Specificity of antibodies from human sera for Naegleria species.

Serum samples from adult humans in North Carolina and Pennsylvania were assayed for antibodies against four Naegleria species: N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis. Agglutinating activities of serum samples from North Carolina subjects were higher for N. fowleri than were those from Pennsylvania subjects. The distributions of agglutination titers of human serum samples for N. australiensis, N. gruberi, and N. lovaniensis were heterogeneous. The agglutination capabilities of selected serum samples absorbed with rounded, killed trophozoites of N. australiensis and N. lovaniensis were distinctly different, as were those of serum samples absorbed with N. fowleri and N. gruberi. N. australiensis and N. gruberi shared some agglutinating antigens, as did N. fowleri and N. lovaniensis. The agglutinating activities of most serum samples correlated with the capability of their immunoglobulin M (IgM) to bind to antigens in extracts of Naegleria species but not with the capabilities of their IgG to bind to antigens of Naegleria species. Absorption of IgM binding capability with rounded, killed trophozoites established that N. gruberi was distinctly different from N. fowleri and N. lovaniensis but that N. fowleri and N. lovaniensis shared surface antigens. The proteins in extracts of the four Naegleria species were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tested for their ability to bind immunoglobulins in a serum sample. The antigens of the four species that bound IgM or IgG in the tested serum sample were separated by SDS-PAGE, and when they were incubated with anti-IgM or anti-IgG, they gave distinct profiles. There was one distinct, shared antigen that had a molecular size of 40,000 daltons. Absorption of the test serum with killed, rounded trophozoites did not markedly change the immunoglobulin binding profile for Naegleria internal antigens separated by SDS-PAGE and did not remove the shared 40,000-dalton protein(s). These results demonstrate that the four Naegleria species have antigenically distinct surfaces and that humans have been individually exposed to antigens of Naegleria species.

Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans.

A chromosomal tetracycline resistance (Tcr) determinant previously cloned from Streptococcus mutans into Streptococcus sanguis (Tobian and Macrina, J. Bacteriol. 152:215-222, 1982) was characterized by using restriction endonuclease mapping, deletion analysis, and Southern blot hybridization. Deletion analysis allowed localization of the Tcr determinant to a 2.8-kilobase region of the originally cloned 10.4-kilobase sequence. This cloned determinant hybridized to a representative of the tetM class of streptococcal Tcr determinants but not to representatives of the tetL and tetN classes and, like other tetM determinants, mediated high-level resistance to tetracycline and low-level resistance to minocycline. A portion (approximately 3 kilobases) of the isolated streptococcal fragment was subcloned into Escherichia coli, where it conferred resistance to tetracycline and minocycline. Two proteins with apparent molecular weights of 33,000 and 35,000, encoded by the S. mutans DNA, were synthesized in E. coli minicells. Insertion of DNA into a unique SstI site of the cloned S. mutans fragment resulted in inactivation of Tcr expression in E. coli and S. sanguis, as well as loss of production of both the 33,000- and 35,000-dalton proteins in E. coli minicells. Incubation of minicells in subinhibitory concentrations of tetracycline did not result in changes in the levels of synthesis of either protein. Our data suggest that at least one of these proteins is involved in the expression of Tcr.