In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2-4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9 -14, 72).The sortase A (SrtA) 7 enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between...
Even after extended treatment with powerful antiretroviral drugs, HIV is not completely eliminated from infected individuals. Latently infected CD4 ؉ T cells constitute one reservoir of replication-competent HIV that needs to be eliminated to completely purge virus from antiretroviral drug-treated patients. However, a major limitation in the development of therapies to eliminate this latent reservoir is the lack of relevant in vivo models that can be used to test purging strategies. Here, we show that the humanized BLT (bone marrow-liver-thymus) mouse can be used as both an abundant source of primary latently infected cells for ex vivo latency analysis and also as an in vivo system for the study of latency. We demonstrate that over 2% of human cells recovered from the spleens of HIV-infected BLT mice can be latently infected and that this virus is integrated, activation inducible, and replication competent. The non-tumor-inducing phorbol esters prostratin and 12-deoxyphorbol-13-phenylacetate can each induce HIV ex vivo from these latently infected cells, indicating that this model can be used as a source of primary cells for testing latency activators. Finally, we show activation-inducible virus is still present following suppression of plasma viral loads to undetectable levels by using the antiretroviral drugs zidovudine, indinavir sulfate, and didanosine, demonstrating that this model can also be used to assess the in vivo efficacy of latency-purging strategies. Therefore, the HIV-infected BLT mouse should provide a useful model for assessment of HIV latency activators and approaches to eliminate persistent in vivo HIV reservoirs.
Many virulence factors in GramSurface proteins on bacteria are frequently virulence factors, promoting bacterial adhesion, resistance to phagocytic killing, and host cell invasion during infection. In Gram-positive bacteria these proteins are often covalently anchored to the cell wall by sortase enzymes, a family of novel cysteine transpeptidases (1-3). The sortase A protein (SrtA) 2 from Staphylococcus aureus has been characterized extensively (4) and anchors proteins bearing a cell wall sorting signal that consists of a conserved LPXTG motif (where X is any amino acid), a hydrophobic domain, and a tail of mostly positively charged residues (4 -6). SrtA cleaves in between the threonine and glycine of the LPXTG motif (7) and catalyzes the formation of a peptide bond between the carboxylgroup of the threonine and the amine-group of the cell-wall precursor lipid II (7-9). The lipid II-linked protein is then incorporated into the peptidoglycan of the cell wall via the transglycosylation and transpeptidation reactions of bacterial cell-wall synthesis. Sortases represent an attractive target for new anti-infective agents, because they are widely distributed among a variety of bacterial pathogens (10, 11) (e.g. Bacillus anthracis, Listeria monocytogenes, Streptococcus pneumoniae, and Streptococcus pyogenes), and have been shown to be required for virulence (12-16).The catalytic domain of SrtA (SrtA ⌬N59 , residues 60 -206) adopts a conserved eight-stranded -barrel fold (17, 18). The active site is organized around the catalytically essential side chain of Cys-184, whose thiolate nucleophilically attacks the threonine carbonyl carbon within the LPXTG sorting signal, forming a thioester linkage between the enzyme and substrate (19). In addition to Cys-184, the hydrophilic side chains of His-120 and Arg-197 are absolutely required for catalysis (20 -22). These residues likely participate in general acid/base catalysis, and one of them must activate the thiol for nucleophilic attack, because it is protonated at neutral pH (23). The indole ring of Trp-194 partially shields the cysteine thiol from the solvent, and its mutation to alanine reduces enzyme activity 4-fold through an unknown mechanism (20). Using NMR and crystallography, the LPXTG sorting signal binding site has recently been localized to a surface formed by strands 4 and 7, and to a proximal loop that connects strands 6 to 7(the 6/7 loop) (18,22). Substrate binding may occur through an induced-fit mechanism involving conformational changes in the 6/7 loop, because it is disordered in the absence of the sorting signal substrate (17,18). Ca 2ϩ stimulates the activity of SrtA ⌬N59 in vitro (17) and may enable S. aureus to increase the rate of surface protein anchoring as it encounters elevated concentrations of this ion at sites of infection. Because many surface proteins function as virulence factors, the stimulatory effect of Ca 2ϩ likely plays an important role in the infection process. Previously we showed that Ca 2ϩ bound to an ordered pocket positio...
Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-infective agents as this enzyme attaches virulence factors to the cell wall. Using highthroughput screening, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure-activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione analogs that inhibit SrtA with IC 50 values in the sub-micromolar range. Many of these molecules also inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase inhibitors.
Surface proteins in Gram-positive bacteria are anchored to the cell wall by the action of sortase enzymes. The Staphylococcus aureus sortase A (SrtA) protein anchors proteins by recognizing a cell wall sorting signal containing the amino acid sequence LPXTG. To understand how SrtA binds this sequence, we carried out NMR studies of new peptidylcyanoalkene and peptidyl-sulfhydryl inhibitors that contain the sorting signal sequence LPAT. These studies combined with amino acid mutagenesis identified a catalytically important and conserved binding surface formed by residues A118, T180, and I182. Compatible with its recently proposed role as a general base, R197 is also shown to be required for catalysis.
Sortase enzymes are attractive targets for the development of new anti-infective agents against Gram-positive pathogens because they covalently anchor virulence factors to the cell wall. Here we review what is known about the mechanism of sortase mediated protein anchoring and discuss recently identified inhibitors of this new important enzyme family.
Fish skin, a by-product from fish processing industries, still contains a significant amount of protein-rich material. Gelatin was extracted from Nile tilapia skin with the yield 20.77±0.80 % wet weight. Gelatin was then separately hydrolyzed by proteases, including bromelain, papain, trypsin, flavourzyme, alcalase and neutrase. Low molecular weight gelatin hydrolysate (<10 kDa) has a great potential as an antioxidant agent. Flavourzyme hydrolysate has potent activity on ABTS radical scavenging (1,413.61±88.74 μg trolox/ mg protein) and also inhibits the oxidation of linoleic acid at a high level (59.74±16.57 % inhibition). The greatest reducing power is in alcalase hydrolysate (4.951±1.577 mM trolox/mg protein). While, bromelain hydrolysate has the highest ferrous ion chelating activity (86.895±0.061 %). Evaluation of the angiotensin-I-converting enzyme's inhibitory activity indicates that all hydrolysates have great potency as an antihypertensive agent. All studied tilapia skin gelatin hydrolysates contain potent antioxidant and anti-hypertensive effects.
In this study, for the first time, a biodegradable poly(L-lactide-co-ε-caprolactone), PLC 67:33 copolymer was developed for use as temporary scaffolds in reconstructive nerve surgery. The effect of the surface topology and pore architecture were studied on the biocompatibility for supporting the growth of human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) and human neuroblastoma cells (hNBCs) as cell models. Porous PLC membranes were prepared by electrospinning and phase immersion precipitation with particulate leaching and nonporous PLC membranes were prepared by solvent casting. From the results, the porous PLC membranes can support hWJ-MSCs and hNBCs cells better than the nonporous PLC membrane, and the interconnected pore scaffold prepared by electrospinning exhibited a more significant supporting attachment of the cells than the open pore and nonporous membranes. We can consider that these electrospun PLC membranes with 3-D interconnecting fiber networks and a high porosity warrant a potential use as nerve guides in reconstructive nerve surgery.
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