[Frontiers in Bioscience 1, e42-54, August 1,1996]


Catherine Brenner, Olivier Neyrolles, Alain Blanchard

Institut Pasteur, Unité d'Oncologie Virale, Département SIDA et Rétrovirus, 28, rue du Dr. Roux, 75724 Paris Cedex 15, France

Received 07/05/96; Accepted 07/09/96; On-line 08/01/96

6. M. Fermentans AND M. Penetrans CYTOPATHOGENICITY

We will not review all the possible mechanisms of mycoplasma virulence which have been discussed elsewhere (26). We will focus on cell invasion and attachment.

6.1. Cell invasion

Initial studies of the interaction between HIV and mycoplasmas were based on the view that the bacteria were exclusively extracellular parasites. However, the work of Lo et al. (28) caused several investigators to reevaluate this view and several mycoplasma species were subsequently found intracellularly. The demonstration of their capacity to survive and to replicate in the host cell cytoplasm was hampered by the fact that classical techniques used for the quantitative study of entry for other eubacteria such as Shigella spp. or Listeria spp. are not directly transposable to mycoplasmas. Indeed, the two classes of antibiotics (tetracyclines and fluoroquinolones) which are the most effective in vitro against mycoplasmas are known to penetrate the cytoplasm of eukaryotic cells, and cannot therefore be used for selective elimination of extracellular microorganisms. In addition, the standard method used for releasing intracellular bacteria from the eukaryotic cells involves Triton X-100. As mycoplasmas are sensitive to this neutral detergent, it is unsuitable for their isolation.

Taylor Robinson et al. (54) performed an electron microscopy study using a combination of ruthenium red staining and immuno-gold labeling to document M. fermentans (strains incognitus and PG18) invasion of Hela cell cultures. The mycoplasma was found in the cytoplasmic fraction or membrane-bound vacuoles of experimentally infected cells. These observations have been confirmed by other investigators (36, 55). This invasive ability could account at least in part for the cytopathic effects of this mycoplasma but it remains unclear if various mycoplasmas found in vacuoles result from microbial replication or from multiple cell invasion. Indeed, it has not been demonstrated that intracellular mycoplasmas are alive and can replicate. In another study in which the M. fermentans cytopathogenicity was evaluated for tracheal tissue in vitro and in vivo, there were large difference between the capacity of different strains of M. fermentans to induce ciliostasis and their cytopathic effect (56). M. fermentans incognitus was found to be more invasive than other strains. However it has been propagated much less than other strains. Therefore, the difference in cytopathogenicity might be due to loss of virulence during in vitro passages.

The invasive properties of M. penetrans were evidenced from its initial characterization, hence its name (36, 35). A novel technique was proposed to quantify M. penetrans cell invasion: a combination of gentamycin and Triton X-100 at low concentration kills extracellular mycoplasmas (8 log decrease) without affecting the intracellular microbes (57). Although it is reported that Triton X-100 treatment greatly potentiates gentamycin activity treatment, the choice of this antibiotic is surprising because M. penetrans has been shown to be resistant to gentamycin (58). However, using this method Andreev et al. (57) reported that M. penetrans internalization, but not cell attachment, was abolished by cytochalasin D, an inhibitor of actin polymerization, and significantly affected by tyrosine kinase inhibitors including staurosporin and genistein. Furthermore, M. penetrans cell entry was associated with the phosphorylation of a cell protein of 145 kDa, but not of the Hp90 protein, as for E. coli entry. M. penetrans cell entry has also been studied by confocal microscopy (59), which reveals that all mycoplasmas are internalized only 2 hours after infection, and M. penetrans attachment and entry induce large local rearrangements of the cellular cytoskeleton. This was confirmed by others who demonstrated that M. penetrans attachment modifies the distribution of tubulin, a-actinin and aggregated phosphorylated cellular proteins (60).

6.2. Mycoplasma adherence to host cells

Both anti-M. penetrans antibodies and trypsin treatment inhibits bacterial attachment to HEp-2 cells, suggesting the involvement of microbial proteins (such as adhesins) in the cytadherence process (60). Bacterial attachment is also strongly reduced by metaperiodate and neuraminidase treatment of mycoplasmas, implicating surface sugars containing sialic acid in bacterial cytadherence, as suggested elsewhere (57). To try to identify the eukaryotic receptor for M. penetrans a cell-blotting technique was used (60). Biotin-labeled M. penetrans were found to bind fibronectin and this binding was not inhibited when mycoplasmas were preincubated with a 70 kDa fragment of fibronectin containing the heparin-gelatin binding domain, nor by the RGD peptide. The mycoplasma factors recognizing this putative receptor were isolated by affinity chromatography with immobilized fibro-nectin. A 65 kDa protein (P65) was thereby obtained. In addition, several other mycoplasma polypeptides (18 kDa, 28 kDa, 32 kDa, 36 kDa, 39 kDa, and 41 kDa) also bound HEp-2 cells on blots.

M. penetrans cell adhesion appears to be cell line-independent as it attaches to HEp-2, Hela, Wi38-VA13 and NCM/SM cells. This is consistent with the possibility that M. penetrans binds common cell surface proteins, such as fibronectin, as has been shown for the syphilis spirochete and Candida spp.

The P36, P39, and P41 HEp2-binding proteins from M. penetrans may be lipid associated membrane proteins (LAMPS, 47). In particular, the lipoprotein P35 which can be extracted with Triton X-114 is a candidate for the P36 described by Giron et al.(60). The attachment of M. penetrans to eukaryotic cell seems to implicate both bacterial adhesins and surface sugars. This is not surprising as M. penetrans cells are surrounded by a polysaccharide capsule-like material that we have partly characterized (61).

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