Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae

AM Berry, J Yother, DE Briles, D Hansman… - Infection and …, 1989 - Am Soc Microbiol
AM Berry, J Yother, DE Briles, D Hansman, JC Paton
Infection and immunity, 1989Am Soc Microbiol
Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative
of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated
strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment
from the middle of the pneumolysin structural gene. DNA was extracted from the resultant
erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform
S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants …
Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.
American Society for Microbiology