Group B Streptococcus CAMP Factor Does Not Contribute to Interactions with the Vaginal Epithelium and Is Dispensable for Vaginal Colonization in Mice

Group B Streptococcus (GBS) remains a pervasive pathogen for pregnant women and their newborns. Maternal screening and intrapartum antibiotic prophylaxis to GBS-positive mothers have reduced, but not eliminated GBS neonatal disease, and have not impacted GBS-associated preterm birth or stillbirth.

CAMP test v1

Group B Streptococcus agalactiae has CAMP factor which allow it to hemolized zones when it is grown on blood agar plates near to Staphylococcus aureus ATCC 25293 colonies, this effect is brought about by Staphylococcus aureus sphingomyelinase.

Genetic Basis Underlying the Hyperhemolytic Phenotype of Streptococcus agalactiae Strain CNCTC10/84

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is a major cause of infections in newborns, pregnant women, and immunocompromised patients. GBS strain CNCTC10/84 is a clinical isolate that has high virulence in animal models of infection and has been used extensively to study GBS pathogenesis. Two unusual features of this strain are hyperhemolytic activity and hypo-CAMP factor activity. These two phenotypes are typical of GBS strains that are functionally deficient in the CovR-CovS two-component regulatory system. A previous whole-genome sequencing study found that strain CNCTC10/84 has intact covR and covS regulatory genes. We investigated CovR-CovS regulation in CNCTC10/84 and discovered that a single-nucleotide insertion in a homopolymeric tract in the covR promoter region underlies the strong hemolytic activity and weak CAMP activity of this strain. Using isogenic mutant strains, we demonstrate that this single-nucleotide insertion confers significantly decreased expression of covR and covS and altered expression of CovR-CovS-regulated genes, including that of genes encoding β-hemolysin and CAMP factor. This single-nucleotide insertion also confers significantly increased GBS survival in human whole blood ex vivo. IMPORTANCE Group B streptococcus (GBS) is the leading cause of neonatal sepsis, pneumonia, and meningitis. GBS strain CNCTC10/84 is a highly virulent blood isolate that has been used extensively to study GBS pathogenesis for over 20 years. Strain CNCTC10/84 has an unusually strong hemolytic activity, but the genetic basis is unknown. In this study, we discovered that a single-nucleotide insertion in an intergenic homopolymeric tract is responsible for the elevated hemolytic activity of CNCTC10/84.

Structure determination of the CAMP factor of Streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment

CAMP factor is a unique α-helical bacterial toxin that is known for its co-hemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Å resolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltose-binding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.