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Bioscience Reports, Vol. 19, No. 5, 1999 MINI REVIEW Adherence of Pathogenic Mycoplasmas to Host Cells Shmuel Razin 1 The significant genome compaction in mycoplasmas was made possible by adoption of a parasitic lifestyle. During their evolution and adaptation to a parasitic mode of life the mycoplasmas have developed various genetic systems enabling their attachment to host tissues as well as a highly plastic set of variable surface proteins. The generation of a versatile surface coat through high-frequency phase and size variation provides the organism with a useful tool for immune system avoidance, allowing the mycoplasmas to escape antibody attack, explaining why these minute organisms are such successful parasites. KEY WORDS: Mycoplasmas; adherence; membrane receptors; adhesins; Mycoplasma pneumoniae; Mycoplasma gallisepticum. ABBREVIATIONS: HMW, High-Molecular Weight proteins; P1, major adhesin of Mycoplasma pneumoniae; MgPa, major adhesin of Mycoplasma genitalium. Mycoplasmas comprise a very large group of prokaryotes widely distributed in nature as parasites and pathogens of humans, animals, plants and insects. There are no free-living mycoplasmas (Razin, 1992). The mycoplasmas have a special appeal for studies of cell biology. They are not only the smallest self-replicating organisms, but, they are also the simplest in ultrastructure. The mycoplasma cell is built of a minimum set of organelles: a plasma membrane, ribosomes and a highly coiled circular double-stranded DNA molecule, the typical prokaryotic genome. The M. genitalium genome is the smallest prokaryotic genome. The number of coding regions, the ORFs for proteins, in the M. genitalium genome is only 479, and 677 in the closely related M. pneumoniae, compared to 1703 in H. influenzae, 4288 in E. coli, 4100 in B. subtilis and 5883 in the unicellular eukaryote Saccharomyces cerevisiae (Razin et al., 1998). The currently dominating hypothesis is that mycoplasmas evolved by degenerate or reductive evolution from Gram-positive bacteria with genomes of low guanine + cytosine content (Woese, 1987). The significant genome compaction that occurred during mycoplasma evolution from their common bacterial ancestor was made possible by adoption of a parasitic mode of life. The supply of many nutrients from their hosts facilitated the loss, during evolution, of the genes for many 1 Department of Membrane and Ultrastructure Research, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. E-mail: [email protected] 367 0144-8463/99/1000-0367$ 16.00/0 c 1999 Plenum Publishing Corporation 368 Razin assimilative processes (Fraser et al., 1995; Himmelreich et al., 1996, 1997; Razin, 1997; Razin et al., 1998). Mycoplasmas are either commensals or cause chronic, generally mild infections, rarely killing their host. In this respect, mycoplasmas may be close to the concept of ideal parasites. The mycoplasmas are usually surface parasites, adhering to and colonizing the epithelial linings of the respiratory and urogenital tracts, rarely invading tissues. M. pneumoniae is a well-established pathogen of the human respiratory tract, causing pneumonia mostly in children and young adults, while M. genitalium may cause nongonococcal urethritis in men, and intrauterine infections of the human fetus. The dominating view for a long time was that mycoplasmas cannot penetrate into eukaryotic cells. While this basic concept still holds, recent findings triggered by the intensive research on the possible role of mycoplasmas as cofactors in AIDS activation, showed mycoplasmas to be located also intracellularly. This feature is particularly prominent in a new human mycoplasma isolated from AIDS patients, and named accordingly M. penetrans (Lo et al., 1993). Penetration of these mycoplasmas into the cells is directed through a tip organelle, differing in shape from the rest of the flask-shaped cell. The intracellular location of even a minor fraction of the mycoplasmal population in the host may contribute to resistance of mycoplasmas towards the immune system, as well as resisting antibiotic treatment. These are factors which may explain chronicity of mycoplasmal infections and the difficulties in eradicating completely mycoplasmas from infected tissue and cell cultures. Current extensive research on the modulation of the immune system by mycoplasmas appears to point out that the pathogenic manifestations of mycoplasma infections are in fact the outcome of the host immune reactions mediated by the production of various cytokines induced by the mycoplasmas, including the proinflammatory cytokines TNF-a, IL-1, and IL-6 (Razin et al., 1998). The lack of a cell wall in mycoplasmas may facilitate the direct contact of the mycoplasma membrane with that of its eukaryotic host, creating a condition which, in principle, could lead to fusion of the two membranes, enabling the transfer or exchange of membrane components, and injection of the mycoplasmal cytoplasmic content, including hydrolytic enzymes, into the host cell cytoplasm. Thus, the potent nucleases of mycoplasmas combined with superoxide radicals may be responsible for clastogenic effects and chromosomal aberrations observed in eukaryotic cells infected by mycoplasmas. These observations are in line with recent claims that the AIDS-associated mycoplasmas M. penetrans and M. fermentans exhibit an oncogenic potential, as reflected by malignant cell transformation following persistent infection of cultured mouse embryo cells with these mycoplasmas (Tsai et al., 1995). Adhesion of mycoplasmas to their target tissue is a prerequisite for colonization by the parasite and for infection. The loss of adhesion capacity by mutation results in loss of infectivity, while reversion to the cytadhering type is accompanied by regaining infectivity and virulence. There is no wonder, therefore, that a significant percentage of genes in the minute mycoplasmal genomes is devoted to adhesion. Some of these genes code for membrane proteins, exposed at least partially on the membrane surface, acting as adhesins. Both M. pneumoniae and M. genitalium and Mycoplasma Adherence 369 several other mycoplasmas have developed a special organelle at the tip of the elongated flask-shaped cell on the surface of which there is a high concentration of adhesin molecules. These mycoplasmas attach to the eukaryotic cells via this tip structure (Razin and Jacobs, 1992; Krause, 1996, 1998). Thus, this tip organelle serves as an attachment organelle. It is bounded by the cell membrane and contains a central rod-like structure. Treatment of the mycoplasmas with 1% Triton X-100 solubilizes the membrane, releasing the rod and a network of filaments associated with it. Proteins of this so-called “Triton shell” are apparently cytoskeleton-forming or cytoskeleton-associated proteins. The cytoskeleton-like structure is thought to function in modulating the peculiar flask shape of the wall-less cells, which would otherwise assume a spherical shape. The genomic analysis of M. pneumoniae has enabled the identification and molecular characterization of major protein building blocks of the cytoskeleton of this mycoplasma. Some of these proteins function as surface exposed adhesins, including proteins P1 and P30, while others, named accessory proteins (designated HMW1, HMW2, and HMW3 and A, B, C) collectively maintain the proper distribution and/ or disposition of the adhesins in the mycoplasma membrane. Loss of one or more of these proteins may lead to the loss of the cytadherence ability of the mycoplasmas. Additional proteins, named P65 and P200, share characteristic structural features with HMW1 and HMW3, suggesting their function as elements of the M. pneumoniae cytoskeleton, consistent with their presumed scaffolding role. Protein HMW2 (about 215 kDa) is predicted to assume a coiled-coil conformation, similar to that of the filamentous portion of the myosin heavy chain, reflecting a likely structural role for this cytoskeletal mycoplasmal protein (Krause, 1996, 1998; Himmelreich et al., 1996). The best characterized adhesin of M. pneumoniae is adhesin P1. As can be seen in Table 1, the P1 gene is part of an operon. The G + C content of the P1 operon is significantly higher than that of the entire genome of M. pneumoniae, leading to the suggestion that the P1 operon is of an exogenous origin. About 60% of the tryptophan codons are of the unusual type UGA, a peculiar property of mycoplasmas, characteristic also of mitochondria. While the P1 operon carries only one copy of the P1 gene, partial sequences of the gene are distributed all over the genome, as repetitive sequences (Himmelreich et al., 1997). These repetitive sequences constitute about 8% of the total M. pneumoniae genome. The finding of the large percentage in the genome of repetitive non-coding regions was somewhat contradictory to our expectations from a minimal compact genome. Only the recent comparison of the genomes of M. pneumoniae and M. genitalium (Himmelreich et al., 1997) have indicated that these repetitive sequences are conducive to homologous recombination and genomic rearrangements and may thus play a role in induction of antigenic variation of the mycoplasmal cell surface and in this way help the parasite to evade the host immune response. The M. genitalium gene homologous to P1 was named MgPa (Table 1). Clearly, these genes show a high degree of similarity, reflected also on comparison of the adhesin proteins encoded by the genes (Table 2). P1 and MgPa are high-molecular weight proteins transversing the cell membrane several times. The molecular properties of a second M. pneumoniae adhesin, having a molecular weight of about 30 mDa, 370 Razin Table 1. Comparison of the Molecular Properties of the P1 and MgPa Genes P1 MgPa 4881 53.5 TGA (21)TGG (16) Part of a 3-gene operon (ORF4–P1–ORF6) Present 4335 39.9 TGA(16)TGG(12) Part of a 3-gene operon (ORFl–MgPa–ORF3) Present Property Number of nucleotides Mol.% G + C Tryptophan codons Organization in genome Repetitive partial gene sequences References in Razin and Jacobs (1992); Baseman, (1993); Himmelreich et al. (1997); and Krause(1996, 1998). the P30 adhesin, are summarized in Table 3. The three adhesins have proline-rich C-terminals exposed on the outer membrane surface and associated with the cytadherence property (Razin and Jacobs, 1992; Baseman, 1993; Krause, 1996, 1998). Interestingly, these adhesins have been found by immunoelectron microscopy to cluster at the surface of the tip organelle. The high concentration of the adhesins at the tip is apparently responsible for the remarkable strength of attachment of the mycoplasmas to erythrocytes through the attachment tip (Razin et al., 1980). While most attention has been given to the adhesins and cytoskeletal elements of M. pneumoniae and M. genitalium, lipid-modified membrane proteins, probably acting as adhesins, have also been characterized in another human mycoplasma— M. hominis (Henrich et al., 1993). The notion that the same lipid-modified proteins responsible for the antigenic variation phenomenon act also as adhesins, has recently gained experimental support. Antigenic variation has become one of the hottest topics in infectious diseases research. During their evolution and adaptation to a Table 2. Comparison of the Molecular Properties of the P1 and MgPa Adhesin Proteins Property Molecular weight (daltons) Cysteine Proline-rich Serological activity Localization in mycoplasma P1 MgPa 169,758 Absent C-terminus Immunodominant Tip organelle 153,134 Absent C-terminus Immunodominant Tip organelle References in Razin and Jacobs (1992); Baseman, (1993); and Krause (1996, 1998). Table 3. Molecular Properties of the P30 Adhesin of M. pneumoniae Gene 825 nucleotides 54.4 mol.% G + C Tryptophan codons: one TGA and one TGG Apparently part of an operon Protein 275 amino acids Mol. wt. 29,743 daltons One cysteine residue Proline-rich C-terminus Immunodominant Localized at tip organelle References in Razin and Jacobs (1992) and Baseman (1993). Mycoplasma Adherence 371 Table 4. Receptors for M. pneumoniae Receptor type Active receptor site Location Sialoglycoproteins (sialoglycolipids?) a-2-3 sialylated poly-Nacetyllactosamine residues Human erythrocytes, bronchial epithelium Sulfated glycolipids (sulfatide, seminolipid) Terminal Gal (3SO4) B-1 residues Human trachea and lung Asialoglycoprotein Not defined MRC-5 human lung fibroblasts References in Razin and Jacobs (1992). parasitic mode of life, the mycoplasmas have developed various genetic systems providing a highly plastic set of variable surface proteins. The uniqueness of the mycoplasmal systems is manifested by the presence of highly mutable modules combined with an ability to expand the antigenic repertoire by generating structural alternatives, all compressed into limited genomic sequences. In the absence of a cell wall and a periplasmic space, the majority of surface proteins involved in generating antigenic variation in mycoplasmas are lipoproteins. These surface components, anchored to the cell membrane via acyl chains, are most dominant antigens and their abundance in the mycoplasma membrane is remarkable. The generation of a versatile surface coat through high-frequency phase and size variation, provides the organism with a useful tool for immune avoidance, allowing the mycoplasmas to escape antibody attack. The subject of antigenic variation in mycoplasmas has been recently reviewed (Razin et al., 1998). A novel approach developed in our Jerusalem laboratory by David Yogev and his collaborators (Athamna et al., 1997) may allow the identification of phasevariable proteins involved in mycoplasma adherence. The approach is based on the linkage between the ability of the mycoplasma cells to attach to erythrocytes (hemadsorption) and the expression of variable surface antigens. One of the most conspicuous ways this heterogeneity takes shape in in vitro studies is colony sectoring. A sector is defined as an immunologically distinct region in which a change in protein expression has occurred within a single colony. Attachment of erythrocytes via variable surface membrane protein(s) could be identified by the selective adherence of the erythrocytes to organisms within a single mycoplasma colony, exhibiting a typical nonhemadsorbing sector, an approach which appears to be a valuable tool in identification and cloning of the corresponding cytadherence variable genes of mycoplasmas. Unfortunately, we know much less about the molecular nature of the receptors for mycoplasmas, located at the surface of the host cell, than about the mycoplasmal adhesins. The fact that neuraminidase treatment of the host cells decreases significantly M. pneumoniae and M. genitalium attachment has been shown by us and by others long ago (Razin, 1985). Thus, Sialoglycoproteins or sialoglycolipids have been long considered as possible receptors for these mycoplasmas. Table 4 summarizes our current knowledge of the different receptors for M. pneumoniae. These consist of Sialoglycoproteins and sialoglycolipids, sulfated glycolipids, and possibly an asialoglycoprotein. Clearly, there is more than one receptor type for M. pneumoniae. 372 Razin This should not, perhaps, come as a surprise, considering that M. pneumoniae carries more than one adhesin type. In fact, it appears that the asialoglycoprotein binds to the P30 adhesin and not to the P1 adhesin of M. pneumoniae. REFERENCES Athamna, A., Rosengarten, R., Levisohn, S., Kahane, I., and Yogev, D. (1997) Adherence of Mycoplasma gallisepticum involves variable surface membrane proteins. Infect. Immun. 65:2468–2471. Baseman, J. B. (1993) The cytadhesins of Mycoplasma pneumoniae and M. genitalium. In: Subcell. Biochem, S. Rottem and I. Kahane, eds., Plenum Press, New York, N.Y., 20:243–259. Fraser, C. M., et al., (1995) The minimal gene complement of Mycoplasma genitalium. Science 270:397– 403. 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