Group B Streptococcus Hyaluronidase Promotes Lung Invasion and Early Onset Disease

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Abstract Rationale: Group B Streptococcus (GBS) infections pose a significant public health risk leading to preterm births and invasive infections in newborns. Early onset disease (EOD) in infants occurs following the inhalation of infected fluids during birth, which results in neonatal pneumonia and sepsis. Previous studies from our group showed that GBS isolated from invasive disease exhibit increased secretion of the bacterial hyaluronidase (HylB) and HylB exacerbates ascending infection and preterm births in mice and non-human primates. Whether HylB is a critical virulence factor for invasion of the airway epithelial cells (AEC), i.e., the initial barrier against GBS entry into the lung, is currently unknown. Objective: Test the hypothesis that HylB is a critical mediator of AEC invasion during GBS infection. Methods: Neonatal mice (n=5/group) at P0 were intranasally inoculated with 5 x 105 CFU of either wild-type (WT) GBS or an isogenic strain deficient in HylB (ΔhylB). Mice were monitored for morbidity. Twenty-four hours post infection, surviving mice were euthanized and bacterial burden was estimated in the neonatal lungs by serial dilution and plating on CHROMagar StrepB. In separate studies, primary human AECs (n=3/group) were isolated from pediatric donors undergoing sedated elective procedures in the operating room using airway brushings. AECs were differentiated at an air-liquid interface (ALI) for 21 days and then infected with either GBS WT or ΔhylB at MOI=10 for 18 hours, in the absence of antibiotics. Outcome measures were assessed by GBS invasion assays, immunofluorescence, and confocal microscopy. Results: Neonatal mouse lungs demonstrated greater GBS invasion following infection with WT compared to ΔhylB (3.2 x 106± 2.5 x 106 vs. 3.2 x 105± 3.0 x 105 CFU/g, p<0.007). In primary differentiated AEC cultures, infection with WT GBS led to greater invasion of AECs compared to ΔhylB (19.2% ± 6.5% vs. 6.6% ± 3.7%, p<0.02). Similarly, GBS invasion as determined by immunofluorescence demonstrated increased GBS staining area in AECs infected with WT vs. ΔhylB (7881 ± 744.0 vs. 1763 ± 411.2 fluorescence units, p<0.05). Conclusion: Using both in vivo neonatal mouse models and ex vivo primary differentiated human AEC models of GBS infection, HylB expression enhanced GBS invasion and promoted GBS infection. Further studies to identify the mechanism(s) by which GBS HylB expression promotes enhanced disease are in progress.

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