Mycoplasma hyorhinis (strain MCLD) was recently isolated from a melanoma cell culture. Growth of MCLD was considerably improved by 24 serial passages in a modified Hayflick's mycoplasma medium. Transmission electron microscopy showed that MCLD exhibits a polymorphic appearance, with ovoid or elongated cells frequently harboring an electron-dense core at one of the poles. Adherence of M. hyorhinis to melanoma cells followed saturation kinetics. Furthermore, although M. hyorhinis has been considered to remain attached to the surface of the host cells, we show for the first time, qualitatively by confocal laser scanning microscopy and quantitatively by a gentamicin resistance assay, that MCLD is able to invade melanoma cells. The ingested mycoplasmas were randomly distributed in the cytoplasm, tending to concentrate near the plasma membrane. Both adherence to and invasion of melanoma cells by M. hyorhinis strain MCLD were dramatically enhanced by mild proteolytic digestion with proteinase K (2.5 g/mg cell protein for 2.5 min at 37°C) that affected the surface-exposed proteins of this organism, mainly the major 47-kDa lipoprotein. We suggest that the intracellular location of M. hyorhinis strain MCLD is a privileged niche, which may explain the survival of M. hyorhinis in tissue cultures. The enhanced binding to and invasion of melanoma cells by protease treatment may be due to either the activation or the enhanced exposure of an adhesin(s) on the mycoplasmal cell surface.Mycoplasmas (class Mollicutes) are the smallest self-replicating bacteria. These bacteria lack a rigid cell wall and are parasites, exhibiting strict host and tissue specificities (2, 22). Many mycoplasmas are pathogenic to humans and animals and are frequent contaminants of cell cultures (24). Almost all human and animal mycoplasmas depend on adhesion to host cells for subsequent colonization and infection (20,24). In these mycoplasmas, adhesion is the major virulence factor, and adherence-deficient mutants are avirulent (2,20). The lack of a cell wall has forced mycoplasmas to develop sophisticated molecular mechanisms to enable their prolonged existence within the host, usually without causing major harm. Mycoplasma hyorhinis was first isolated from the respiratory tract of young pigs and has been implicated in pleuritis, peritonitis, pericarditis, arthritis, and otitis media in swine (9, 18). Interest in M. hyorhinis has recently increased after the detection of this organism in human gastric cancer tissues, suggesting a possible association between M. hyorhinis and tumorigenesis (12,14). Another important property of M. hyorhinis is its effectiveness in contaminating cell cultures, which impinges on many aspects of biological research (16). A mycoplasma identified as M. hyorhinis has recently been identified in the melanoma cell line LB33mel A1 (to be referred to as strain MCLD) (11). Although M. hyorhinis is considered a typical extracellular microorganism that is able to adhere to epithelial cells (16,20), ultrastructural studies performed w...

Thede novosynthesized polar lipids ofMycoplasmaspecies are rather simple, comprising primarily of the acidic glycerophospholipids PG and CL. In addition, when grown in a medium containing serum, significant amounts of PC and SPM are incorporated into the mycoplasma cell membrane although these lipids are very uncommon in wall-covered bacteria. The exogenous lipids are either incorporated unchanged or the PC incorporated is modified by a deacylation-acylation enzymatic cycle to form disaturated PC. Although their small genome, in someMycoplasmaspecies, other genes involved in lipid biosynthesis were detected, resulting in the synthesis of a variety of glycolipis, phosphoglycolipids and ether lipids. We suggest that analyses and comparisons of mycoplasma polar lipids may serve as a novel and useful tool for classification. Nonetheless, to evaluate the importance of polar lipids in mycoplasma, further systematic and extensive studies on moreMycoplasmaspecies are needed. While studies are needed to elucidate the role of lipids in the mechanisms governing the interaction of mycoplasmas with host eukaryotic cells, the finding that a terminal phosphocholine containing glycolipids ofM. fermentansserves both as a major immune determinants and as a trigger of the inflammatory responses, and the findings that the fusogenicity ofM. fermentanswith host cells is markedly stimulated by lyso-ether lipids, are important steps toward understanding the molecular mechanisms ofM. fermentanspathogenicity.

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The complete genome of Mycoplasma hyorhinis strain MCLD has been sequenced and annotated. This genome differs by the inversion of a 14.4-kb and a 3.7-kb fragment and the deletion of a 9.9-kb fragment from M. hyorhinis strain HUB-1, isolated from swine respiratory tract. The genome revealed 778 coding sequences (CDSs), with a limited number of vlp genes encoding variable surface lipoproteins.Mycoplasma hyorhinis is a swine pathogen causing respiratory diseases and arthritis. Additionally, it is a frequent cell culture contaminant and has recently been detected in human gastric carcinoma tissues (13). M. hyorhinis MCLD has been isolated from a primary human melanoma cell line (5). Although mycoplasmas were considered adherent extracellular microorganisms (10), it has been shown that M. hyorhinis MCLD invades host cells (7). Furthermore, host cells infected with M. hyorhinis MCLD showed elevated expression of a CD99 ligand on melanoma cells (5) and a marked increase in the cellular concentration of the protease inhibitor calpastatin within infected neuroblastoma cells (4).The M. hyorhinis MCLD genome was sequenced by using GS FLX Titanium technology and annotated using RAST (1) and PGAAP with manual curation. The fully assembled circular chromosome has 829,709 bp and an average GϩC content of 25.9%. We predicted 778 coding sequences (CDSs) with a coding density of 89.2%, of which 273 CDSs were hypothetical or conserved hypothetical proteins.Genome alignment of M. hyorhinis MCLD with the M. hyorhinis HUB-1 sequence (8), starting at the dnaA gene (SRH_2175) in the reverse complement orientation and using MAUVE (3), revealed 18 locally colinear blocks (LCBs). Unlike recent data on M. mycoides (12), no function-specific LCBs were identified. The MAUVE progressive mode utilized to identify conserved large segments revealed that each chromosome was composed of 4 regions and that differences may be explained by 2 inversions (14.4 kb, SRH_02645 to SRH_02545, and 3.7 kb, SRH_2605 to SRH_2635).Similar to other mycoplasmas, 10 putative transcriptional regulators were detected in the M. hyorhinis MCLD genome, among them two sigma factors and the transcriptional repressor HrcA. Unlike other mycoplasma species, no homologs of the RelA and SpoT families were found.The bacterial translational system components are conserved and included 30 tRNAs and 21 tRNA synthetases, a single copy of the 16S-23S rRNA operon, and a separate 5S rRNA gene. Three protein initiation factors, 4 elongation factors, and a single peptide release factor (RF-1) were also identified. However, the peptide chain release factor 2 (RF-2) was not detected.It has been proposed that the variable surface lipoprotein (vlp) locus in M. hyorhinis contains seven distinct vlp genes (2). Comparative analysis of the vlp locus among five M. hyorhinis strains (MCLD, GDL, SK76, HUB-1, and a clonal variant of SK76) revealed that the vlp genes in M. hyorhinis MCLD are reduced, containing only 4 genes, vlpD (SRH_00185), vlpE (SRH_00180), vlpB (SRH_00175), and vlpC (SRH_00170),...

We demonstrated that when M. pneumoniae was grown on an abiotic surface of either glass or polystyrene with a serum-containing medium, the bacteria adhered to the surface and formed highly differentiated volcano-like biofilm structures. As adherence to the surface and/or biofilm formation was totally inhibited by anti-P1 polyclonal monospecific antibodies, we suggest that the adherence of M. pneumoniae to the abiotic surface and/or biofilm formation is associated with P1, the major tip organelle protein of this organism. Furthermore, adherence and/or biofilm formation was markedly inhibited by treating the serum component of the growth medium with neuraminidase or by growing the bacteria in the presence of sialyllactose, suggesting that the initial step in the adherence to and/or biofilm formation by M. pneumoniae on an abiotic surface is the interaction of the bacterium through its tip organelle with sialic acid residues of serum glycoproteins.