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The evolving saga of Mycoplasma genitalium
Clinical Microbiology Newsletter Vol. 28, No. 6
March 15, 2006
The Evolving Saga of Mycoplasma genitalium Murry A. Stein, Ph.D. and Joel B. Baseman, Ph.D., University of Texas Health Science Center San Antonio, Department of Microbiology & Immunology, San Antonio, Texas
Abstract Mycoplasma genitalium has the smallest genome of any organism capable of independent growth. Sequence comparisons among completed bacterial genomes indicate that the 580-kb genome of M. genitalium arose by minimization of the 816-kb genome of the human respiratory pathogen Mycoplasma pneumoniae. Thus, the selective pressure that has led to genome minimization renders M. genitalium an ideal starting organism to define determinants essential for life. Further, the advent of molecular and serological diagnostic approaches has established M. genitalium as a significant cause of human infection, although its highly fastidious nature has made the study of its clinical significance and pathogenesis very challenging. As such, the virulence determinants that allow this minimal organism to establish human infections, circumvent host responses, and potentially contribute to chronic infections are just beginning to be elucidated. The following review will focus on recent advances in our understanding of the biology and pathogenesis of this smallest of all bacterial pathogens.
Basic Biology of M. genitalium and the Minimal-Genome Concept Mycoplasma genitalium is a descendent of gram-positive bacteria, most likely a clostridial ancestor, and is a member of the fermentative branch of the class Mollicutes. Like all mollicutes, this mycoplasma has a single cytoplasmic membrane and lacks a peptidoglycan cell wall. M. genitalium shares similarity with other mycoplasma pathogens by having a highly differentiated apical structure that is involved in adherence (1). This specialized tip with its core-like structure of electron-dense parallel tracts forms a neck-like extension from the main body of the mycoplasma (reviewed in reference 2). M. genitalium attaches to glass and plastic surfaces during medium cultiva-
Mailing address: Joel B. Baseman, Ph.D., University of Texas Health Science Center San Antonio, Department of Microbiology & Immunology, 7703 Floyd Curl Dr., San Antonio, Texas 78229-3900. Tel.: 210-567-3939. Fax: 210-567-6491. E-mail: [email protected] Clinical Microbiology Newsletter 28:6,2005
tion (1) and to epithelial cells in their natural environment, the human host (3,4). The property of clinical isolates of M. genitalium of adhering to red blood cells has been used to identify mutants deficient for adherence as well as factors that mediate this property (3,5). In addition, M. genitalium possesses homologues of all adherence, and adherence accessory proteins identified in the better-characterized M. pneumoniae attachment organelle (6). Since M. genitalium and M. pneumoniae are so closely related, inferences from studies that established the molecular basis for M. pneumoniae adherence (7) are commonly applied to M. genitalium. In both mycoplasmas, adherencerelated proteins are contained in three comparable operons (8). In this review, we first refer to the mycoplasma adherence-related factors using designations given by the original investigators and by the genetic loci assigned during annotation of the genome database (available at http:// www.tigr.org/tigrscripts/CMR2/ CMRHomePage.spl). Later in the text, we use gene annotation only. © 2006 Elsevier
The major determinant implicated as the M. genitalium adhesin is a 140-kDa surface membrane protein originally termed P140 or MgPa (3) (annotation, MG191). This adhesin is a homologue of the M. pneumoniae major adhesin P1 (annotation, MPN141). In addition, it is likely that the 110-kDa protein originally designated MgPc or P110 (annotation, MG192) serves as a cytadherence-associated protein, like the related M. pneumoniae Orf6 product (9) (annotation, MPN142). However, MPN142 is cleaved into two proteins of 90 and 40 kDa, but this processing does not occur with MG192 (3,9). Additionally, the 30-kDa adhesin (P30; annotation, MPN453) of M. pneumoniae has its homologue in the 32-kDa adhesin of M. genitalium (P32; annotation, MG318) (10). In a remarkable instance of prokaryotic differentiation and protein targeting, mycoplasma adhesins localize almost
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exclusively to the apical tip or neck region of the attachment organelle (11), and a variety of cytadherence accessory proteins are involved in assembly and stabilization of the apical tip (12). Events related to assembly of the attachment organelle require sequential proteinprotein interactions that are likely to involve coiled-coil domains. In addition, M. genitalium is mobile by virtue of a gliding motility that, based on studies with M. pneumoniae, is mediated by the attachment organelle (13). M. genitalium has evolved to live as a parasite of human tissues but has retained sufficient biochemical capability to permit growth in very rich media. M. genitalium directly obtains many essential components, including the cholesterol found in its membrane, from the host (reviewed in reference 14). The tricarboxylic acid (TCA) cycle is absent, as are cytochrome pigments. However, M. genitalium has retained a subset of enzymes involved in glycolysis and generates energy by substrate level phosphorylation of both glucose and fructose. These substrates are transported in the bacterium by group translocation via a phosphotransferase system. In addition, a flavin-terminated respiratory chain is also present, allowing oxidative phosphorylation, but the mechanism is not entirely understood (15). Evidence is also emerging that M. genitalium has evolved multifunctional enzymes that replace lost biochemical capabilities. For example, the absence of the TCA component malate dehydrogenase in M. genitalium is compensated for by the biochemical promiscuity of the lactate dehydrogenase enzyme (16). As detailed below, the expansion of functions by metabolic enzymes in M. genitalium has provided unexpected biochemical versatility related to host cell colonization. Also, potentially relevant to M. genitalium virulence, hydrogen peroxide is produced by metabolic processes, which may contribute to
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host cell damage (17). The genome of M. genitalium is strongly A-T biased, and the UGA codon, recognized as a transcriptional stop by non-mollicutes, is used as a codon for tryptophan (18). This feature presents an additional challenge to expressing recombinant mycoplasma proteins, as UGA codons must be changed to the universal UGG tryptophan codon. The M. genitalium genome is largely devoid of redundancy and is efficiently organized into operons with very limited intragenic sequences (19, 20). The few instances of functional redundancy are the presence of three genes of the DnaJ family of heat shock proteins and multiple, apparently nontranscribed fragments of the MG191 and MG192 genes (6). The M. genitalium genome is 1/10th the size of the largest prokaryotic genomes but shares similar coding density with other prokaryotes, about 1 gene per kb (21). Current M. genitalium genome annotations predict that 484 proteins and 39 RNA species are generated (22); about one-third of these products have been visualized by proteome studies. Mechanisms for potential transcriptional or translational regulation are largely unexplored, but proteome analysis provides evidence for growth-phaseregulated, differential protein production. Proteins that appear unregulated include the cytadherence accessory protein MG218 (9) and the protein chaperone GroEL (23). The identification of the M. genitalium genome as the smallest of all genomes allowing autonomous growth has also provided biologists with an ideal starting point to address the most fundamental question in biology: what are the essential components needed for life? To this end, by using the already minimized M. genitalium bacterium as a starting point, investigators combined biochemical and molecular approaches to determine the minimal number of
© 2006 Elsevier
genes that sustain life. One initial strategy used bioinformatics, based on comparisons between the genomes of the human bacterial pathogens Haemophilus influenzae (1,830 kb) and M. genitalium (580 kb). The identification of common orthologous proteins led to the proposal that 256 genes comprised the minimal set for independent growth (24). Further refinement of this approach was possible as more genomes became available (21). The second strategy took an experimental approach. A pool of M. genitalium transposon mutants was generated, and mycoplasma variants capable of growth in media were used to define non-essential genes. Specifically, those genes that could not be mutagenized were viewed as essential (25). Combination of the experimentally and theoretically derived minimal-genome strategies predicted that 338 proteincoding sequences comprised the minimal genome (21). Since conditions of mycoplasma growth in media are not necessarily reflective of the normal association between M. genitalium and host cells, it is unclear how many of the gene products that constitute the minimal genome might also be considered indispensable for in vivo colonization, growth, persistence, and virulence.
Disease and Diagnosis Two strains of M. genitalium were initially isolated by cultivation of urethral material from two men with nongonococcal urethritis (NGU) nearly 25 years ago (1). Urethritis is associated with penile irritation and the presence of elevated numbers of polymorphonuclear leukocytes in exudates. Currently, NGU is among the most common infectious diseases found in men who attend genitourinary clinics, and an estimated 2 million cases per year occur in the United States (26). Evidence for M. genitalium as a causative agent for NGU was provided when cultures of the initial mycoplasma
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isolates were used to fulfill Koch’s postulates in a primate model system (27). Chimpanzees challenged with these clinical strains by the urogenital route displayed clinical symptoms and pathological findings consistent with urethritis. Shedding of mycoplasmas was detected for several weeks after symptoms manifested, and a greater-thanfourfold seroconversion against M. genitalium was observed. In addition, evidence for hematogenous spread was obtained, raising the possibility of disseminated infections (27). Despite the initial identification of M. genitalium by cultivation from clinical specimens, isolation has proven to be an infrequent event. M. genitalium is a very slow-growing, highly fastidious organism that is cultivated axenically in serum-containing SP-4 medium, arguably among the richest of all bacterial culture media (1). The history of culture from clinical samples subsequent to the first isolation of this species follows. Five strains were cultured from extragenital sites (respiratory tract [28] and synovial fluid [29]). Eight strains were isolated from urogenital specimens from populations at high-risk for sexually transmitted infections (STI) in China (30), and most recently, M. genitalium was isolated from cervical specimens from 31 individual high-risk women (31). In the latter case, restriction fragment length polymorphisms in the gene encoding MG192 revealed DNA sequence divergences, establishing the usefulness of this gene in epidemiological typing of clinical strains (32). This recent success was partly attributed to the use of medium components selected for optimal growth of the initial M. genitalium isolates and inclusion of low levels of ciprofloxacin to prevent overgrowth by other microbes (31). The scope of the effort probably also contributed, as nearly 3,670 samples from 838 individual patients were cultivated. An alternative approach to axenic culture has also been used to isolate M. genitalium from clinical specimens. Blind passage by inoculation of Vero cells with samples from male NGU patients led to the isolation of four additional strains (33). Collectively, it is apparent that current cultivation approaches are not suitable methods to diagnose M. genitalium infection. The advent of PCR-based detection methods and seroconversion assays has Clinical Microbiology Newsletter 28:6,2005
greatly facilitated detection of M. genitalium in the clinical setting. Presently, several diagnostic methodologies are used at a limited number of research institutions, and the resultant data implicate M. genitalium as a leading human pathogen that requires broad screening efforts (34). Patient samples that are helpful for molecular identification of M. genitalium in a clinical setting include clean catch urine (35) and urogenital-swab samples (36). Recent studies have concluded that first-voided urine is the most appropriate starting material for molecular diagnosis of M. genitalium infections in males. However, both cervical swabs and urine should be analyzed from female patients. Freezing of samples was also found to moderately reduce the accuracy of diagnosis, as about one-fifth of formerly positive urine samples were negative after a freeze-thaw (37). PCR-based methods for M. genitalium most commonly employ specific oligonucleotides directed against either 16S rRNA (35) or unique portions of the MG191 adhesin (36). More recently, real-time PCR approaches have been introduced, which increase the speed of detection and reduce the number of required sample manipulations (38-41). A comparison between conventional PCR and real-time PCR indicates that the former shows slightly greater sensitivity, but the latter provides greater ease of screening and facilitates analysis of more samples (38). A recently described real-time PCR method provides the added advantage of an internal control for interfering substances, which commonly contribute to false negatives with urogenital samples (41). Finally, to facilitate the high-throughput screening needed in clinical settings, a microwell plate-based PCR assay has been devised (42). Detection of antibodies directed against the lipid-associated membrane protein (LAMP) fraction of M. genitalium has also proven suitable for clinical diagnosis. Low levels of antibodies that react against M. genitalium LAMP antigens are found in low-risk populations, and minimal cross-reactivity against other human mycoplasmal pathogens was observed (43). Serological screening by both ELISA format and immunoblotting has been performed. The latter method demonstrates that MG191 and © 2006 Elsevier
MG192 are major targets for antibody production, and reactivity to a 66-kDa protein is also commonly observed (31,44,45). Recently, a recombinant immunogen derived from a portion of MG191 was found to display the same immunoreactivity profiles as the bacterially derived adhesin (46) and to be devoid of cross-reactivity with M. pneumoniae-reactive sera. Numerous clinical studies have compared symptomatic male outpatients at STI clinics with asymptomatic controls. Collectively, these studies implicate M. genitalium as a major human pathogen accounting for 14 to 33% of all acute NGU in men (reviewed in reference 34). Infection by M. genitalium appears to be largely independent of Chlamydia trachomatis, another major cause of NGU (47). Evidence also indicates that infection of males by M. genitalium routinely results in symptomatic urethritis (73% of PCR-positive males), in contrast to C. trachomatis infections, in which over half of the infected males did not display clinical symptoms (48). Also, the M. genitalium infection rate in males in the 18- to 45-year age range is similar, while chlamydia infection is found more commonly in the youngest age groups examined (37). A frequent complication of NGU that occurs in 20 to 60% of all cases is the recurrence of the infection following completion of an appropriate course of tetracycline, erythromycin, or related drugs. While chlamydia is rarely found in recurrent NGU, mounting evidence points to M. genitalium persistence as a leading cause of this problem. Recent studies have raised the possibility that standard therapies used to treat patients with NGU may not be sufficient to eradicate M. genitalium. Some studies observed mycoplasma recurrence when erythromycin treatment is used (49), while others found that recurrence was associated only with tetracycline treatment but not with azithromycin therapy (50). Further investigation is warranted to determine if modification of NGU treatment could reduce the incidence of recurrent M. genitalium infections. Recent studies also provide evidence that M. genitalium is sexually transmitted, as nearly 70% of female partners of infected men were found to be infected with this mycoplasma (48). An increasing number of reports on female patients 0196-4399/00 (see frontmatter)
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support a role for M. genitalium in female urogenital maladies, but a clear picture of clinical symptomology and incidence is just emerging. There is convincing evidence that links M. genitalium to mucopurulent cervicitis (51,52), endometritis (53,54), pelvic inflammatory disease (54), and possibly tubal infection with sequelae in the form of ectopic pregnancy or tubal infertility (45). Given the caveat that STIs in women are commonly asymptomatic, studies of M. genitalium infection in women have not focused exclusively on symptomatic patients but have examined cross sections of attendees at STI clinics to ascertain the frequency of carriage. For example, a prospective study of patients attending STI clinics in Seattle reported an M. genitalium incidence of ~7% of attendees compared to ~11% for C. trachomatis infections (51), and M. genitalium-positive women displayed a threefold-higher risk for mucopurulent cervicitis. A report from Sweden found the incidence of M. genitalium infections in female STI clinic attendees to be ~6% compared to ~10% for C. trachomatis infections (52), and about 30% of women infected with M. genitalium exhibited symptoms. A recent study showed that infection in M. genitalium culture-positive women was independently associated with increased genitourinary symptoms (55). Clearly, additional studies are required to ascertain the association of M. genitalium with symptomatic and asymptomatic female infections. However, current evidence suggests that female populations are more predisposed toward asymptomatic carriage than males, as has been established for other major causes of NGU. As presented above, recent evidence strongly implicates M. genitalium as an important human sexually transmitted infectious agent that already approaches the incidence of leading STIs, such as C. trachomatis. Additionally, sporadic cases of M. genitalium at extragenital infection sites and the association of M. genitalium with sequelae have been reported (56). M. genitalium has been isolated along with M. pneumoniae in respiratory tract infections (28), alone in synovial fluid from arthritis patients (29,57), and from the brain stem of a child with encephalitis (58). Experimental verification of its pneumonia44
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Figure 1. Six representative serological patterns from M. genitalium culture-positive patients evaluated over multiple visits. Antibody levels to M. genitalium LAMP were determined by ELISA, and values for optical density at 404 nm above 0.28 were deemed to demonstrate seroconversion. The asterisks indicate the time of M. genitalium isolation. Peak antibody levels appeared at the time of mycoplasma isolation (A), 4 months after mycoplasma isolation (B), 3 months prior to mycoplasma isolation with a second peak at 18 months (C), and at the time of isolation with a second peak between 23 and 28 months (D). Patterns E and F show no obvious peak antibody titer throughout the 30-month period. This figure was previously presented (31) and is used with permission from ASM Press.
causing potential has been provided in the guinea pig model and showed that M. genitalium respiratory infections are pathologically very similar to M. pneumoniae infections (59). In the absence of routine screening for M. genitalium in settings other than STI clinics, it is unclear how common extra-genital infections need to be addressed. Mycoplasma infections have also been invoked as potential triggers for AIDS, and evidence has been presented that does suggest a role for M. genitalium as an independent risk factor in HIV infection progression (60). Finally, efforts to link mycoplasma infections to Gulf War illnesses have been unconvincing (61). Clearly, the ability to fully address the significance of M. genitalium as a human pathogen requires more widespread clinical awareness and suitable commercial tests. However, recent studies suggest that no single methodological approach may be sufficient to obtain the highest frequency of detection in female populations. Using 31 women who were productively infected as shown by positive M. genitalium cultures, the effectiveness of commonly used diagnostic tests was assessed. Interestingly, comparisons between the times at which culture-positive samples
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from women were obtained and peak serological reactivity to LAMP antigens revealed a range of host antibody responses to infection. As depicted in Fig. 1, these responses included peak reactivity that coincided with M. genitalium isolation (A and D), appeared several months after (B) or before (C) isolation, and failed to result despite a productive infection (E and F). These findings underscore the complex interactions seemingly occurring between M. genitalium and susceptible hosts and suggest that, in certain instances, the bacteria may delay or completely evade humoral responses. The diagnostic assessment of these culture-positive patients further demonstrated that neither PCR nor LAMP-based serological detection alone was suitable to identify infected individuals at the time when bacteria were cultivated (31). The analysis by both PCR and serological methods of multiple samples collected over repeated patient visits increased the incidence of detection. Additionally, it was found that the combination of a quantitative PCR assay that determined the infectious load in culture-positive women and a novel confocal immunoanalysis that detected M. genitalium in clinically derived vaginal cells was an effective diagnostic approach (62). As
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indicated above, the need to examine more than one clinical sample source from female patients is supported by a recent study in which M. genitalium was often detected only in two types of samples (urine and cervical swab) (37).
Pathogenic Mechanisms The exact mechanisms used by M. genitalium to cause inflammation and pathological changes associated with urethritis or extra-genital infections remain largely unknown. However, a number of recent insights into M. genitalium factors involved in colonization, resistance to host defenses, and potential strategies for immune evasion have emerged. Infection of the human host begins with colonization of mucosal surfaces. The best-characterized M. genitalium colonization mechanism is cytadherence mediated by the apical tip (5). The MG191 adhesin domain that facilitates host cell binding is located within the C-terminal portion. Antibodies raised against this C-terminal domain bind to intact MG191 and block adherence to epithelial cells (46). This binding domain shares limited identity with the corresponding region of the M. pneumoniae adhesin homologue and therefore may contribute to cellular tropism. The nature of the cellular receptor recognized by MG191 is not established, but studies on M. pneumoniae implicate the involvement of sialic acid residues (63,64). Therefore, it is possible that cellular carbohydrates also serve as M. genitalium receptors. The MG192 protein is also likely to play a role in cytadherence, since antibodies to surface-exposed portions of the protein partially block M. pneumoniae cytadherence (46). Therefore, it is possible that the immune response directed against these epitopes could serve to block M. genitalium cytadherence and permit clearance of the pathogen. Recently, M. genitalium has been shown to bind vaginal/cervical (v/c) mucin, and this interaction with a major component of uroepithelial surfaces is envisioned to play a role in host colonization (65). In a remarkable example of expanded functionality for metabolic enzymes, the three proteins implicated in mucin binding are all components of the glycolytic pathway. These proteins were identified as pyruvate dehydrogeClinical Microbiology Newsletter 28:6,2005
nase alpha and beta and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The contribution of GAPDH to mucin binding revealed that a subfraction (10%) of the total GAPDH cellular protein is surface associated, and anti-GAPDH antibodies blocked nearly 70% of M. genitalium binding to v/c mucin (65). These data suggest that M. genitalium has compensated for genome minimization by expanding the functions served by certain essential housekeeping genes to include colonization functions. The surface localization of metabolic enzymes and their involvement in colonization may be a common theme in human mycoplasma virulence. A subpopulation of M. pneumoniae elongation factor Tu and pyruvate dehydrogenase beta subunit were found to be surface localized and to mediate binding to fibronectin (66). Future studies to address how surface-associated metabolic enzymes contribute to a given tropism for urogenital mucosal surfaces are important considerations for investigation. One putative M. genitalium virulence factor that, in part, provides protection against oxidants associated with host innate immunity has been identified (67). The protein, methionine sulfoxide reductase A (MsrA), was deleted by allelic exchange, and the mutant exhibited increased sensitivity to hydrogen peroxide. This ∆msrA strain was markedly attenuated for infection in the hamster respiratory model. This observation demonstrates that the oxidantscavenging capacity of this enzyme contributes to both in vitro and in vivo oxidant resistance and compensates for the loss of catalase and superoxide dismutase enzymes. Surprisingly, the ∆msrA mutant also displayed reduced adherence to sheep erythrocytes. It was postulated that the loss of MsrA could prevent appropriate processing of MG191, which contains 13 methionine residues (67). M. genitalium has been observed to induce cytopathological changes in cultured cells via an ill-defined mechanism (4,68). A role for cellular damage by mycoplasma-generated hydrogen peroxide is postulated, since this oxidant contributes to the hemolytic activity of mycoplasmas (69). Other factors that are envisioned to contribute to the establishment of infections are stress responses, such as heat shock, involved © 2006 Elsevier
in adaptation to environmental changes encountered in the host. M. pneumoniae has been shown to regulate a variety of genes in response to heat shock (70), and similar proteins and regulatory elements are found in M. genitalium (71). The ability of M. genitalium to persist in infected individuals and recur after antibiotic treatment suggests that these mycoplasmas can evade the immune system. One possible strategy supported by several studies is that M. genitalium can enter and reside within host epithelial cells (4,72). Intracellular bacteria have an enduring ability to persist within host cells in the presence of antibiotics that selectively kill extracellular bacteria. An intriguing finding, potentially related to difficulties associated with culturing mycoplasma from clinical samples, is the inability to grow M. genitalium from persistently infected cultures despite microscopic and molecular evidence of its presence, viability, and apparent multiplication (73). Recent findings have provided in vivo evidence for the invasive potential of M. genitalium (62). Confocal analysis of vaginal epithelial cells from culture-positive women routinely found that most detectable bacteria were within epithelial cells (Fig. 2). Electron-microsopic analysis of these samples demonstrated intact mycoplasma plasma membranes consistent with viable M. genitalium (62). However, many intracellular mycoplasmas failed to exhibit the classic flask-shaped morphology, raising the possibility that a distinct mycoplasma cell differentiation program may be associated with persistence. Another potential contributor to immune evasion is the antigenic variation displayed by adhesin proteins. The precedent for sequence variation within mycoplasma adhesins in a clinical setting comes from analysis of the M. pneumoniae MPN141 adhesin protein, which is homologous to the MG191 product. Clinical M. pneumoniae isolates displayed one of two distinct MPN141 variants that differed from each other within repetitive regions of the genome; these regions represented partial, apparently untranscribed variants of the MPN141 gene (74). M. genitalium, despite the strong selective pressure to reduce its genome, has also retained repetitive genome regions that include partial variants of MG191 and 0196-4399/00 (see frontmatter)
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MG192 genes (19,75). Examination of the polymorphisms found within the MG191 genes from four M. genitalium strains isolated by blind passage demonstrated that these polymorphisms could be explained by recombination of the full-length gene with portions of the repeat sequences (76). Therefore, different MG191 types may emerge from among variants, which would facilitate circumvention of immune responses and promote entry into a protected niche. Also, as mentioned earlier, polymorphisms in the MG192 gene have been detected (32). Presently, the mechanism for gene conversion is not known and has not been observed for M. genitalium grown axenically or in cell culture systems. Other significant aspects of M. genitalium pathogenesis remain largely unexplored. The contributions of proinflammatory lipids (77) have not been addressed, and little is known about aspects of gene regulation that occur during pathogenesis. The signal transduction consequences of the intimate association between mycoplasmas and host membranes and mechanisms for cellular invasion also remain unknown.
Conclusions The increasing clarity of the significance of this pathogen as an emerging human health problem should drive efforts to understand its molecular pathogenesis. Tools to facilitate this effort are now available. M. genitalium is amenable to global gene inactivation by transposition and site-specific inactivation by allelic exchange (9,25). Unfortunately, animal models to parallel human infections are sub-optimal (27,59,78,79), and improved in vivo assessment of virulence determinants of M. genitalium is needed, especially in the identification of variants with reduced virulence potential. Finally, studies facilitated by gene array methodologies should facilitate exploration of potential regulatory events related to interactions between mycoplasmas and host tissues. The mechanisms that underlie the pathogenesis of this beguiling “little pathogen” hold the potential to be as remarkable as the minimized organism.
Acknowledgements Preparation of this review was supported by NIH grants U19 A145429-06 and RO1 AI041010-06A. We are grate46
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Figure 2. Sequential visualization of a representative clinically derived vaginal cell using confocal immunoanalysis. Both surface-associated and internalized immunolabeled M. genitalium cells are readily observed using 1-mm-diameter z-series optical sections (A to F). Mycoplasmas can be readily visualized as intense, discrete white fluorescing bodies, while the cell nucleus can be visualized using propidium iodide (B to E). This figure was previously presented (62) and is used with permission from ASM Press.
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29. Tully, J.G. et al. 1995. Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate. J. Clin. Microbiol. 33:1851-1855. 30. Luo, D. et al. 1999. Isolation and identification of Mycoplasma genitalium from high risk populations of sexually transmitted diseases in China. Chin. Med. J. 112:489-492. 31. Baseman, J.B. et al. 2004. Diagnostic assessment of Mycoplasma genitalium in culture-positive women. J. Clin. Microbiol. 42:203-211. 32. Musatovova, O.C. et al. 2006. Proximal region of gene encoding cytadherencerelated protein permits molecular typing of Mycoplasma genitalium clinical strains by PCR-restriction fragment length polymorphism. J. Clin. Microbiol. 44:598-603. 33. Jensen, J.S. et al. 1996. Isolation of Mycoplasma genitalium strains from the male urethra. J. Clin. Microbiol. 34:286-291. 34. Deguchi, T. and S. Maeda. 2002. Mycoplasma genitalium: another important pathogen of nongonococcal urethritis. J. Urol. 167:1210-1217. 35. Eastick, K. et al. 2003. A novel polymerase chain reaction assay to detect Mycoplasma genitalium. Mol. Pathol. 56:25-28. 36. Jensen, J.S. et al. 1991. Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples. J. Clin. Microbiol. 29:46-50. 37. Jensen, J.S. et al. 2004. Comparison of first void urine and urogenital swab specimens for detection of Mycoplasma genitalium and Chlamydia trachomatis by polymerase chain reaction in patients attending a sexually transmitted disease clinic. Sex. Transm. Dis. 31:499-507. 38. Jurstrand, M. et al. 2005. Detection of Mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay. J. Med. Microbiol. 54:23-29. 39. Yoshida, T. et al. 2002. Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR. J. Clin. Microbiol. 40:1451-1455. 40. Deguchi, T. et al. 2002. Longitudinal quantitative detection by real-time PCR of Mycoplasma genitalium in first-pass urine of men with recurrent nongonococcal urethritis. J. Clin. Microbiol. 40:3854-3856. 41. Jensen, J.S. et al. 2004. 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 sexu-
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55. Korte, J.E. et al. 2006. Cervicitis and genitourinary symptoms in women culture positive for Mycoplasma genitalium. Am. J. Reprod. Immun. In press. 56. Baseman, J.B., and J.G. Tully. 1997. Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. Emerg. Infect. Dis. 3:21-32. 57. Taylor-Robinson, D. et al. 1994. Mycoplasma genitalium in the joints of two patients with arthritis. Eur. J. Clin. Microbiol. Infect. Dis. 13:1066-1069. 58. Sakata, H. et al. 1993. Brain stem encephalitis due to Mycoplasma genitalium. Kansenshogaku Zasshi 67:500504. 59. Jacobs, E. et al. 1991. Comparison of host responses after intranasal infection of guinea-pigs with Mycoplasma genitalium or with Mycoplasma pneumoniae. Microb. Pathog. 10:221-229. 60. Perez, G. et al. 1998. Herpes simplex type II and Mycoplasma genitalium as risk factors for heterosexual HIV transmission: report from the heterosexual HIV transmission study. Int. J. Infect. Dis. 3:5-11. 61. Donta, S.T. et al. 2004. Benefits and harms of doxycycline treatment for Gulf War veterans’ illnesses: a randomized, double-blind, placebo-controlled trial. Ann. Intern. Med. 141:85-94. 62. Blaylock, M.W. et al. 2004. Determination of infectious load of Mycoplasma genitalium in clinical samples of human vaginal cells. J. Clin. Microbiol. 42:746752. 63. Chandler, D.K. et al. 1982. Mycoplasma pneumoniae attachment: competitive
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Clinical Microbiology Newsletter 28:6,2005
March 15, 2006
The Evolving Saga of Mycoplasma genitalium Murry A. Stein, Ph.D. and Joel B. Baseman, Ph.D., University of Texas Health Science Center San Antonio, Department of Microbiology & Immunology, San Antonio, Texas
Abstract Mycoplasma genitalium has the smallest genome of any organism capable of independent growth. Sequence comparisons among completed bacterial genomes indicate that the 580-kb genome of M. genitalium arose by minimization of the 816-kb genome of the human respiratory pathogen Mycoplasma pneumoniae. Thus, the selective pressure that has led to genome minimization renders M. genitalium an ideal starting organism to define determinants essential for life. Further, the advent of molecular and serological diagnostic approaches has established M. genitalium as a significant cause of human infection, although its highly fastidious nature has made the study of its clinical significance and pathogenesis very challenging. As such, the virulence determinants that allow this minimal organism to establish human infections, circumvent host responses, and potentially contribute to chronic infections are just beginning to be elucidated. The following review will focus on recent advances in our understanding of the biology and pathogenesis of this smallest of all bacterial pathogens.
Basic Biology of M. genitalium and the Minimal-Genome Concept Mycoplasma genitalium is a descendent of gram-positive bacteria, most likely a clostridial ancestor, and is a member of the fermentative branch of the class Mollicutes. Like all mollicutes, this mycoplasma has a single cytoplasmic membrane and lacks a peptidoglycan cell wall. M. genitalium shares similarity with other mycoplasma pathogens by having a highly differentiated apical structure that is involved in adherence (1). This specialized tip with its core-like structure of electron-dense parallel tracts forms a neck-like extension from the main body of the mycoplasma (reviewed in reference 2). M. genitalium attaches to glass and plastic surfaces during medium cultiva-
Mailing address: Joel B. Baseman, Ph.D., University of Texas Health Science Center San Antonio, Department of Microbiology & Immunology, 7703 Floyd Curl Dr., San Antonio, Texas 78229-3900. Tel.: 210-567-3939. Fax: 210-567-6491. E-mail: [email protected] Clinical Microbiology Newsletter 28:6,2005
tion (1) and to epithelial cells in their natural environment, the human host (3,4). The property of clinical isolates of M. genitalium of adhering to red blood cells has been used to identify mutants deficient for adherence as well as factors that mediate this property (3,5). In addition, M. genitalium possesses homologues of all adherence, and adherence accessory proteins identified in the better-characterized M. pneumoniae attachment organelle (6). Since M. genitalium and M. pneumoniae are so closely related, inferences from studies that established the molecular basis for M. pneumoniae adherence (7) are commonly applied to M. genitalium. In both mycoplasmas, adherencerelated proteins are contained in three comparable operons (8). In this review, we first refer to the mycoplasma adherence-related factors using designations given by the original investigators and by the genetic loci assigned during annotation of the genome database (available at http:// www.tigr.org/tigrscripts/CMR2/ CMRHomePage.spl). Later in the text, we use gene annotation only. © 2006 Elsevier
The major determinant implicated as the M. genitalium adhesin is a 140-kDa surface membrane protein originally termed P140 or MgPa (3) (annotation, MG191). This adhesin is a homologue of the M. pneumoniae major adhesin P1 (annotation, MPN141). In addition, it is likely that the 110-kDa protein originally designated MgPc or P110 (annotation, MG192) serves as a cytadherence-associated protein, like the related M. pneumoniae Orf6 product (9) (annotation, MPN142). However, MPN142 is cleaved into two proteins of 90 and 40 kDa, but this processing does not occur with MG192 (3,9). Additionally, the 30-kDa adhesin (P30; annotation, MPN453) of M. pneumoniae has its homologue in the 32-kDa adhesin of M. genitalium (P32; annotation, MG318) (10). In a remarkable instance of prokaryotic differentiation and protein targeting, mycoplasma adhesins localize almost
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exclusively to the apical tip or neck region of the attachment organelle (11), and a variety of cytadherence accessory proteins are involved in assembly and stabilization of the apical tip (12). Events related to assembly of the attachment organelle require sequential proteinprotein interactions that are likely to involve coiled-coil domains. In addition, M. genitalium is mobile by virtue of a gliding motility that, based on studies with M. pneumoniae, is mediated by the attachment organelle (13). M. genitalium has evolved to live as a parasite of human tissues but has retained sufficient biochemical capability to permit growth in very rich media. M. genitalium directly obtains many essential components, including the cholesterol found in its membrane, from the host (reviewed in reference 14). The tricarboxylic acid (TCA) cycle is absent, as are cytochrome pigments. However, M. genitalium has retained a subset of enzymes involved in glycolysis and generates energy by substrate level phosphorylation of both glucose and fructose. These substrates are transported in the bacterium by group translocation via a phosphotransferase system. In addition, a flavin-terminated respiratory chain is also present, allowing oxidative phosphorylation, but the mechanism is not entirely understood (15). Evidence is also emerging that M. genitalium has evolved multifunctional enzymes that replace lost biochemical capabilities. For example, the absence of the TCA component malate dehydrogenase in M. genitalium is compensated for by the biochemical promiscuity of the lactate dehydrogenase enzyme (16). As detailed below, the expansion of functions by metabolic enzymes in M. genitalium has provided unexpected biochemical versatility related to host cell colonization. Also, potentially relevant to M. genitalium virulence, hydrogen peroxide is produced by metabolic processes, which may contribute to
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host cell damage (17). The genome of M. genitalium is strongly A-T biased, and the UGA codon, recognized as a transcriptional stop by non-mollicutes, is used as a codon for tryptophan (18). This feature presents an additional challenge to expressing recombinant mycoplasma proteins, as UGA codons must be changed to the universal UGG tryptophan codon. The M. genitalium genome is largely devoid of redundancy and is efficiently organized into operons with very limited intragenic sequences (19, 20). The few instances of functional redundancy are the presence of three genes of the DnaJ family of heat shock proteins and multiple, apparently nontranscribed fragments of the MG191 and MG192 genes (6). The M. genitalium genome is 1/10th the size of the largest prokaryotic genomes but shares similar coding density with other prokaryotes, about 1 gene per kb (21). Current M. genitalium genome annotations predict that 484 proteins and 39 RNA species are generated (22); about one-third of these products have been visualized by proteome studies. Mechanisms for potential transcriptional or translational regulation are largely unexplored, but proteome analysis provides evidence for growth-phaseregulated, differential protein production. Proteins that appear unregulated include the cytadherence accessory protein MG218 (9) and the protein chaperone GroEL (23). The identification of the M. genitalium genome as the smallest of all genomes allowing autonomous growth has also provided biologists with an ideal starting point to address the most fundamental question in biology: what are the essential components needed for life? To this end, by using the already minimized M. genitalium bacterium as a starting point, investigators combined biochemical and molecular approaches to determine the minimal number of
© 2006 Elsevier
genes that sustain life. One initial strategy used bioinformatics, based on comparisons between the genomes of the human bacterial pathogens Haemophilus influenzae (1,830 kb) and M. genitalium (580 kb). The identification of common orthologous proteins led to the proposal that 256 genes comprised the minimal set for independent growth (24). Further refinement of this approach was possible as more genomes became available (21). The second strategy took an experimental approach. A pool of M. genitalium transposon mutants was generated, and mycoplasma variants capable of growth in media were used to define non-essential genes. Specifically, those genes that could not be mutagenized were viewed as essential (25). Combination of the experimentally and theoretically derived minimal-genome strategies predicted that 338 proteincoding sequences comprised the minimal genome (21). Since conditions of mycoplasma growth in media are not necessarily reflective of the normal association between M. genitalium and host cells, it is unclear how many of the gene products that constitute the minimal genome might also be considered indispensable for in vivo colonization, growth, persistence, and virulence.
Disease and Diagnosis Two strains of M. genitalium were initially isolated by cultivation of urethral material from two men with nongonococcal urethritis (NGU) nearly 25 years ago (1). Urethritis is associated with penile irritation and the presence of elevated numbers of polymorphonuclear leukocytes in exudates. Currently, NGU is among the most common infectious diseases found in men who attend genitourinary clinics, and an estimated 2 million cases per year occur in the United States (26). Evidence for M. genitalium as a causative agent for NGU was provided when cultures of the initial mycoplasma
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isolates were used to fulfill Koch’s postulates in a primate model system (27). Chimpanzees challenged with these clinical strains by the urogenital route displayed clinical symptoms and pathological findings consistent with urethritis. Shedding of mycoplasmas was detected for several weeks after symptoms manifested, and a greater-thanfourfold seroconversion against M. genitalium was observed. In addition, evidence for hematogenous spread was obtained, raising the possibility of disseminated infections (27). Despite the initial identification of M. genitalium by cultivation from clinical specimens, isolation has proven to be an infrequent event. M. genitalium is a very slow-growing, highly fastidious organism that is cultivated axenically in serum-containing SP-4 medium, arguably among the richest of all bacterial culture media (1). The history of culture from clinical samples subsequent to the first isolation of this species follows. Five strains were cultured from extragenital sites (respiratory tract [28] and synovial fluid [29]). Eight strains were isolated from urogenital specimens from populations at high-risk for sexually transmitted infections (STI) in China (30), and most recently, M. genitalium was isolated from cervical specimens from 31 individual high-risk women (31). In the latter case, restriction fragment length polymorphisms in the gene encoding MG192 revealed DNA sequence divergences, establishing the usefulness of this gene in epidemiological typing of clinical strains (32). This recent success was partly attributed to the use of medium components selected for optimal growth of the initial M. genitalium isolates and inclusion of low levels of ciprofloxacin to prevent overgrowth by other microbes (31). The scope of the effort probably also contributed, as nearly 3,670 samples from 838 individual patients were cultivated. An alternative approach to axenic culture has also been used to isolate M. genitalium from clinical specimens. Blind passage by inoculation of Vero cells with samples from male NGU patients led to the isolation of four additional strains (33). Collectively, it is apparent that current cultivation approaches are not suitable methods to diagnose M. genitalium infection. The advent of PCR-based detection methods and seroconversion assays has Clinical Microbiology Newsletter 28:6,2005
greatly facilitated detection of M. genitalium in the clinical setting. Presently, several diagnostic methodologies are used at a limited number of research institutions, and the resultant data implicate M. genitalium as a leading human pathogen that requires broad screening efforts (34). Patient samples that are helpful for molecular identification of M. genitalium in a clinical setting include clean catch urine (35) and urogenital-swab samples (36). Recent studies have concluded that first-voided urine is the most appropriate starting material for molecular diagnosis of M. genitalium infections in males. However, both cervical swabs and urine should be analyzed from female patients. Freezing of samples was also found to moderately reduce the accuracy of diagnosis, as about one-fifth of formerly positive urine samples were negative after a freeze-thaw (37). PCR-based methods for M. genitalium most commonly employ specific oligonucleotides directed against either 16S rRNA (35) or unique portions of the MG191 adhesin (36). More recently, real-time PCR approaches have been introduced, which increase the speed of detection and reduce the number of required sample manipulations (38-41). A comparison between conventional PCR and real-time PCR indicates that the former shows slightly greater sensitivity, but the latter provides greater ease of screening and facilitates analysis of more samples (38). A recently described real-time PCR method provides the added advantage of an internal control for interfering substances, which commonly contribute to false negatives with urogenital samples (41). Finally, to facilitate the high-throughput screening needed in clinical settings, a microwell plate-based PCR assay has been devised (42). Detection of antibodies directed against the lipid-associated membrane protein (LAMP) fraction of M. genitalium has also proven suitable for clinical diagnosis. Low levels of antibodies that react against M. genitalium LAMP antigens are found in low-risk populations, and minimal cross-reactivity against other human mycoplasmal pathogens was observed (43). Serological screening by both ELISA format and immunoblotting has been performed. The latter method demonstrates that MG191 and © 2006 Elsevier
MG192 are major targets for antibody production, and reactivity to a 66-kDa protein is also commonly observed (31,44,45). Recently, a recombinant immunogen derived from a portion of MG191 was found to display the same immunoreactivity profiles as the bacterially derived adhesin (46) and to be devoid of cross-reactivity with M. pneumoniae-reactive sera. Numerous clinical studies have compared symptomatic male outpatients at STI clinics with asymptomatic controls. Collectively, these studies implicate M. genitalium as a major human pathogen accounting for 14 to 33% of all acute NGU in men (reviewed in reference 34). Infection by M. genitalium appears to be largely independent of Chlamydia trachomatis, another major cause of NGU (47). Evidence also indicates that infection of males by M. genitalium routinely results in symptomatic urethritis (73% of PCR-positive males), in contrast to C. trachomatis infections, in which over half of the infected males did not display clinical symptoms (48). Also, the M. genitalium infection rate in males in the 18- to 45-year age range is similar, while chlamydia infection is found more commonly in the youngest age groups examined (37). A frequent complication of NGU that occurs in 20 to 60% of all cases is the recurrence of the infection following completion of an appropriate course of tetracycline, erythromycin, or related drugs. While chlamydia is rarely found in recurrent NGU, mounting evidence points to M. genitalium persistence as a leading cause of this problem. Recent studies have raised the possibility that standard therapies used to treat patients with NGU may not be sufficient to eradicate M. genitalium. Some studies observed mycoplasma recurrence when erythromycin treatment is used (49), while others found that recurrence was associated only with tetracycline treatment but not with azithromycin therapy (50). Further investigation is warranted to determine if modification of NGU treatment could reduce the incidence of recurrent M. genitalium infections. Recent studies also provide evidence that M. genitalium is sexually transmitted, as nearly 70% of female partners of infected men were found to be infected with this mycoplasma (48). An increasing number of reports on female patients 0196-4399/00 (see frontmatter)
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support a role for M. genitalium in female urogenital maladies, but a clear picture of clinical symptomology and incidence is just emerging. There is convincing evidence that links M. genitalium to mucopurulent cervicitis (51,52), endometritis (53,54), pelvic inflammatory disease (54), and possibly tubal infection with sequelae in the form of ectopic pregnancy or tubal infertility (45). Given the caveat that STIs in women are commonly asymptomatic, studies of M. genitalium infection in women have not focused exclusively on symptomatic patients but have examined cross sections of attendees at STI clinics to ascertain the frequency of carriage. For example, a prospective study of patients attending STI clinics in Seattle reported an M. genitalium incidence of ~7% of attendees compared to ~11% for C. trachomatis infections (51), and M. genitalium-positive women displayed a threefold-higher risk for mucopurulent cervicitis. A report from Sweden found the incidence of M. genitalium infections in female STI clinic attendees to be ~6% compared to ~10% for C. trachomatis infections (52), and about 30% of women infected with M. genitalium exhibited symptoms. A recent study showed that infection in M. genitalium culture-positive women was independently associated with increased genitourinary symptoms (55). Clearly, additional studies are required to ascertain the association of M. genitalium with symptomatic and asymptomatic female infections. However, current evidence suggests that female populations are more predisposed toward asymptomatic carriage than males, as has been established for other major causes of NGU. As presented above, recent evidence strongly implicates M. genitalium as an important human sexually transmitted infectious agent that already approaches the incidence of leading STIs, such as C. trachomatis. Additionally, sporadic cases of M. genitalium at extragenital infection sites and the association of M. genitalium with sequelae have been reported (56). M. genitalium has been isolated along with M. pneumoniae in respiratory tract infections (28), alone in synovial fluid from arthritis patients (29,57), and from the brain stem of a child with encephalitis (58). Experimental verification of its pneumonia44
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Figure 1. Six representative serological patterns from M. genitalium culture-positive patients evaluated over multiple visits. Antibody levels to M. genitalium LAMP were determined by ELISA, and values for optical density at 404 nm above 0.28 were deemed to demonstrate seroconversion. The asterisks indicate the time of M. genitalium isolation. Peak antibody levels appeared at the time of mycoplasma isolation (A), 4 months after mycoplasma isolation (B), 3 months prior to mycoplasma isolation with a second peak at 18 months (C), and at the time of isolation with a second peak between 23 and 28 months (D). Patterns E and F show no obvious peak antibody titer throughout the 30-month period. This figure was previously presented (31) and is used with permission from ASM Press.
causing potential has been provided in the guinea pig model and showed that M. genitalium respiratory infections are pathologically very similar to M. pneumoniae infections (59). In the absence of routine screening for M. genitalium in settings other than STI clinics, it is unclear how common extra-genital infections need to be addressed. Mycoplasma infections have also been invoked as potential triggers for AIDS, and evidence has been presented that does suggest a role for M. genitalium as an independent risk factor in HIV infection progression (60). Finally, efforts to link mycoplasma infections to Gulf War illnesses have been unconvincing (61). Clearly, the ability to fully address the significance of M. genitalium as a human pathogen requires more widespread clinical awareness and suitable commercial tests. However, recent studies suggest that no single methodological approach may be sufficient to obtain the highest frequency of detection in female populations. Using 31 women who were productively infected as shown by positive M. genitalium cultures, the effectiveness of commonly used diagnostic tests was assessed. Interestingly, comparisons between the times at which culture-positive samples
© 2006 Elsevier
from women were obtained and peak serological reactivity to LAMP antigens revealed a range of host antibody responses to infection. As depicted in Fig. 1, these responses included peak reactivity that coincided with M. genitalium isolation (A and D), appeared several months after (B) or before (C) isolation, and failed to result despite a productive infection (E and F). These findings underscore the complex interactions seemingly occurring between M. genitalium and susceptible hosts and suggest that, in certain instances, the bacteria may delay or completely evade humoral responses. The diagnostic assessment of these culture-positive patients further demonstrated that neither PCR nor LAMP-based serological detection alone was suitable to identify infected individuals at the time when bacteria were cultivated (31). The analysis by both PCR and serological methods of multiple samples collected over repeated patient visits increased the incidence of detection. Additionally, it was found that the combination of a quantitative PCR assay that determined the infectious load in culture-positive women and a novel confocal immunoanalysis that detected M. genitalium in clinically derived vaginal cells was an effective diagnostic approach (62). As
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indicated above, the need to examine more than one clinical sample source from female patients is supported by a recent study in which M. genitalium was often detected only in two types of samples (urine and cervical swab) (37).
Pathogenic Mechanisms The exact mechanisms used by M. genitalium to cause inflammation and pathological changes associated with urethritis or extra-genital infections remain largely unknown. However, a number of recent insights into M. genitalium factors involved in colonization, resistance to host defenses, and potential strategies for immune evasion have emerged. Infection of the human host begins with colonization of mucosal surfaces. The best-characterized M. genitalium colonization mechanism is cytadherence mediated by the apical tip (5). The MG191 adhesin domain that facilitates host cell binding is located within the C-terminal portion. Antibodies raised against this C-terminal domain bind to intact MG191 and block adherence to epithelial cells (46). This binding domain shares limited identity with the corresponding region of the M. pneumoniae adhesin homologue and therefore may contribute to cellular tropism. The nature of the cellular receptor recognized by MG191 is not established, but studies on M. pneumoniae implicate the involvement of sialic acid residues (63,64). Therefore, it is possible that cellular carbohydrates also serve as M. genitalium receptors. The MG192 protein is also likely to play a role in cytadherence, since antibodies to surface-exposed portions of the protein partially block M. pneumoniae cytadherence (46). Therefore, it is possible that the immune response directed against these epitopes could serve to block M. genitalium cytadherence and permit clearance of the pathogen. Recently, M. genitalium has been shown to bind vaginal/cervical (v/c) mucin, and this interaction with a major component of uroepithelial surfaces is envisioned to play a role in host colonization (65). In a remarkable example of expanded functionality for metabolic enzymes, the three proteins implicated in mucin binding are all components of the glycolytic pathway. These proteins were identified as pyruvate dehydrogeClinical Microbiology Newsletter 28:6,2005
nase alpha and beta and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The contribution of GAPDH to mucin binding revealed that a subfraction (10%) of the total GAPDH cellular protein is surface associated, and anti-GAPDH antibodies blocked nearly 70% of M. genitalium binding to v/c mucin (65). These data suggest that M. genitalium has compensated for genome minimization by expanding the functions served by certain essential housekeeping genes to include colonization functions. The surface localization of metabolic enzymes and their involvement in colonization may be a common theme in human mycoplasma virulence. A subpopulation of M. pneumoniae elongation factor Tu and pyruvate dehydrogenase beta subunit were found to be surface localized and to mediate binding to fibronectin (66). Future studies to address how surface-associated metabolic enzymes contribute to a given tropism for urogenital mucosal surfaces are important considerations for investigation. One putative M. genitalium virulence factor that, in part, provides protection against oxidants associated with host innate immunity has been identified (67). The protein, methionine sulfoxide reductase A (MsrA), was deleted by allelic exchange, and the mutant exhibited increased sensitivity to hydrogen peroxide. This ∆msrA strain was markedly attenuated for infection in the hamster respiratory model. This observation demonstrates that the oxidantscavenging capacity of this enzyme contributes to both in vitro and in vivo oxidant resistance and compensates for the loss of catalase and superoxide dismutase enzymes. Surprisingly, the ∆msrA mutant also displayed reduced adherence to sheep erythrocytes. It was postulated that the loss of MsrA could prevent appropriate processing of MG191, which contains 13 methionine residues (67). M. genitalium has been observed to induce cytopathological changes in cultured cells via an ill-defined mechanism (4,68). A role for cellular damage by mycoplasma-generated hydrogen peroxide is postulated, since this oxidant contributes to the hemolytic activity of mycoplasmas (69). Other factors that are envisioned to contribute to the establishment of infections are stress responses, such as heat shock, involved © 2006 Elsevier
in adaptation to environmental changes encountered in the host. M. pneumoniae has been shown to regulate a variety of genes in response to heat shock (70), and similar proteins and regulatory elements are found in M. genitalium (71). The ability of M. genitalium to persist in infected individuals and recur after antibiotic treatment suggests that these mycoplasmas can evade the immune system. One possible strategy supported by several studies is that M. genitalium can enter and reside within host epithelial cells (4,72). Intracellular bacteria have an enduring ability to persist within host cells in the presence of antibiotics that selectively kill extracellular bacteria. An intriguing finding, potentially related to difficulties associated with culturing mycoplasma from clinical samples, is the inability to grow M. genitalium from persistently infected cultures despite microscopic and molecular evidence of its presence, viability, and apparent multiplication (73). Recent findings have provided in vivo evidence for the invasive potential of M. genitalium (62). Confocal analysis of vaginal epithelial cells from culture-positive women routinely found that most detectable bacteria were within epithelial cells (Fig. 2). Electron-microsopic analysis of these samples demonstrated intact mycoplasma plasma membranes consistent with viable M. genitalium (62). However, many intracellular mycoplasmas failed to exhibit the classic flask-shaped morphology, raising the possibility that a distinct mycoplasma cell differentiation program may be associated with persistence. Another potential contributor to immune evasion is the antigenic variation displayed by adhesin proteins. The precedent for sequence variation within mycoplasma adhesins in a clinical setting comes from analysis of the M. pneumoniae MPN141 adhesin protein, which is homologous to the MG191 product. Clinical M. pneumoniae isolates displayed one of two distinct MPN141 variants that differed from each other within repetitive regions of the genome; these regions represented partial, apparently untranscribed variants of the MPN141 gene (74). M. genitalium, despite the strong selective pressure to reduce its genome, has also retained repetitive genome regions that include partial variants of MG191 and 0196-4399/00 (see frontmatter)
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MG192 genes (19,75). Examination of the polymorphisms found within the MG191 genes from four M. genitalium strains isolated by blind passage demonstrated that these polymorphisms could be explained by recombination of the full-length gene with portions of the repeat sequences (76). Therefore, different MG191 types may emerge from among variants, which would facilitate circumvention of immune responses and promote entry into a protected niche. Also, as mentioned earlier, polymorphisms in the MG192 gene have been detected (32). Presently, the mechanism for gene conversion is not known and has not been observed for M. genitalium grown axenically or in cell culture systems. Other significant aspects of M. genitalium pathogenesis remain largely unexplored. The contributions of proinflammatory lipids (77) have not been addressed, and little is known about aspects of gene regulation that occur during pathogenesis. The signal transduction consequences of the intimate association between mycoplasmas and host membranes and mechanisms for cellular invasion also remain unknown.
Conclusions The increasing clarity of the significance of this pathogen as an emerging human health problem should drive efforts to understand its molecular pathogenesis. Tools to facilitate this effort are now available. M. genitalium is amenable to global gene inactivation by transposition and site-specific inactivation by allelic exchange (9,25). Unfortunately, animal models to parallel human infections are sub-optimal (27,59,78,79), and improved in vivo assessment of virulence determinants of M. genitalium is needed, especially in the identification of variants with reduced virulence potential. Finally, studies facilitated by gene array methodologies should facilitate exploration of potential regulatory events related to interactions between mycoplasmas and host tissues. The mechanisms that underlie the pathogenesis of this beguiling “little pathogen” hold the potential to be as remarkable as the minimized organism.
Acknowledgements Preparation of this review was supported by NIH grants U19 A145429-06 and RO1 AI041010-06A. We are grate46
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Figure 2. Sequential visualization of a representative clinically derived vaginal cell using confocal immunoanalysis. Both surface-associated and internalized immunolabeled M. genitalium cells are readily observed using 1-mm-diameter z-series optical sections (A to F). Mycoplasmas can be readily visualized as intense, discrete white fluorescing bodies, while the cell nucleus can be visualized using propidium iodide (B to E). This figure was previously presented (62) and is used with permission from ASM Press.
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