economic consequences worldwide. Recent outbreaks and epidemiological studies predict that the incidence of human and animal mycoplasma diseases might increase which indicates the urgent need to develop new approaches for prevention and therapy. Development of such reagents, however, requires a solid understanding of the molecular biology of mycoplasma infections. Knowledge in this field has considerably increased during the past decade since new techniques have been developed and adapted to mycoplasmas that allow these organisms to be studied at the molecular level. Research on the two human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium of which the genome sequences have recently been completed as well as the substantial number of studies carried out on the AIDS-associated mycoplasmas, Mycoplasma penetrans and Mycoplasma fermentans, has led the way, but a number of animal mycoplasmas are becoming increasingly appreciated as models for the study of the molecular basis of mycoplasma diseases. This review summarizes and highlights some of the recent findings concerning the molecular interactions that occur between pathogenic mycoplasmas and their hosts, both the common strategies as well as some unique approaches evolved by particular mycoplasma pathogens, including adherence to and uptake into non-phagocytic host cells, as well as mechanisms of escaping the host immune system.     

Cross-hybridization between the cytadhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium and genomic DNA of Mycoplasma gallisepticum

Immunological cross-reactivity was observed between the cytadhesin proteins of Mycoplasma pneumoniae and Mycoplasma genitalium and a 155 kDa protein of Mycoplasma gallisepticum. Furthermore, the cytadhesin genes of M. pneumoniae and M. genitalium were used to demonstrate homology with M. gallisepticum genomic DNA under low stringency conditions suggesting that a family of adhesin-related genes exists among these pathogenic mycoplasmas.     

Comparison of host responses after intranasal infection of guinea-pigs with Mycoplasma genitalium or with Mycoplasma pneumoniae

Guinea-pigs were infected intranasally with Mycoplasma genitalium or Mycoplasma pneumoniae. The lung lesions produced by the two mycoplasmas were comparable in extent and histological pattern. Sera of both animal groups taken 2 weeks after infection reacted strongly in the complement fixation test with the M. pneumoniae glycolipid extract. In an ELISA using the respective adherence proteins (P1-protein of M. pneumoniae and MgPa of M. genitalium), strong specific activity, but also considerable cross-reactions were found. Epitope analysis by using overlapping octapeptides of a P1-region immunologically active in human M. pneumoniae infections and of the corresponding MgPa-region revealed six common epitopes but also one M. genitalium and two M. pneumoniae specific determinants. For analysis of a possible pathogenicity of M. genitalium in the human respiratory tract species-specific tests have to be developed.     

DNA and protein sequence homologies between the adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae.

Mycoplasma genitalium and Mycoplasma pneumoniae are morphologically and serologically related pathogens that colonize the human host. Their successful parasitism appears to be dependent on the product, an adhesin protein, of a gene that is carried by each of these mycoplasmas. Here we describe the cloning and determine the sequence of the structural gene for the putative adhesin of M. genitalium and compare its sequence to the counterpart P1 gene of M. pneumoniae. Regions of homology that were consistent with the observed serological cross-reactivity between these adhesins were detected at both DNA and protein levels. However, the degree of homology between these two genes and their products was much higher than anticipated. Interestingly, the A + T content of the M. genitalium adhesin gene was calculated as 60.1%, which is substantially higher tham that of the P1 gene (46.5%). Comparisons of codon usage between the two organisms revealed that M. genitalium preferentially used A- and T-rich codons. A total of 65% of positions 3 and 56% of positions 1 in M. genitalium codons were either A or T, whereas M. pneumoniae utilized A or T for positions 3 and 1 at a frequency of 40 and 47%, respectively. The biased choice of the A- and T-rich codons in M. genitalium could also account for the preferential use of A- and T-rich codons in conservative amino acid substitutions found in the M. genitalium adhesin. These facts suggest that M. genitalium might have evolved independently of other human mycoplasma species, including M. pneumoniae.     

Hemolytic and hemoxidative activities in Mycoplasma penetrans

Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H(2)O(2)-mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.     

Determination of infectious load of Mycoplasma genitalium in clinical samples of human vaginal cells

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.     

Mycoplasma penetrans and other mycoplasmas in urine of human immunodeficiency virus-positive children

Urine samples from children with human immunodeficiency virus (HIV) infection and healthy controls were examined for mycoplasmas by culture. Standard biochemical assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and PCR (16S and 16S-23S spacer rRNA region) were used for identification of isolates. Mycoplasmas were identified from 13 (87%) of 15 HIV-positive patients and 3 (20%) of 15 HIV-negative control patients. The frequency and type of mycoplasma varied with the severity of HIV infection. Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma fermentans, and Mycoplasma genitalium were isolated from patients with severe immunodeficiency. Mycoplasma hominis and Ureaplasma urealyticum were isolated more frequently from children in the early stages of HIV infection and from HIV-negative patients. Mycoplasma penetrans was isolated from one (50%) of two patients in Centers for Disease Control and Prevention (CDC) group B and from five (55.5%) of nine pediatric patients with AIDS (CDC group C). This is the first report that indicates that "AIDS-associated" mycoplasmas are more common in HIV-infected children than in HIV-negative controls.     

Failure of Mycoplasma pneumoniae infection to confer protection against Mycoplasma genitalium: observations from a mouse model

Mycoplasma pneumoniae and M. genitalium are genomically distinct but share antigens that induce some serological cross-reactivity. Therefore, the possibility that M. pneumoniae infection of the human respiratory tract might provide immunity to M. genitalium infection of the genital tract was considered. Because of the difficulty of assessing this proposition in man, it was evaluated experimentally in a mouse model. Female BALB/c mice were susceptible to infection of the vagina with M. pneumoniae, whereas those infected previously in the oropharynx with M. pneumoniae were completely immune to infection of the vagina with this mycoplasma. However, all mice with such a respiratory tract infection were susceptible to infection of the vagina with M. genitalium. The findings suggest that an M. pneumoniae infection of the human respiratory tract is unlikely to influence infection of the genital tract by M. genitalium.     

Amplified-Fragment Length Polymorphism Fingerprinting of Mycoplasma Species

Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneumoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5). AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and MfeI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp. The method was able to discriminate the analyzed strains at species and intraspecies levels as well. Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis. Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma species analyzed. The extent of polymorphism varied markedly between the analyzed mycoplasmas, comprising pattern similarity levels from 61.7% detected among M. dispar strains to 95.9% detected among M. genitalium strains. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of mycoplasmas.     

Antibiotic susceptibilities of AIDS-associated mycoplasmas.

Because mycoplasmas may be a cofactor in the progression of human immunodeficiency virus infection to AIDS, their susceptibilities to antibiotics need to be known in the event that appropriate therapy is required. The mycoplasmas studied were a stock culture strain of Mycoplasma fermentans, two strains of M. fermentans isolated from patients with AIDS, M. fermentans var. incognitus, Mycoplasma penetrans, and Mycoplasma pirum. The antibiotics tested were doxycycline, tetracycline, clindamycin, ofloxacin, erythromycin, azithromycin, and clarithromycin at levels consistent with the attainable levels in serum. By the macrodilution metabolic inhibition method, all six mycoplasma strains were susceptible to doxycycline, tetracycline, clindamycin, ofloxacin, azithromycin, and clarithromycin. M. penetrans was susceptible to erythromycin. The M. fermentans strains and M. pirum were resistant to erythromycin. The macrodilution metabolic inhibition method results showed agreement with the Sensititre Gram Positive MIC Panel results for tetracycline, clindamycin, and erythromycin. MICs of clarithromycin for all six mycoplasma isolates tested were low, indicating susceptibility.     

Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract.

Mycoplasma genitalium, an organism first isolated from the urethras of two men with nongonococcal urethritis, has been found in throat specimens from military recruits participating in an inactivated Mycoplasma pneumoniae vaccine field trial in 1974-1975. Four of 16 preserved throat isolates, previously identified as strains of M. pneumoniae, have now been shown to be mixtures of M. pneumoniae and M. genitalium. Purification of these mixed mycoplasmas by selection of single colonies confirmed the presence of M. genitalium. Identification of M. genitalium was based upon the occurrence of a species-specific 140-kilodalton protein adhesin in these isolates and their serologic reactivity to an M. genitalium antiserum. The frequent occurrence of both M. pneumoniae and M. genitalium in a number of these throat specimens, in combination with their shared antigenic cross-reactivities, suggests the likelihood that M. genitalium strains are easily missed in the usual laboratory identification procedures. What role M. genitalium may play in human respiratory disease remains to be determined.     

Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans.

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.     

Shared epitopes between Mycoplasma pneumoniae major adhesin protein P1 and a 140-kilodalton protein of Mycoplasma genitalium

Previous serological data have demonstrated cross-reactive antigens between two pathogenic species of mycoplasmas, M. pneumoniae and M. genitalium. Preliminary analysis of sera and monoclonal antibodies (MAbs) to protein antigens of these species showed an immunodominance of adhesin P1 (165 kilodaltons [kDa]) of M. pneumoniae in mice and hamsters and a 140-kDa protein of M. genitalium in mice and experimentally infected chimpanzees. To further characterize these two proteins, we assayed multiple anti-P1 and anti-140-kDa protein MAbs by enzyme-linked immunosorbent assay, immunoblot, and radioimmunoprecipitation techniques. The 140-kDa M. genitalium protein was shown to be surface accessible and insensitive to levels of trypsin which readily degrade protein P1. Peptide mapping was used to identify a unique class of MAbs which bound a cross-reactive molecule common to both the major adhesin protein P1 of M. pneumoniae and the 140-kDa protein of M. genitalium. MAbs generated against both M. pneumoniae and M. genitalium which were reactive with this determinant blocked M. pneumoniae attachment to chicken erythrocytes.     

Parasitism of hamster trachea epithelial cells by Mycoplasma pneumoniae.

Hamster trachea epithelial (HTE) cells were employed as a model system for Mycoplasma pneumoniae pathogenesis. To more closely mimic natural infection, M. pneumoniae was forced to rely upon host cells (as opposed to the growth medium) for nutrients, and infections were initiated with relatively low mycoplasma doses and monitored for extended time periods. A time- and dose-dependent decline in the viability of infected cells was observed; however, viability never declined below 50% of that in uninfected controls. Protein and RNA synthesis actually increased above control levels in infected cells, despite a concomitant decrease in viability. This response was pronounced at higher multiplicities of infection but was only transient at lower doses. In parallel studies in which a culture medium capable of supporting M. pneumoniae growth was used, loss of viability was accelerated. With a low-dose infection a transient increase followed by a precipitous decline in macromolecular synthesis was observed, relative to that in uninfected controls. At higher doses, however, macromolecular synthesis decreased dramatically and in proportion to the loss of viability. The requirement for HTE cells for mycoplasma growth under the experimental culture conditions was demonstrated by quantitating viable mycoplasmas in the culture medium in the presence or absence of HTE cells over 4 days. The increase in mycoplasma number was negligible in the absence of HTE cells, while a 30-fold increase was observed in the presence of HTE cells. These findings demonstrate the feasibility of long-term, low-dose studies of M. pneumoniae pathogenesis with trachea epithelial cells and a nonpermissive culture medium. This experimental system should facilitate the elucidation of the mechanism(s) responsible for host cell injury, and perhaps reveal how host cells respond to infection.     

Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.     

Regulation of proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1 beta mRNA also increased after infection and IL-1 beta protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytoadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level.     

Absence of Mycoplasma pneumoniae cytadsorption protein P1 in Mycoplasma genitalium and Mycoplasma gallisepticum

Polyclonal and monoclonal antibodies to Mycoplasma pneumoniae protein P1 were nonreactive with whole-cell or soluble preparations of M. genitalium and M. gallisepticum. However, radioimmunoprecipitation performed with hyperimmune rabbit sera raised against each mycoplasma species indicated antigenic cross-reactivity between M. pneumoniae and M. genitalium.     

Interaction of Mycoplasma pneumoniae with human lung fibroblasts: characterization of the in vitro model.

The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 37 degrees C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of less than or equal to 10(6) colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.     

Use of TaqMan 5' nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic

Mycoplasma genitalium is a cause of nongonococcal urethritis, particularly in patients not infected with Chlamydia trachomatis. A quantitative 5' nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from M. genitalium-positive men were examined, and the M. genitalium DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher M. genitalium DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.