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Background: Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial for optimizing antimicrobial therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). Gene expression changes in S. aureus strain Newman exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two are derived from frog, temporin L and dermaseptin K4-S4(1-16), and the ovispirin-1 is obtained from sheep.

SummaryThe PrsA protein is a membrane-anchored peptidylprolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin-binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross-linkage index. Misfolded PBP2a was detected in PrsA-depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.

Transcription profiling of all protein-encoding genes of Bacillus subtilis was carried out under several secretion stress conditions in the exponential growth phase. Cells that secreted AmyQ alpha-amylase at a high level were stressed only moderately: seven genes were induced, most significantly htrA and htrB, encoding quality control proteases, and yqxL, encoding a putative CorA-type Mg(2+) transporter. These three genes were induced more strongly by severe secretion stress (prsA3 mutant secreting AmyQ), suggesting that their expression responds to protein misfolding. In addition, 17 other genes were induced, including the liaIHGFSR (yvqIHGFEC) operon, csaA and ffh, encoding chaperones involved in the pretranslocational phase of secretion, and genes involved in cell wall synthesis/modification. Severe secretion stress caused downregulation of 23 genes, including the prsA paralogue yacD. Analysis of a cssS knockout mutant indicated that the absence of the CssRS two-component system, and consequently the absence of the HtrA and HtrB proteases, caused secretion stress. The results also suggest that the htrA and htrB genes comprise the CssRS regulon. B. subtilis cells respond to secretion/folding stress by various changes in gene expression, which can be seen as an attempt to combat the stress condition.