scholarly journals Stable Signal Peptides and the Response to Secretion Stress in Staphylococcus aureus

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Arryn Craney ◽  
Floyd E. Romesberg
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Death ◽  
Cell Surface ◽  
Signal Peptide ◽  
Signal Peptidase ◽  
Type I ◽  
Signal Peptides ◽  
Mature Protein ◽  
Bacterial Response ◽  
Secretion Stress

ABSTRACT Protein secretion is essential, but how it is managed is poorly understood. In bacteria, most secreted proteins require release from the outer surface of the cytoplasmic membrane by type I signal peptidase (SPase), which cleaves the mature protein from its membrane-bound N-terminal signal peptide. As the first step that occurs outside the protected cytoplasmic environment and because insufficient activity can rapidly result in the toxic accumulation of preproteins, the activity of SPase is expected to be closely monitored and perhaps supplemented when insufficient. Indeed, we previously demonstrated that inhibition of SPase in Staphylococcus aureus results in derepression of the ayrRABC operon, which encodes an alternate mechanism to release proteins. However, in this case, the proteins are released with partially intact signal peptides, with the exception of IsaA, which is released with a virtually intact signal peptide. Here we show that mutation of AyrA [ayrA(R233K)] results in constitutive derepression of ayrRABC and that mutation of IsaA’s signal peptide [isaA(K2Q)] results in hyperderepression upon SPase inhibition, which also requires AyrA. Further studies demonstrate that the inducing signal for ayrRABC derepression is accumulation of a subset of preproteins with signal peptides that are stable toward further processing and that the signal is critically amplified by the K2Q mutation and relayed to AyrR by AyrA. These results elucidate the mechanism by which S. aureus monitors and responds to secretion stress. The presence of ayrRA in other bacteria suggests that it may represent a general strategy linking membrane stress to appropriate transcriptional responses. IMPORTANCE Bacteria interact with their environment by secreting proteins that perform a myriad of functions, and the final step is the release of the mature protein from the cell surface via the activity of type I signal peptidase (SPase). While the bacterial response to many stresses is understood in some detail, almost nothing is known about how cells respond to secretion stress, such as insufficient SPase activity, which would eventually result in cell death. We previously demonstrated that the inhibition of SPase in Staphylococcus aureus results in the derepression of the ayrRABC operon, which can functionally replace SPase, but which is normally repressed by AyrR. We now demonstrate that the inducing signal for derepression is accumulation of a subset of preproteins with signal peptides that are stable to further processing and that the signal is relayed to AyrR via AyrA. IMPORTANCE Bacteria interact with their environment by secreting proteins that perform a myriad of functions, and the final step is the release of the mature protein from the cell surface via the activity of type I signal peptidase (SPase). While the bacterial response to many stresses is understood in some detail, almost nothing is known about how cells respond to secretion stress, such as insufficient SPase activity, which would eventually result in cell death. We previously demonstrated that the inhibition of SPase in Staphylococcus aureus results in the derepression of the ayrRABC operon, which can functionally replace SPase, but which is normally repressed by AyrR. We now demonstrate that the inducing signal for derepression is accumulation of a subset of preproteins with signal peptides that are stable to further processing and that the signal is relayed to AyrR via AyrA.

scholarly journals A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
J. Hiroshi Morisaki ◽  
Peter A. Smith ◽  
Shailesh V. Date ◽  
Kimberly K. Kajihara ◽  
Chau Linda Truong ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Abc Transporter ◽  
Signal Peptide ◽  
Transcriptional Repressor ◽  
Signal Peptidase ◽  
Clinical Strain ◽  
Type I ◽  
Atpase Gene ◽  
Associated Proteins

ABSTRACT The type I signal peptidase of Staphylococcus aureus , SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro / cI , a homolog of the lambda phage transcriptional repressor cro . These cro / cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB . Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo . IMPORTANCE The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor ( cro / cI ), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro . However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.

scholarly journals A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomycesoris

2016 ◽  
Vol 198 (15) ◽  
pp. 2064-2073 ◽  
Author(s):  
Sara D. Siegel ◽  
Chenggang Wu ◽  
Hung Ton-That
Keyword(s):  
Experimental Model ◽  
Signal Peptide ◽  
Biofilm Formation ◽  
Signal Peptidase ◽  
Type I ◽  
Signal Peptides ◽  
Gram Positive ◽  
Content Type ◽  
Lpxtg Motif ◽  
Gram Positive Bacteria

ABSTRACTThe Gram-positive bacteriumActinomycesoris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria,A. orisexpresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal “glyco-stress,” caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortasesrtAis genetically disrupted. Consistent with this finding, we show here that a mutant lackinglepB2andsrtAwas unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neitherlepB1norlepB2abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, thelepB2deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, thelepB2mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely,lepB1was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity.IMPORTANCESec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria,A. orisexpresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal “glyco-stress” caused by membrane accumulation of glycosylated GspA when the housekeeping sortasesrtAis genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus,A. orisprovides an experimental model with which to investigate the specificity mechanism of type I SPases.

scholarly journals A Putative Cro-Like Repressor Contributes to Arylomycin Resistance in Staphylococcus aureus

2015 ◽  
Vol 59 (6) ◽  
pp. 3066-3074 ◽  
Author(s):  
Arryn Craney ◽  
Floyd E. Romesberg
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Transcriptional Regulator ◽  
Signal Peptidase ◽  
Health Concern ◽  
Resistant Bacteria ◽  
Type I ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Wide Range

ABSTRACTAntibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogenStaphylococcus aureus. To understand the origins of the limited activity againstS. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand howS. aureusresponds to the arylomycins, we profiled the transcriptional response ofS. aureusNCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistanceex vivodemonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate howS. aureuscopes with secretion stress and how it evolves resistance to the inhibition of SPase.

scholarly journals Evaluation of signal peptide prediction algorithms for identification of mycobacterial signal peptides using sequence data from proteomic methods

Microbiology ◽  
2009 ◽  
Vol 155 (7) ◽  
pp. 2375-2383 ◽  
Author(s):  
Nils Anders Leversen ◽  
Gustavo A. de Souza ◽  
Hiwa Målen ◽  
Swati Prasad ◽  
Inge Jonassen ◽  
...  
Keyword(s):  
Markov Model ◽  
Signal Peptide ◽  
Cleavage Site ◽  
Hidden Markov ◽  
Signal Peptidase ◽  
Secreted Proteins ◽  
Signal Peptides ◽  
Sanger Institute ◽  
Signal Peptidase I ◽  
Peptide Prediction

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5 % of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway.

Lipopeptide substrates for SpsB, the Staphylococcus aureus type I signal peptidase: design, conformation and conversion to α-ketoamide inhibitors

2003 ◽  
Vol 38 (4) ◽  
pp. 351-356 ◽  
Author(s):  
Gordon Bruton ◽  
Anthony Huxley ◽  
Peter O'Hanlon ◽  
Barry Orlek ◽  
Drake Eggleston ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Signal Peptidase ◽  
Type I

scholarly journals Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

2008 ◽  
Vol 191 (21) ◽  
pp. 6732-6740 ◽  
Author(s):  
Sandy Y. M. Ng ◽  
David J. VanDyke ◽  
Bonnie Chaban ◽  
John Wu ◽  
Yoshika Nosaka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Signal Peptide ◽  
Signal Peptidase ◽  
Signal Peptides ◽  
Peptidase Activity ◽  
Vitro Assay ◽  
Type Iv ◽  
Peptide Length ◽  
Short Signal

ABSTRACT In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.

scholarly journals Mechanism of Action of the Arylomycin Antibiotics and Effects of Signal Peptidase I Inhibition

2012 ◽  
Vol 56 (10) ◽  
pp. 5054-5060 ◽  
Author(s):  
Peter A. Smith ◽  
Floyd E. Romesberg
Keyword(s):  
Secretory Pathway ◽  
Signal Peptidase ◽  
Model Organisms ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Secretion Pathway ◽  
Secretion Stress ◽  
Signal Peptidase I ◽  
Essential Enzyme

ABSTRACTClinically approved antibiotics inhibit only a small number of conserved pathways that are essential for bacterial viability, and the physiological effects of inhibiting these pathways have been studied in great detail. Likewise, characterizing the effects of candidate antibiotics that function via novel mechanisms of action is critical for their development, which is of increasing importance due to the ever-growing problem of resistance. The arylomycins are a novel class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), which is an essential enzyme that functions as part of the general secretory pathway and is not the target of any clinically deployed antibiotic. Correspondingly, little is known about the effects of SPase inhibition or how bacteria may respond to mitigate the associated secretion stress. Using genetically sensitizedEscherichia coliandStaphylococcus aureusas model organisms, we examine the activity of arylomycin as a function of its concentration, bacterial cell density, target expression levels, and bacterial growth phase. The results reveal that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. Interestingly, this has profoundly different effects onE. coliandS. aureus. Finally, we examine the activity of arylomycin in combination with distinct classes of antibiotics and demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.

scholarly journals A role for type I signal peptidase in Staphylococcus aureus quorum sensing

2007 ◽  
Vol 65 (3) ◽  
pp. 780-798 ◽  
Author(s):  
Jeffrey S. Kavanaugh ◽  
Matthew Thoendel ◽  
Alexander R. Horswill
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Signal Peptidase ◽  
Type I

scholarly journals Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 3031-3040 ◽  
Author(s):  
Ding Chen ◽  
Lei Lei ◽  
Chunxue Lu ◽  
Rhonda Flores ◽  
Matthew P. DeLisa ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Signal Peptide ◽  
Chlamydial Infection ◽  
Specific Inhibitor ◽  
Signal Peptidase ◽  
Leader Peptide ◽  
Type I ◽  
Terminal Sequence ◽  
Inner Membrane ◽  
Essential Information

The chlamydial protease/proteasome-like activity factor (CPAF) is secreted into the host cytosol to degrade various host factors that benefit chlamydial intracellular survival. Although the full-length CPAF is predicted to contain a putative signal peptide at its N terminus, the secretion pathway of CPAF is still unknown. Here, we have provided experimental evidence that the N-terminal sequence covering the M1–G31 region was cleaved from CPAF during chlamydial infection. The CPAF N-terminal sequence, when expressed in a phoA gene fusion construct, was able to direct the export of the mature PhoA protein across the inner membrane of wild-type Escherichia coli. However, E. coli mutants deficient in SecB failed to support the CPAF signal-peptide-directed secretion of PhoA. Since native PhoA secretion was known to be independent of SecB, this SecB dependence must be rendered by the CPAF leader peptide. Furthermore, lack of SecY function also blocked the CPAF signal-peptide-directed secretion of PhoA. Most importantly, CPAF secretion into the host cell cytosol during chlamydial infection was selectively inhibited by an inhibitor specifically targeting type I signal peptidase but not by a type III secretion-system-specific inhibitor. Together, these observations have demonstrated that the chlamydial virulence factor CPAF relies on Sec-dependent transport for crossing the chlamydial inner membrane, which has provided essential information for further delineating the pathways of CPAF action and understanding chlamydial pathogenic mechanisms.

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