Cytoplasmic Control of Premature Activation of a Secreted Protease Zymogen: Deletion of Staphostatin B (SspC) in Staphylococcus aureus 8325-4 Yields a Profound Pleiotropic Phenotype

Abstract

The cytoplasmic protein SspC of Staphylococcus aureus, referred to as staphostatin B, is a very specific, tightly binding inhibitor of the secreted protease staphopain B (SspB). SspC is hypothesized to protect intracellular proteins against proteolytic damage by prematurely folded and activated staphopain B (M. Rzychon, A. Sabat, K. Kosowska, J. Potempa, and A. Dubin, Mol. Microbiol. 49:1051-1066, 2003). Here we provide evidence that elimination of intracellular staphopain B activity is indeed the function of SspC. An isogenic sspC mutant of S. aureus 8325-4 exhibits a wide range of striking pleiotropic alterations in phenotype, which distinguish it from the parent. These changes include a defect in growth, a less structured peptidoglycan layer within the cell envelope, severely decreased autolytic activity, resistance to lysis by S. aureus phages, extensively diminished sensitivity to lysis by lysostaphin, the ability to form a biofilm, and a total lack of extracellular proteins secreted into the growth media. The same phenotype was also engineered by introduction of sspB into an 8325-4 sspBC mutant. In contrast, sspC inactivation in the SH1000 strain did not yield any significant changes in the mutant phenotype, apparently due to strongly reduced expression of sspB in the sigma B-positive background. The exact pathway by which these diverse aberrations are exerted in 8325-4 is unknown, but it is apparent that a very small amount of staphopain B (less than 20 ng per 200 g of cell proteins) is sufficient to bring about these widespread changes. It is proposed that the effects observed are modulated through the proteolytic degradation of several cytoplasmic proteins within cells lacking the inhibitor. Seemingly, some of these proteins may play a role in protein secretion; hence, their proteolytic inactivation by SspB has pleiotropic effects on the SspCdeficient mutant.

Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing

“…These features together with a repetitive disordered segment of unknown function at the C terminus of zSspA are uniquely associated with S. aureus and one species of coagulase-negative Staphylococcus where SspA is in an operon and serves to activate an adjacently encoded cysteine protease. Others have found that expression and secretion of zSspB is toxic to S. aureus unless accompanied a third gene in the ssp operon encoding a cytoplasmic protein, SspC (Staphostatin B), that binds to and inhibits mature SspB but reportedly has no interaction with zSspB (12,14,15). Our present study has described additional safety features incorporated into the zSspA propeptide that appear to have evolved to minimize the likelihood of either protease being activated prematurely during protein export.…”

“…The need for an inhibitor is supported by the finding that expression of SspB in Escherichia coli is lethal unless accompanied by Staphostatin B or a Cys 3 Ser active site mutant in SspB. Moreover inactivation of sspC in S. aureus promotes a gross disturbance in growth and protein export, supporting a role for Staphostatin B in protecting against activation of zSspB during protein export (13)(14)(15). One means by which this could occur would be if zSspA were also activated prematurely.…”

An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation

“…It has been reported that GluV8 is processed by aureolysin, a thermolysin-family metalloprotease [6,7]. This processing was faithfully reproduced by thermolysin treatment of the recombinant proteins in vitro (Fig.…”

“…Drapeau [6] reported that activation of the GluV8 precursor is achieved by a neutral metalloprotease. Shaw et al [7] further demonstrated that the cascade reactions in processing of major extracellular pathogenic proteases of S. aureus from metalloprotease/aureolysin, GluV8/SspA, and finally, to cysteine protease/SspB. We previously purified [8] and cloned [9] the glutamyl endopeptidase from Staphylococcus epidermidis (designated GluSE), which consists of 282 amino acids composed of a preprosequence (Met 1 -Ser 66 ) and mature portion (Val 67 -Gln 282 ).…”

Enzybiotics and their Potential Applications in Medicine

“…While no cases of bacterial resistance to lysins have been reported as yet, four mechanisms inducing resistance to lysostaphin have been identifi ed (Shaw et al 2005 ;Gr ü ndling et al 2006 ;Kusuma et al 2007 ). These are described in more detail in Chapter 7 .…”