Abstract
Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.
Highlights
Invasive bacterial infections remain a significant cause of morbidity and mortality in humans [1]
To identify novel factors that regulate hemolysin expression in S. aureus, we screened a transposon insertion library for mutants that showed either an increase or decrease in hemolytic activity compared to the wild type (WT) strain
Given that AgrA positively regulates transcription of hla and hld, and negatively regulates spa transcription during post-exponential phase growth of S. aureus, our results suggest that Stp1 and Stk1 regulation of a toxin transcription is independent of global Agr regulation
Summary
Invasive bacterial infections remain a significant cause of morbidity and mortality in humans [1]. The a toxin encoded by the hla gene is important for S. aureus pneumonia, sepsis, septic arthritis, brain abscess and corneal infections [8,9,10,11,12,13]. This 33kDa pore forming toxin is secreted by majority of S. aureus clinical isolates and is active against a wide range of mammalian cells, with especially marked activity against rabbit erythrocytes [14,15]. These observations emphasize the importance of a toxin in S. aureus infections
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