Joel B. Baseman

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium, are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn4001 was not localized in or near the hmw1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.

Growth requirements of ferret tracheal epithelial cells in primary culture.

Abstract In mass cell culture conditions, protease dissociated ferret tracheal epithelial cells (FTE) proliferated in growth factor-supplemented F12 medium to high cell densities (0.5 × 105 cells/cm2) with an average population doubling time of 24 hr. The growth factor constituents of the F12 medium included epidermal growth factor (25 ng/ml), insulin (1 μg/ml), transferrin (10 μg/ml), hydrocortisone (18 ng/ml), hypothalamus extract (30-100 μg/ml), and conditioned medium from mouse 3T3 fibroblasts. Growth of these cells under clonal conditions was achieved by the partial replacement of F12 medium with M199 medium which was attributed, in part, to the presence of vitamin A in M199 medium. Serum did not stimulate the growth of FTE cells. The epithelial cell nature of these cells in culture was confirmed by ultrastructural features and by immunofluorescent staining for fibronectin.

MOLECULAR BASIS FOR FIBRONECTIN-MEDIATED ADHERENCE IN THE SYPHILIS SPIROCHETES

Nearly five centuries after Fracastorius named the disease syphilis, based upon the escapades of a mythical shepherd, the pathogenesis of syphilis remains a conundrum. How does one explain the propensity of the highly virulent syphilis spirochete to be found in tissues throughout the body, yet remain so dependent on the human host?