Group B streptococcal beta-hemolysin promotes injury of lung microvascularendothelial cells

Group B streptococci (GBS) are the leading cause of pneumonia and sepsis in human newborns. Exudative pulmonary edema and alveolar hemorrhage seen in GBS pneumonia indicate vascular damage, and we reported that GBS injure lun g microvascular endothelial cells (LMvEC) both in vivo and in vitro. The sp ecific GBS factors causing LMvEC injury are uncertain, but GBS beta-hemolys in activity is associated with lung epithelial cell injury. We hypothesized that GBS beta-hemolysin contributes to LMvEC injury and exudative pulmonar y edema. To test this hypothesis we used isogenic nonhemolytic and hyperhem olytic GBS mutants derived by transposon insertional mutagenesis from three different wild-type strains. Hemolytic titers for each strain were calcula ted using live GBS and Tween 80/starch-stabilized extracts of log-phase GBS . All nonhemolytic mutants lacked detectable hemolytic activity, whereas hy perhemolytic mutants produced 4-16 times the hemolytic activity of their pa rent strains. LMvEC injury was assayed by light microscopy, the release of lactate dehydrogenase, trypan blue nuclear staining, and Evans blue-albumin flux. Compared with the parent strains, all nonhemolytic mutants caused si gnificantly reduced, and all hyperhemolytic mutants caused significantly gr eater, lactate dehydrogenase release from and trypan blue nuclear staining of LMvEC. Moreover, a nonhemolytic mutant caused reduced and a hyperhemolyt ic mutant caused increased Evans-blue albumin flux across polar LMvEC monol ayers. These findings were corroborated by light microscopic evidence of he molysin-associated damage to the LMvEC monolayers. We conclude that GBS bet a-hemolysin promotes LMvEC injury and increases permeability in vitro, and speculate that GBS beta-hemolysin contributes to the pathogenesis of alveol ar edema and hemorrhage in early onset GBS pneumonia.