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.     

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.     

Homologous regions shared by adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium

A lambda gt11 library of Mycoplasma genitalium genomic DNA was generated, and clones were identified using a pool of monoclonal antibodies directed against different epitopes of the 140 kDa adhesin protein. Because the 140 kDa protein of M. genitalium and the 170 kDa P1 adhesin of M. pneumoniae share biological properties such as a tip-associated location, cytadherence function and immunologic crossreactivity, we performed Southern blot analysis using these cloned partial 140 kDa gene fragments and 14 subclones that span the P1 structural gene of M. pneumoniae. Homologous regions of the two genes were identified.     

DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium.

DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 10(5) CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.     

Sequence divergency of the cytadhesin gene of Mycoplasma pneumoniae.

The Mycoplasma pneumoniae cytadhesin P1 genes from two groups of clinical isolates that display restriction fragment length polymorphisms were cloned and sequenced. Within each group the nucleotide sequences were identical, but two major differences were detected between the groups. These two stretches of sequence divergence were located in multiple-copy regions of the P1 gene and resulted in considerable amino acid changes.     

A Mycoplasma genitalium protein resembling the Mycoplasma pneumoniae attachment protein.

In previous studies with hyperimmune rabbit sera and monoclonal antibodies against the P1 protein of Mycoplasma pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of Mycoplasma genitalium. Because of biologic and morphologic similarities between these two human Mycoplasma species, attempts were made to characterize this cross-reacting protein of M. genitalium (designated MgPa). The protein was surface exposed and had an estimated molecular size of 140 kilodaltons. Electron microscopy with monoclonal antibodies produced against either MgPa or P1 demonstrated that MgPa is located over the surface of the terminal structure of M. genitalium which is covered by a nap layer. These immunologic and morphologic findings suggest that the MgPa protein of M. genitalium could be the counterpart of the P1 protein of M. pneumoniae.     

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.     

Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein

A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.     

Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae.

Mycoplasma pneumoniae cytadhesin P1 was purified by monoclonal antibody affinity chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal 18-amino-acid sequence of P1 was determined and used to design two synthetic oligonucleotides, a 14-mer corresponding to amino acids 1 to 5 and an 18-mer corresponding to amino acids 7 to 12. These oligonucleotides served as hybridization probes for the identification of the P1 gene by Southern blot analysis of M. pneumoniae DNA. The P1 gene was cloned into plasmid pUC19 and mapped by using appropriate restriction endonucleases. The DNA sequence of the entire P1 gene was determined by subcloning appropriate DNA fragments into bacteriophage M13 and sequencing the DNA by the dideoxy-chain-termination method. The P1 gene contains an open reading frame of 4,881 nucleotides coding for a protein of 1,627 amino acids with a calculated molecular weight of 176,288. Properties of the amino-terminal sequence suggest that protein P1 may be synthesized as a precursor with subsequent processing to a mature protein of a calculated molecular weight of 169,758. Potential antigenic sites were determined by hydrophilicity plots. A computer search revealed that part of the predicted P1 sequence is homologous to cytoskeletal keratin of mammalian species and human fibrinogen alpha chain precursor. These results demonstrate the uniqueness of P1 as a cytadhesin and virulence determinant.     

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.     

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

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

Regions of Mycoplasma pneumoniae cytadhesin P1 structural gene exist as multiple copies.

The Mycoplasma pneumoniae cytadhesin P1 structural gene with flanking regions was labeled by nick translation and used as a probe to analyze gene copy number in M. pneumoniae. Multiple bands of genomic DNA were hybridized by the probe. To establish what part of the P1 gene existed as multiple copies, the P1 gene and regions adjacent to the 3' and 5' ends were divided with restriction enzymes into 14 segments ranging in size from 174 to 651 base pairs. These pieces were purified on agarose gels, subcloned into pUC19, purified, labeled by nick translation, and used to probe the entire M. pneumoniae genome. Several regions near the middle and carboxy end of the P1 structural gene hybridized to single copies. The remaining P1 subclones hybridized to multiple bands under stringent hybridization conditions, indicating extensive homology with other parts of the M. pneumoniae genome. The single- versus multiple-copy nature of P1 structural gene domains is discussed.     

Mycoplasma gallisepticum species and strain-specific recombinant DNA probes

Genomic libraries of vaccine (F-K810) and wild type (S6) Mycoplasma gallisepticum were constructed in Escherichia coli (strain JM83) using the plasmid vector pUC8. Recombinant clones were screened by colony, dot and Southern hybridisations using 32P-labelled genomic DNA from M. gallisepticum strains K810 and S6. Eight clones were identified which contained DNA sequences specific to M. gallisepticum and one clone was identified which contained a DNA fragment unique to the vaccine strain (F-K810) of M. gallisepticum. When labelled and used as a probe in dot hybridisation assays, one of the M. gallisepticum species-specific recombinant plasmids differentiated standard reference cultures, atypical strains and wild type isolates of M. gallisepticum from other avian Mycoplasma species. In similar assays, the plasmid containing vaccine strain-specific sequences differentiated vaccine strains of M. gallisepticum from several other wild type strains of M. gallisepticum. Recombinant DNA probes provided sensitive and specific detection of M. gallisepticum strains and the vaccine-specific probe will be useful for determining if the vaccine strain can replace wild type M. gallisepticum in commercial layer facilities.     

Amino Acid Changes in Elongation Factor Tu of Mycoplasma pneumoniae and Mycoplasma genitalium Influence Fibronectin Binding

Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu_Mp) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu_Mg), in spite of sharing 96% identity with EF-Tu_Mp, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu_Mp that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu_Mg. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu_Mp. Synthetic peptides corresponding to this region of EF-Tu_Mp (EF-Tu_Mp 340-358) blocked both recombinant EF-Tu_Mp and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu_Mg 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.Many pathogens express surface proteins that facilitate colonization and cellular invasion (12, 39, 44, 49, 55). The human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma genitalium, have genome sizes of 816,394 bp (20) and 580,070 bp (12), respectively, with the latter considered the smallest self-replicating biological cell (14, 38). These bacterial pathogens possess terminal tip-like structures comprised of specific membrane adhesins and adherence-related accessory proteins that mediate surface parasitism of target cells (5) and are essential for virulence (4). While adherence of virulent M. pneumoniae is mediated primarily by tip organelle-associated adhesins (10, 24), the absence of these proteins in hemadsorption-negative mutants (HA⁻ class II mutants) (17) still permits detectable adherence (18), suggesting the involvement of alternative mechanisms by which mycoplasmas bind to host cells.Recently, we showed that M. pneumoniae surface-associated elongation factor Tu (EF-Tu_Mp; MPN665) and the pyruvate dehydrogenase E1 beta subunit (MPN392) interact with fibronectin (Fn) (11). In addition, we demonstrated that HA⁻ class II mutants also bind Fn through EF-Tu (11). Fn is an abundantly available pathogen target (22) that exists in soluble form in blood fluids and plasma and in fibrillar form in the extracellular matrix (56). M. pneumoniae could readily access the extracellular matrix through virulence-related determinants following epithelial cell damage (29) and could directly bind to subepithelial tissue targets through EF-Tu interactions with Fn. Furthermore, these distinct pathogenic pathways may also contribute to the ability of M. pneumoniae to invade and to establish intracellular and perinuclear residence (9, 57).Detailed analyses of EF-Tu_Mp-Fn interactions revealed the critical role of the carboxyl region of EF-Tu (amino acids 192 to 219 and 314 to 394) in Fn recognition (3). Other mycoplasmas with tip organelles, such as Mycoplasma penetrans and Mycoplasma gallisepticum, have been reported to bind Fn through a 65-kDa protein (13) and the PlpA and Hlp3 proteins (34).Following our initial findings of EF-Tu_Mp-Fn interactions, surface-associated EF-Tu proteins from other microorganisms, including Lactobacillus johnsonii, Listeria monocytogenes, and Pseudomonas aeruginosa, were reported to bind mucin (16), fibrinogen (43), plasminogen, and factor H (32). Since EF-Tu is one of the most highly conserved proteins in mycoplasmas, it has been used to create an EF-Tu sequence-based mycoplasma phylogeny tree. This allows the classification of the human pathogens, M. genitalium and M. pneumoniae, along with M. gallisepticum, a poultry pathogen, in the same group (28). M. pneumoniae is an established pathogen of the respiratory tract (54) but has also been isolated from the urogenital tract (15). M. genitalium, an emerging sexually transmitted disease pathogen (27, 51), has also been associated with respiratory (6) and joint (50) pathologies. It has been suggested that the tissue-specific tropisms and pathogenic mechanisms of these two mycoplasmas are determined by genetic distinctions between them (19). Most of the open reading frames proposed for M. genitalium are present in M. pneumoniae. Overall, M. pneumoniae and M. genitalium share 67.4% average identity at the amino acid level, while conserved housekeeping proteins exhibit 70 to 97% identity (19). Among the latter proteins, EF-Tu displays a high sequence identity (96%).In this study, we compared EF-Tu-Fn binding between M. pneumoniae and M. genitalium and discovered biological and biochemical differences that facilitated the identification of key amino acids responsible for these interactions. Such distinctions provide evidence of unique colonization capabilities of these bacteria.     

Spontaneous mutation results in loss of the cytadhesin (P1) of Mycoplasma pneumoniae.

The cytadhesin (P1) structural gene of a spontaneous mutant of Mycoplasma pneumoniae which displayed a P1-negative phenotype was analyzed. An extra adenine was discovered in a stretch of normally seven adenines near the N-terminal region of the mutant P1 structural gene. The frameshift mutation resulted in the early termination of protein translation. Possible causes of the mutation are discussed.     

Immunological cross-reactivity of a Mycoplasma pneumoniae membrane-associated protein antigen with Mycoplasma genitalium and Acholeplasma laidlawii.

A murine monoclonal antibody, OC2F5, reacts with a Mycoplasma pneumoniae antigen with an approximate Mr of 43,000. This antigen is trypsin and proteinase K sensitive and partitions in the detergent phase of a Triton X-114 solution. The monoclonal antibody cross-reacts with an antigen from both Mycoplasma genitalium and Acholeplasma laidlawii with a similar molecular weight. This cross-reactivity should be considered in the development of M. pneumoniae antigen detection systems based on the use of antibodies directed to this protein antigen.     

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.     

Duplex PCR to differentiate between Mycoplasma synoviae and Mycoplasma gallisepticum on the basis of conserved species-specific sequences of their hemagglutinin genes

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.