Staphylococcal Major Autolysin (Atl) Is Involved in Excretion of Cytoplasmic Proteins

Abstract

Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as "nonclassical protein secretion." Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.

SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion

“…Studying S. aureus isolate SA113, Pasztor et al reported that atl mutation caused defects in the excretion of 22 cytoplasmic proteins (61). We note that in S. aureus Newman, the sagB mutation causes increased expression of Atl ( Fig.…”

“…Mutations that perturb protein secretion across the staphylococcal cell wall envelope have heretofore not been identified. We demonstrate that sagB mutations diminish the secretion of signal peptide-bearing precursors across the staphylococcal cell wall envelope while simultaneously increasing the excretion of cytoplasmic proteins, a class of proteins first described by Pasztor and colleagues (61). The mechanisms underlying these phenotypes are not yet understood, and we propose two models that may be useful for future experimental testing.…”

Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus

“…How these CPs are excreted is still not known. Cell lysis has been postulated, and indeed when the cell wall structure is altered in such a way that the cross-linking is affected, a higher release of CPs is observed (17); in a major autolysin (Atl) mutant there is almost no ECP (18). There are, however, good arguments speaking against the hypothesis that ECP is caused only by cell lysis.…”

“…There are, however, good arguments speaking against the hypothesis that ECP is caused only by cell lysis. One of the main arguments is that only certain CPs were excreted, while others, although highly abundant in the cytoplasm, were not found extracellularly, suggesting that there is a selection principle at work (18). Studying the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase (FbaA) and enolase (Eno), showed that they are excreted mainly during the exponential growth phase in S. aureus and that the exit site is the septal cleft of dividing cells (19).…”

“…Studying the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase (FbaA) and enolase (Eno), showed that they are excreted mainly during the exponential growth phase in S. aureus and that the exit site is the septal cleft of dividing cells (19). Another typical excreted CP is the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is, like FbaA, an essential enzyme in glycolysis of S. aureus (18,20,21). Here, we investigated the role of the excreted glycolytic enzymes FbaA and GAPDH in staphylococci.…”