Jean A. Tharpe

Distinguishing Chlamydia species by restriction analysis of the major outer membrane protein gene

Clinical isolates of Chlamydia pneumoniae from diverse geographic locations and strains of other Chlamydia species were typed by polymerase chain reaction (PCR) amplification of the major outer membrane protein (MOMP) gene followed by restriction fragment length polymorphism analysis of the product. Use of synthetic primers corresponding to highly conserved regions of the MOMP gene resulted in amplification of a 1070 bp product in laboratory strains and clinical isolates of C. pneumoniae, C. trachomatis and C. psittaci. PCR products were digested with restriction enzymes Alu I and Mbo I and separated by polyacrylamide gel electrophoresis. Restriction fragment patterns varied in length from 8-12 bands of 30-400 bp in size in Alu I digests, and 6-7 bands of 50-400 bp in size in Mbo I digests. Strains representing different chlamydia species were easily distinguishable by this method, as were different serovars of C. trachomatis. Strains of C. pneumoniae tested include laboratory strain TW-183 and recent clinical isolates from Atlanta, Brooklyn, Wisconsin and Norway. One combination of primers reacted with C. psittaci strains and C. pneumoniae strain TW-183, but not with other strains of C. pneumoniae tested regardless of the concentration of DNA in the sample. With use of a pan-reactive primer combination, however, restriction patterns were similar in all strains of C. pneumoniae tested. This gene typing technique can be valuable for distinguishing the three chlamydial species and potentially strains of C. pneumoniae in clinical and epidemiologic studies.

Comparison of a Pneumococcal Common Protein (PsaA) Antibody ELISA and a PsaA Immune Complex ELISA for Detection of Pneumococcal Serum Antibody

We examined and compared results from three assays, an enzyme-linked immunosorbent assay (ELISA) and two immune complex ELISAs for analysis of the serum antibody response to a native pneumococcal 37-kD common cell-wall protein by using acute- and convalescent-phase sera from 56 patients with community-acquired pneumonia. The sensitivities of the ELISA, the undissociated and dissociated immune complex assays were 85% (23 of 27), 78% (21 of 27) and 67% (18 of 27), respectively. To determine specificity, paired sera from patients with pneumonia of other bacterial etiologies were tested. The specificities were 83, 83 and 72% for the ELISA, undissociated immune complex, and dissociated immune complex, respectively. Based on this study, the sensitivities of the three assays were not statistically different. These tests could be used retrospectively to confirm invasive pneumococcal disease.

BACULOVIRUS EXPRESSION, PURIFICATION AND EVALUATION OF RECOMBINANT PNEUMOCOCCAL SURFACE ADHESIN A OF STREPTOCOCCUS PNEUMONIAE

Pneumococcal surface adhesin A (PsaA), with a molecular mass of ∼37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA sequence and was cloned into a baculovirus expression system. We expressed either cell-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound ‘hydrophobic rPsaA’ (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of ∼38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed ‘soluble rPsaA’ (srPsaA) with a molecular mass of ∼37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 µl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.