Type I signal peptidase and protein secretion in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen whose virulence relies on the secretion of many different proteins. In general, the secretion of most proteins in S. aureus, as well as other bacteria, is dependent on the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein to the general secretory pathway. The arylomycins are a class of natural product antibiotics that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. While wild-type S. aureus (NCTC 8325) is naturally resistant to the arylomycins, sensitivity is conferred via a point mutation in its SPase. Here, we use a synthetic arylomycin along with a sensitized strain of S. aureus and multidimensional protein identification technology (MudPIT) mass spectrometry to identify 46 proteins whose extracellular accumulation requires SPase activity. Forty-four possess identifiable Sec-type signal peptides and thus are likely canonically secreted proteins, while four also appear to possess cell wall retention signals. We also identified the soluble C-terminal domains of two transmembrane proteins, lipoteichoic acid synthase, LtaS, and O-acyteltransferase, OatA, both of which appear to have noncanonical, internal SPase cleavage sites. Lastly, we identified three proteins, HtrA, PrsA, and SAOUHSC_01761, whose secretion is induced by arylomycin treatment. In addition to elucidating fundamental aspects of the physiology and pathology of S. aureus, the data suggest that an arylomycin-based therapeutic would reduce virulence while simultaneously eradicating an infection.

Type I signal peptidase and protein secretion in Staphylococcus epidermidis.

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the ß-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

Plasminogen has a broad extrahepatic distribution.

Plasmin is the major enzyme that dissolves fibrin in the vasculature and the predominant source of its zymogen, plasminogen, is liver. However, plasmin has a broad substrate spectrum and, if present in other tissues, may perform additional functions. We tested the hypothesis that plasminogen is expressed broadly extrahepatically. A sensitive and specific isotopic quantitative RT-PCR assay was developed to detect plasminogen mRNA from total RNA isolated from C57BL/6J mice tissues. Plasminogen mRNA was detected in adrenal, kidney, brain, testis, heart, lung, uterus, spleen, thymus and gut. Of these tissues, adrenal had the highest plasminogen mRNA content. In situ hybridization was utilized to localize plasminogen mRNA expressing cell types. Besides hepatocytes, positive cells were identified in both adrenal and kidney medullae and cortexes. Plasminogen mRNA expression was detected in cerebral, hippocampal and cerebellar neurons. Plasminogen mRNA was detected in cells in early stages of spermatogenesis in testis, present in the cortex and medulla of the thymus and in splenic white and red pulps. Our results suggest that the plasminogen gene is expressed broadly in extrahepatic tissues. Thus, tissues separated by local anatomic barriers as well as tissues accessible to circulating plasminogen have the capacity to provide local sources of plasminogen.

Localization of regulatory elements mediating constitutive and cytokine-stimulated plasminogen gene expression.

The activity of plasmin, the major enzyme responsible for dissolving fibrin clots, is regulated by plasminogen activators, plasminogen activator inhibitors, alpha(2)-antiplasmin, and inflammatory mediators. Recent studies suggest that plasmin activity can be regulated also at the level of plasminogen gene expression. In this study, we characterized the murine plasminogen promoter and 5'-flanking region. The major transcription start site was identified at -83 bp relative to the ATG translational initiation codon. A series of 5'-flanking sequences up to 2400 bp upstream of the transcription initiation site were fused to the luciferase reporter gene and transfected into hepatocytic cells. A 106-bp 5'-flanking region of the murine plasminogen gene demonstrated sufficient functional promoter activity in plasminogen-expressing cells. IL-6 treatment stimulated luciferase activity driven by the 5'-flanking region and an intact consensus IL-6-responsive element at -791, was required for maximal stimulation by this cytokine. These results indicate the presence of regulatory elements in the 5'-flanking region of the murine plasminogen promoter that may regulate murine plasminogen gene expression and, hence, plasmin activity.