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 Inactivation of the srtA Gene inStreptococcus gordonii Inhibits Cell Wall Anchoring of Surface Proteins and Decreases In Vitro and In Vivo Adhesion

2001 ◽  
Vol 69 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Tové C. Bolken ◽  
Christine A. Franke ◽  
Kevin F. Jones ◽  
Gloria O. Zeller ◽  
C. Hal Jones ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein A ◽  
Surface Protein ◽  
Surface Proteins ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Human Fibronectin ◽  
Gram Positive Cocci

ABSTRACT The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium Staphylococcus aureus. There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013–5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine ifsrtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordoniiexhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci.

Covalent Sortase A Inhibitor ML346 Prevents Staphylococcus aureus Infection of Galleria mellonella

2021 ◽  
Author(s):  
Xiang-Na Guan ◽  
Tao Zhang ◽  
Teng Yang ◽  
Ze Dong ◽  
Song Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Galleria Mellonella ◽  
Surface Proteins ◽  
Sortase A ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Aureus Infection ◽  
Cell Wall Peptidoglycan

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation...

scholarly journals A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

2004 ◽  
Vol 72 (5) ◽  
pp. 2710-2722 ◽  
Author(s):  
David Comfort ◽  
Robert T. Clubb
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Proteins ◽  
Sequence Motifs ◽  
Comparative Genome Analysis ◽  
Gram Positive ◽  
Microbial Genomes ◽  
Searchable Database ◽  
Gram Positive Bacteria ◽  
Related Proteins

ABSTRACT Surface proteins in gram-positive bacteria are frequently required for virulence, and many are attached to the cell wall by sortase enzymes. Bacteria frequently encode more than one sortase enzyme and an even larger number of potential sortase substrates that possess an LPXTG-type cell wall sorting signal. In order to elucidate the sorting pathways present in gram-positive bacteria, we performed a comparative analysis of 72 sequenced microbial genomes. We show that sortase enzymes can be partitioned into five distinct subfamilies based upon their primary sequences and that most of their substrates can be predicted by making a few conservative assumptions. Most bacteria encode sortases from two or more subfamilies, which are predicted to function nonredundantly in sorting proteins to the cell surface. Only ∼20% of sortase-related proteins are most closely related to the well-characterized Staphylococcus aureus SrtA protein, but nonetheless, these proteins are responsible for anchoring the majority of surface proteins in gram-positive bacteria. In contrast, most sortase-like proteins are predicted to play a more specialized role, with each anchoring far fewer proteins that contain unusual sequence motifs. The functional sortase-substrate linkage predictions are available online (http://www.doe-mbi.ucla.edu/Services/Sortase/ ) in a searchable database.

scholarly journals Novel Sortase A Inhibitors to Counteract Gram-Positive Bacterial Biofilms

Proceedings ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 23
Author(s):  
Maria Valeria Raimondi ◽  
Roberta Listro ◽  
Maria Grazia Cusimano ◽  
Mery La Franca ◽  
Teresa Faddetta ◽  
...  
Keyword(s):  
Cell Wall ◽  
Surface Proteins ◽  
Bacterial Biofilms ◽  
Sortase A ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Membrane Enzyme

Sortase A (SrtA) is a membrane enzyme responsible for the covalent anchoring of surface proteins on the cell wall of Gram-positive bacteria. [...]

scholarly journals Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara D. Siegel ◽  
Brendan R. Amer ◽  
Chenggang Wu ◽  
Michael R. Sawaya ◽  
Jason E. Gosschalk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Disulfide Bond ◽  
Cell Envelope ◽  
Teichoic Acid ◽  
Surface Proteins ◽  
Wall Teichoic Acid ◽  
Gram Positive ◽  
Content Type ◽  
X Ray Crystallography ◽  
Gram Positive Bacteria

ABSTRACT The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-β-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM. Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms. IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

scholarly journals Cell wall sorting signals in surface proteins of gram-positive bacteria.

1993 ◽  
Vol 12 (12) ◽  
pp. 4803-4811 ◽  
Author(s):  
O. Schneewind ◽  
D. Mihaylova-Petkov ◽  
P. Model
Keyword(s):  
Cell Wall ◽  
Surface Proteins ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Sorting Signals

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 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 PREDICTION OF CELL WALL SORTING SIGNALS IN GRAM-POSITIVE BACTERIA WITH A HIDDEN MARKOV MODEL: APPLICATION TO COMPLETE GENOMES

2008 ◽  
Vol 06 (02) ◽  
pp. 387-401 ◽  
Author(s):  
ZOI I. LITOU ◽  
PANTELIS G. BAGOS ◽  
KONSTANTINOS D. TSIRIGOS ◽  
THEODORE D. LIAKOPOULOS ◽  
STAVROS J. HAMODRAKAS
Keyword(s):  
Cell Wall ◽  
Markov Model ◽  
Hidden Markov Model ◽  
Hidden Markov ◽  
Surface Proteins ◽  
Data Sets ◽  
Cell Wall Proteins ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Sorting Signals

Surface proteins in Gram-positive bacteria are frequently implicated in virulence. We have focused on a group of extracellular cell wall-attached proteins (CWPs), containing an LPXTG motif for cleavage and covalent coupling to peptidoglycan by sortase enzymes. A hidden Markov model (HMM) approach for predicting the LPXTG-anchored cell wall proteins of Gram-positive bacteria was developed and compared against existing methods. The HMM model is parsimonious in terms of the number of freely estimated parameters, and it has proved to be very sensitive and specific in a training set of 55 experimentally verified LPXTG-anchored cell wall proteins as well as in reliable data sets of globular and transmembrane proteins. In order to identify such proteins in Gram-positive bacteria, a comprehensive analysis of 94 completely sequenced genomes has been performed. We identified, in total, 860 LPXTG-anchored cell wall proteins, a number that is significantly higher compared to those obtained by other available methods. Of these proteins, 237 are hypothetical proteins according to the annotation of SwissProt, and 88 had no homologs in the SwissProt database — this might be evidence that they are members of newly identified families of CWPs. The prediction tool, the database with the proteins identified in the genomes, and supplementary material are available online at .

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