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 Inhibition of Protein Secretion inEscherichia coliand Sub-MIC Effects of Arylomycin Antibiotics

2018 ◽  
Vol 63 (2) ◽  
pp. e01253-18 ◽  
Author(s):  
Shawn I. Walsh ◽  
David S. Peters ◽  
Peter A. Smith ◽  
Arryn Craney ◽  
Melissa M. Dix ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Protein Identification ◽  
Secretory Pathway ◽  
Signal Peptidase ◽  
Type I ◽  
Content Type ◽  
Multidimensional Protein Identification Technology ◽  
Bacterial Type

ABSTRACTAt sufficient concentrations, antibiotics effectively eradicate many bacterial infections. However, during therapy, bacteria are unavoidably exposed to lower antibiotic concentrations, and sub-MIC exposure can result in a wide variety of other effects, including the induction of virulence, which can complicate therapy, or horizontal gene transfer (HGT), which can accelerate the spread of resistance genes. Bacterial type I signal peptidase (SPase) is an essential protein that acts at the final step of the general secretory pathway. This pathway is required for the secretion of many proteins, including many required for virulence, and the arylomycins are a class of natural product antibiotics that target SPase. Here, we investigated the consequences of exposingEscherichia colicultures to sub-MIC levels of an arylomycin. Using multidimensional protein identification technology mass spectrometry, we found that arylomycin treatment inhibits the proper extracytoplasmic localization of many proteins, both those that appear to be SPase substrates and several that do not. The identified proteins are involved in a broad range of extracytoplasmic processes and include a number of virulence factors. The effects of arylomycin on several processes required for virulence were then individually examined, and we found that, at even sub-MIC levels, the arylomycins potently inhibit flagellation, motility, biofilm formation, and the dissemination of antibiotic resistance via HGT. Thus, we conclude that the arylomycins represent promising novel therapeutics with the potential to eradicate infections while simultaneously reducing virulence and the dissemination of resistance.

scholarly journals Characterization of a Novel Microcin That Kills Enterohemorrhagic Escherichia coli O157:H7 and O26

2012 ◽  
Vol 78 (18) ◽  
pp. 6592-6599 ◽  
Author(s):  
Lauren J. Eberhart ◽  
James R. Deringer ◽  
Kelly A. Brayton ◽  
Ashish A. Sawant ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterohemorrhagic Escherichia Coli ◽  
Logarithmic Phase ◽  
Type I ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
E Coli ◽  
Secretion Pathway ◽  
Dependent Inhibition

ABSTRACTA novel phenotype was recently identified in which specific strains ofEscherichia coliinhibit competingE. colistrains via a mechanism that was designated “proximity-dependent inhibition” (PDI). PDI-expressing (PDI+)E. coliis known to inhibit susceptible (PDI−)E. colistrains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC)E. colistrains. In this study, every strain from a genetically diverse panel ofE. coliO157:H7 (n= 25) and additional strains ofE. coliserovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI−strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encodedtolCalso resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity ofE. colistrains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens.

scholarly journals An Alternative Terminal Step of the General Secretory Pathway in Staphylococcus aureus

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Arryn Craney ◽  
Melissa M. Dix ◽  
Ramkrishna Adhikary ◽  
Benjamin F. Cravatt ◽  
Floyd E. Romesberg
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Signal Peptidase ◽  
Type I ◽  
Wild Type ◽  
Content Type ◽  
Intrinsic Signals ◽  
Translocated Proteins ◽  
A Site

ABSTRACT Type I signal peptidase (SPase) is essential for viability in wild-type bacteria because the terminal step of the bacterial general secretory pathway requires its proteolytic activity to release proteins from their membrane-bound N-terminal leader sequences after translocation across the cytoplasmic membrane. Here, we identify the Staphylococcus aureus operon ayrRABC (SA0337 to SA0340) and show that once released from repression by AyrR, the protein products AyrABC together confer resistance to the SPase inhibitor arylomycin M131 by providing an alternate and novel method of releasing translocated proteins. Thus, the derepression of ayrRABC allows cells to bypass the essentiality of SPase. We demonstrate that AyrABC functionally complements SPase by mediating the processing of the normally secreted proteins, albeit in some cases with reduced efficiency and either without cleavage or via cleavage at a site N-terminal to the canonical SPase cleavage site. Thus, ayrRABC encodes a secretion stress-inducible alternate terminal step of the general secretory pathway. IMPORTANCE Addressing proteins for proper localization within or outside a cell in both eukaryotes and prokaryotes is often accomplished with intrinsic signals which mediate membrane translocation and which ultimately must be removed. The canonical enzyme responsible for the removal of translocation signals is bacterial type I signal peptidase (SPase), which functions at the terminal step of the general secretory pathway and is thus essential in wild-type bacteria. Here, we identify a four-gene operon in S. aureus that encodes an alternate terminal step of the general secretory pathway and thus makes SPase nonessential. The results have important implications for protein secretion in bacteria and potentially for protein trafficking in prokaryotes and eukaryotes in general.

scholarly journals Cloning and Characterization of Archaeal Type I Signal Peptidase from Methanococcus voltae

2003 ◽  
Vol 185 (20) ◽  
pp. 5936-5942 ◽  
Author(s):  
Sandy Y. M. Ng ◽  
Ken F. Jarrell
Keyword(s):  
Gene Product ◽  
Signal Peptidase ◽  
Type I ◽  
Peptidase Activity ◽  
E Coli ◽  
Methanococcus Voltae ◽  
Signal Peptidase I ◽  
Archaeal Gene ◽  
Enzyme Source

ABSTRACT Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.

scholarly journals Origins of Yersinia pestis Sensitivity to the Arylomycin Antibiotics and the Inhibition of Type I Signal Peptidase

2015 ◽  
Vol 59 (7) ◽  
pp. 3887-3898 ◽  
Author(s):  
Danielle B. Steed ◽  
Jian Liu ◽  
Elizabeth Wasbrough ◽  
Lynda Miller ◽  
Stephanie Halasohoris ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Protein Secretion ◽  
Lipid A ◽  
Etiologic Agent ◽  
Signal Peptidase ◽  
Type I ◽  
Small Contribution ◽  
Content Type ◽  
Lipopeptide Antibiotics ◽  
Signal Peptidase I

ABSTRACTYersinia pestisis the etiologic agent of the plague. Reports ofY. pestisstrains that are resistant to each of the currently approved first-line and prophylactic treatments point to the urgent need to develop novel antibiotics with activity against the pathogen. We previously reported thatY. pestisstrain KIM6+, unlike mostEnterobacteriaceae, is susceptible to the arylomycins, a novel class of natural-product lipopeptide antibiotics that inhibit signal peptidase I (SPase). In this study, we show that the arylomycin activity is conserved against a broad range ofY. pestisstrains and confirm that it results from the inhibition of SPase. We next investigated the origins of this unique arylomycin sensitivity and found that it does not result from an increased affinity of theY. pestisSPase for the antibiotic and that alterations to each component of theY. pestislipopolysaccharide—O antigen, core, and lipid A—make at most only a small contribution. Instead, the origins of the sensitivity can be traced to an increased dependence on SPase activity that results from high levels of protein secretion under physiological conditions. These results highlight the potential of targeting protein secretion in cases where there is a heavy reliance on this process and also have implications for the development of the arylomycins as an antibiotic with activity againstY. pestisand potentially other Gram-negative pathogens.

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 Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

scholarly journals Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Homer Pantua ◽  
Elizabeth Skippington ◽  
Marie-Gabrielle Braun ◽  
Cameron L. Noland ◽  
Jingyu Diao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Susceptibility ◽  
Pathogenic Bacteria ◽  
Susceptibility Testing ◽  
Regulatory Elements ◽  
Signal Peptidase ◽  
Type Ii ◽  
Mechanisms Of Resistance ◽  
Content Type ◽  
E Coli

ABSTRACT Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the Escherichia coli type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic E. coli strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of lspA that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in E. coli and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use. IMPORTANCE Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.

scholarly journals Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
G. J. Staton ◽  
S. D. Carter ◽  
S. Ainsworth ◽  
J. Mullin ◽  
R. F. Smith ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Signal Peptidase ◽  
Effective Vaccine ◽  
Digital Dermatitis ◽  
Diverse Range ◽  
Content Type ◽  
Signal Peptidase I

ABSTRACT Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

scholarly journals Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

2014 ◽  
Vol 80 (17) ◽  
pp. 5542-5550 ◽  
Author(s):  
Zihe Liu ◽  
Lifang Liu ◽  
Tobias Österlund ◽  
Jin Hou ◽  
Mingtao Huang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Secretory Pathway ◽  
Single Point ◽  
Point Mutations ◽  
Model Organisms ◽  
Cell Factory ◽  
Amylase Secretion ◽  
Content Type ◽  
Inverse Metabolic Engineering

ABSTRACTThe increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeastSaccharomyces cerevisiaeis widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in theVTA1gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.

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