scholarly journals Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria

2009 ◽  
Vol 191 (18) ◽  
pp. 5603-5612 ◽  
Author(s):  
Irene K. Guttilla ◽  
Andrew H. Gaspar ◽  
Arlene Swierczynski ◽  
Anu Swaminathan ◽  
Prabhat Dwivedi ◽  
...  
Keyword(s):  
Cytoplasmic Membrane ◽  
Nucleophilic Attack ◽  
Electron Microscopic ◽  
Gram Positive ◽  
Lpxtg Motif ◽  
Extracellular Milieu ◽  
Gram Positive Bacteria ◽  
Enzyme Intermediates ◽  
Electron Microscopic Data ◽  
Covalently Linked

ABSTRACT In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.

scholarly journals Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

2006 ◽  
Vol 70 (1) ◽  
pp. 192-221 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Andrea C. DeDent ◽  
Olaf Schneewind
Keyword(s):  
Cell Wall ◽  
Surface Protein ◽  
Surface Proteins ◽  
Nucleophilic Attack ◽  
Gram Positive ◽  
Bacterial Physiology ◽  
Gram Positive Bacteria ◽  
Anchoring Proteins ◽  
Enzyme Intermediates ◽  
Cross Bridges

SUMMARY The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.

scholarly journals Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria

2000 ◽  
Vol 44 (8) ◽  
pp. 2086-2092 ◽  
Author(s):  
Carol L. Friedrich ◽  
Dianne Moyles ◽  
Terry J. Beveridge ◽  
Robert E. W. Hancock
Keyword(s):  
Amino Acid ◽  
Mechanism Of Action ◽  
Cytoplasmic Membrane ◽  
Membrane Depolarization ◽  
Membrane Permeabilization ◽  
Cationic Peptides ◽  
Nuclear Condensation ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Helical Peptide

ABSTRACT Antimicrobial cationic peptides are ubiquitous in nature and are thought to be a component of the first line of defense against infectious agents. It is widely believed that the killing mechanism of these peptides on bacteria involves an interaction with the cytoplasmic membrane. Cationic peptides from different structural classes were used in experiments withStaphylococcus aureus and other medically important gram-positive bacteria to gain insight into the mechanism of action. The membrane potential-sensitive fluorophore dipropylthiacarbocyanine was used to assess the interactions of selected antimicrobial peptides with the cytoplasmic membrane of S. aureus. Study of the kinetics of killing and membrane depolarization showed that, at early time points, membrane depolarization was incomplete, even when 90% or more of the bacteria had been killed. CP26, a 26-amino-acid α-helical peptide with a high MIC against S. aureus, still had the ability to permeabilize the membrane. Cytoplasmic-membrane permeabilization was a widespread ability and an action that may be necessary for reaching an intracellular target but in itself did not appear to be the killing mechanism. Transmission electron microscopy of S. aureus andStaphylococcus epidermidis treated with CP29 (a 26-amino-acid α-helical peptide), CP11CN (a 13-amino-acid, proline- and tryptophan-rich peptide), and Bac2A-NH2 (a linearized version of the 12-amino-acid loop peptide bactenecin) showed variability in effects on bacterial structure. Mesosome-like structures were seen to develop in S. aureus, whereas cell wall effects and mesosomes were seen with S. epidermidis. Nuclear condensation and abherrent septation were occasionally seen in S. epidermidis. Our experiments indicated that these peptides vary in their mechanisms of action and that the mechanism of action likely does not solely involve cytoplasmic-membrane permeabilization.

scholarly journals Antibacterial Properties of Some Cyclic Organobismuth(III) Compounds

2005 ◽  
Vol 49 (7) ◽  
pp. 2729-2734 ◽  
Author(s):  
Toshiaki Kotani ◽  
Daisuke Nagai ◽  
Kensuke Asahi ◽  
Hitomi Suzuki ◽  
Fumiaki Yamao ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibacterial Properties ◽  
Cell Number ◽  
Viable Cell Number ◽  
Electron Microscopic ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Ring Compounds ◽  
Medical Disorders

ABSTRACT Bismuth compounds are known for their low levels of toxicity in mammals, and various types of bismuth salts have been used to treat medical disorders. As part of our program to probe this aspect of bismuth chemistry, cyclic organobismuth compounds 1 to 8 bearing a nitrogen or sulfur atom as an additional ring member have been synthesized, and their antimicrobial activities against five standard strains of gram-negative and gram-positive bacteria were assessed. The eight-membered-ring compounds, compounds 1 to 3, exhibited MICs of less than 0.5 μg/ml against Staphylococcus aureus and were more active than the six-membered ones, compounds 5 to 8 (MICs, 4.0 to 16 μg/ml). The gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis) were more susceptible to both types of ring compounds than the gram-negative ones (Escherichia coli and Pseudomonas aeruginosa). Treatment with polymyxin B nonapeptide increased the susceptibility of E. coli to cyclic organobismuth compounds, indicating the low permeability of the outer membrane of gram-negative bacteria to the compounds. Compound 1 also had activity against methicillin-resistant S. aureus, which had an MIC for 90% of the hospital stock strains of 1.25 μg/ml. The killing curves for S. aureus treated with compound 1 or 3 revealed a static effect at a low dose (2× the MIC). However, when S. aureus was treated with 10× the MIC of compound 1 or 3, there was an approximately 3-log reduction in the viable cell number after 48 h of treatment. Electron microscopic inspection demonstrated a considerable increase in the size of S. aureus and the proportion of cells undergoing cell division after treatment with compound 1 at 0.5× the MIC.

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 The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

2002 ◽  
Vol 70 (3) ◽  
pp. 1382-1390 ◽  
Author(s):  
Caroline Garandeau ◽  
Hélène Réglier-Poupet ◽  
Iharilalao Dubail ◽  
Jean-Luc Beretti ◽  
Patrick Berche ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Protein A ◽  
Knockout Mutant ◽  
Reporter Protein ◽  
Gram Positive ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
Covalently Linked

ABSTRACT Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.

scholarly journals Different subcellular locations of secretome components of Gram-positive bacteria

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2867-2874 ◽  
Author(s):  
Girbe Buist ◽  
Anja N. J. A. Ridder ◽  
Jan Kok ◽  
Oscar P. Kuipers
Keyword(s):  
Cell Wall ◽  
Cytoplasmic Membrane ◽  
Secretion System ◽  
Transport Systems ◽  
Cell Wall Synthesis ◽  
Secretion Systems ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Different Types ◽  
Folded Proteins

Gram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various locations in the cell. The translocons of the general Sec secretion system in the rod-shaped bacterium Bacillus subtilis have been shown to localize in spirals along the cytoplasmic membrane, whereas the translocons in the coccoid Streptococcus pyogenes are located in a microdomain near the septum. In both bacteria the Sec translocons appear to be located near the sites of cell wall synthesis. The Tat secretion system, which is used for the transport of folded proteins, probably localizes in the cytoplasmic membrane and at the cell poles of B. subtilis. In Lactococcus lactis the ABC transporter dedicated to the transport of a small antimicrobial peptide is distributed throughout the membrane. Possible mechanisms for maintaining the localization of these secretion machineries involve their interaction with proteins of the cytoskeleton or components of the cell wall synthesis machinery, or the presence of lipid subdomains surrounding the transport systems.

scholarly journals Characterization of Streptococcus suis Genes Encoding Proteins Homologous to Sortase of Gram-Positive Bacteria

2002 ◽  
Vol 184 (4) ◽  
pp. 971-982 ◽  
Author(s):  
Makoto Osaki ◽  
Daisuke Takamatsu ◽  
Yoshihiro Shimoji ◽  
Tsutomu Sekizaki
Keyword(s):  
Cell Wall ◽  
Streptococcus Suis ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Gram Positive ◽  
Two Dimensional Gel Electrophoresis ◽  
Gram Positive Bacteria ◽  
Genes Encoding ◽  
Covalently Linked ◽  
Sorting System

ABSTRACT Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptideglycan by a specific enzyme called sortase. Using the type strain of Streptococcus suis, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated srtA, was linked to gyrA, as were the sortase and sortase-like genes of other streptococci. Three genes, designated srtB, srtC, and srtD, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated srtE, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of Streptococcus gordonii and Staphylococcus aureus, except that SrtA of S. suis had 65% identity with that of S. gordonii. Isogenic mutants deficient for srtA, srtBCD, or srtE were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with N-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the srtA mutant compared with that of the parent strain, and this phenotype was completely complemented by srtA cloned from S. suis. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the srtA mutant appeared in the profiles of the srtBCD and srtE mutants. These results provide evidence that the cell wall sorting system involving srtA is also present in S. suis.

scholarly journals The antibiotic negamycin crosses the bacterial cytoplasmic membrane by multiple routes

2021 ◽  
Author(s):  
Daniel Hörömpöli ◽  
Catherine Ciglia ◽  
Karl-Heinz Glüsenkamp ◽  
Lars Ole Haustedt ◽  
Hildegard Falkenstein-Paul ◽  
...  
Keyword(s):  
Cytoplasmic Membrane ◽  
Binding Mode ◽  
Blood Levels ◽  
Gram Positive ◽  
Gram Negative ◽  
Bacterial Membranes ◽  
Target Interaction ◽  
Gram Positive Bacteria ◽  
Multiple Routes ◽  
Resistance Rates

Negamycin is a natural pseudo-dipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli. Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of DtpD, a proton-dependent oligopeptide transporter. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions point to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multi-faceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

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