Invasion of Melanoma Cells byMycoplasma hyorhinis: Enhancement by Protease Treatment

Abstract

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...

Contamination of Tissue Cultures by Mycoplasmas

“…Changes in host cell lipid turnover occur as a result of M. penetrans binding and/or invasion of Molt-3 lymphocytes [40]. These changes include the accumulation of DAG and the release of unsaturated fatty acids, predominantly long-chain polyunsaturated ones such as docosahexanoic acid (C22: 6,40]. Nonetheless, metabolites of phosphatidylinositol were not detected.…”

“…Imaging single infected HeLa cells revealed that invasion is both time and temperature dependent. Penetration of melanoma cells by M. hyorhinis has been observed as early as 30 min after infection [6], whereas invasion of cultured HEp-2 cells by M. penetrans has been shown to begin after 2 h of infection [36].…”

“…Plg and uPA are two proteins that play an important role in the invasion of several human malignant tumors [28], therefore it is not surprising that the same system stimulates M. fermentans invasion. Other mycoplasmas known to be surface parasites such as M. pneumoniae [29], M. genitalium [30], M. gallisepticum [31], and M. hyorhinis [6] were also found under certain circumstances to reside within host cells.…”

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“…Namiki and colleagues reported the occurrence of M. hyorhinis infection in malignant of the benign human prostate (BPH-1) in cell culture (10, [17][18]. Besides, Urbanek and his colleagues, using serological test showed positive tests of M. hyorhinis for more than thirty percent benign prostatic hyperplasia (BPH) and more than fifty percent prostatic cancer tissues (19).…”

“…Dong and his research group in 1980 performed studies on monoclonal PD4 antibody via mice immunization with the gastric cancer cell, then the mycoplasma antigen was detected by PD4, which is a lipoprotein of M. hyorhinis called P37 and does not have any homology with human proteins (17)(18)(19)(20)(21).…”