Analysis of a novel prophage-encoded group A Streptococcus extracellular phospholipase A(2).
Group A Streptococcus (GAS) is an important human pathogen that causes many types of infections, including pharyngitis and severe invasive diseases. We recently sequenced the genome of a serotype M3 strain and identified a prophage-encoded secreted phospholipase A(2) designated SlaA. To study SlaA structure-activity relationships, 20 site-specific mutants were constructed by alanine-replacement mutagenesis and purified to apparent homogeneity. Enzymatic activity was greatly reduced by alanine replacement of amino acid residues previously described as crucial in the catalytic mechanism of secreted phospholipase A(2). Similarly, substitution of five residues in an inferred Ca(2+)-binding loop and three residues in the inferred active site region resulted in loss of activity of 76.5% or greater relative to the wild-type enzyme. Analysis of enzyme substrate specificity confirmed SlaA as a phospholipase A(2), with activity against multiple phospholipid head groups and acyl chains located at the sn-2 position. PCR analysis of 1,189 GAS strains representing 48 M protein serotypes commonly causing human infections identified the slaA gene in 129 strains of nine serotypes (M1, M2, M3, M4, M6, M22, M28, M75, and st3757). Expression of SlaA by strains of these serotypes was confirmed by Western immunoblot. SlaA production increased rapidly and substantially on co-culture with Detroit 562 human pharyngeal epithelial cells. Together, these data provide new information about a novel extracellular enzyme that participates in GAS-human interactions.
Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Michal J
MJ
Nagiec
Benfang
B
Lei
Sarah K
SK
Parker
Michael L
ML
Vasil
Masakado
M
Matsumoto
Robin M
RM
Ireland
Stephen B
SB
Beres
Nancy P
NP
Hoe
James M
JM
Musser
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D039002:Prophages / Q000235:genetics; D013297:Streptococcus pyogenes / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010741:Phospholipases A