Group B streptococci (GBS) are the leading cause of serious bacterial infection in human newborns. Early-onset disease is characteristically heralded by pneumonia with dense bacterial infiltration, epithelial cell damage, alveolar hemorrhage and inflammatory exudate. Premature infants are at greatly increased risk for acquiring GBS pneumonia and invasive disease. We have demonstrated that expression of β-hemolytic activity by GBS is associated with injury of lung epithelial cells in vitro. Using transposon Tn916 and Tn917 mutagenesis, we have created single-insertion isogenic mutants of GBS which exhibit either a nonhemolytic (NH) or hyperhemolytic (HH) phenotype. NH mutants are noninjurious, and HH mutants hyperinjurious, to monolayers of the A549 human pneumocyte cell line, as measured by release of lactate dehydrogenase (LDH) and trypan blue nuclear staining. Electron microscopic studies of injured pneumocytes suggest the GBSβ-hemolysin is a pore-forming cytolysin, producing discrete membrane disruptions, cellular swelling, loss of intracytoplasmic density and changes in organelles and chromatin consistent with hypoosmotic damage from water influx. We have shown that GBS β-hemolysin-induced pneumocyte injury is inhibited in a dose-dependent fashion by the major component of human surfactant, dipalmotyl phosphatidylcholine (DPPC), providing a rationale for the increased susceptibility of premature, surfactant-deficient infants to severe GBS pneumonia. The GBS β-hemolysin has never been isolated, but our analysis of protein extracts from isogenic hemolysin mutants reveal an 11-kd putative hemolysin protein absent in NH mutants and overexpressed by HH mutants. Finally, we have used a neonatal rat model to test the role ofβ-hemolysin in animal virulence. A NH single-insertion transposon mutant possessed a 1000-fold increased LD50 compared to wild-type (5 × 106 cfu vs. 5 × 103 cfu) following intrathoracic inoculation, whereas a HH single-insertion transposon mutant appears to produce a more rapid time to death and increased lung injury compared to wild-type at high inocula (> 106 cfu). We conclude that the GBSβ-hemolysin is a possible virulence factor important in early-onset GBS pneumonia. Transposon mutants with altered β-hemolysin phenotype are currently being analyzed to identify the genes required for β-hemolysin expression by GBS.
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Nizet, V., Gibson, R. & Rubens, C. GROUP B STREPTOCCAL β-HEMOLYSIN EXPRESSION AND INJURY OF NEWBORN LUNG.• 1781. Pediatr Res 39, 299 (1996) doi:10.1203/00006450-199604001-01805