Natural and Synthetic Sortase A Substrates Are Processed by Staphylococcus aureus via Different Pathways.

Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation.

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 µM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 µM, 100 µM and 250 µM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.

Sortase-mediated backbone cyclization of proteins and peptides.

Backbone cyclization has a profound impact on the biological activity and thermal and proteolytic stability of proteins and peptides. Chemical methods for cyclization are not always feasible, especially for large peptides or proteins. Recombinant Staphylococcus aureus sortase A shows potential as a new tool for the cyclization of both proteins and peptides. In this review, the scope and background of the sortase-mediated cyclization are discussed. High efficiency, versatility, and easy access make sortase A a promising cyclization tool, both for recombinant and chemo-enzymatic production methods.

Sortase A as a tool for high-yield histatin cyclization.

Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research. Here, we describe a novel and efficient cyclization method that uses the peptidyl-transferase activity of the Staphylococcus aureus enzyme sortase A to cyclize linear synthetic precursor peptides. As a model, we used histatin 1, a 38-mer salivary peptide with motogenic activity. Chemical cyclization of histatin 1 resulted in ≤ 3% yields, whereas sortase-mediated cyclization provided a yield of >90%. The sortase-cyclized peptide displayed a maximum wound closure activity at 10 nM, whereas the linear peptide displayed maximal activity at 10 µM. Circular dichroism and NMR spectroscopic analysis of the linear and cyclic peptide in solution showed no evidence for conformational changes, suggesting that structural differences due to cyclization only became manifest when these peptides were located in the binding domain of the receptor. The sortase-based cyclization technology provides a general method for easy and efficient manufacturing of large cyclic peptides.

Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides.

The innate immunity factor lactoferrin harbours two antimicrobial moieties, lactoferricin and lactoferrampin, situated in close proximity in the N1 domain of the molecule. Most likely they cooperate in many of the beneficial activities of lactoferrin. To investigate whether chimerization of both peptides forms a functional unit we designed a chimerical structure containing lactoferricin amino acids 17-30 and lactoferrampin amino acids 265-284. The bactericidal activity of this LFchimera was found to be drastically stronger than that of the constituent peptides, as was demonstrated by the need for lower dose, shorter incubation time and less ionic strength dependency. Likewise, strongly enhanced interaction with negatively charged model membranes was found for the LFchimera relative to the constituent peptides. Thus, chimerization of the two antimicrobial peptides resembling their structural orientation in the native molecule strikingly improves their biological activity.

Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania.

Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On these premises, Hst5 activity was assayed on both stages of its life cycle, promastigotes and amastigotes (LC(50)=7.3 and 14.4 microM, respectively). In a further step, its lethal mechanism was studied. The main conclusions drawn were as follows: 1) Hst5 causes limited and temporary damage to the plasma membrane of the parasites, as assessed by electron microscopy, depolarization, and entrance of the vital dye SYTOX Green; 2) Hst5 translocates into the cytoplasm of Leishmania in an achiral receptor-independent manner with accumulation into the mitochondrion, as shown by confocal microscopy; and 3) Hst5 produces a bioenergetic collapse of the parasite, caused essentially by the decrease of mitochondrial ATP synthesis through inhibition of F(1)F(0)-ATPase, with subsequent fast ATP exhaustion. By using the Hst5 enantiomer, it was found that the key steps of its lethal mechanism involved no chiral recognition. Hst5 thus constitutes the first leishmanicidal peptide with a defined nonstereospecific intracellular target. The prospects of its development, by its own or as a carrier molecule for other leishmanicidal molecules, into a novel anti-Leishmania drug with a preferential subcellular accumulation are discussed.

Distinct bactericidal activities of bovine lactoferrin peptides LFampin 268-284 and LFampin 265-284: Asp-Leu-Ile makes a difference.

Two lactoferrampin (LFampin) peptides derived from bovine lactoferrin were compared with respect to their bactericidal activities. LFampin 265-284 killed a set of Gram-positive bacteria that were resistant to LFampin 268-284. The presence of 265Asp-Leu-267Ile did not simply lead to an overall increased potency, since higher concentrations of LFampin 265-284 than LFampin 268-284 were needed to kill the Gram-negative bacteria that were tested. The Asp-Leu-Ile sequence enhances the propensity of LFampin to adopt an alpha-helix, as shown by circular dichroism spectroscopy. These results suggest that the helical conformation of the peptide is an important determinant of the susceptibility of Gram-positive bacteria.

Effect of amino acid substitutions on the candidacidal activity of LFampin 265-284.

LFampin 265-284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells.

Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains.

The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.

Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5.

The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.

Internalisation and degradation of histatin 5 by Candida albicans.

Histatins, salivary antimicrobial peptides, are susceptible to proteolytic degradation, often ascribed to host proteinases. In this study, we addressed the question whether proteolytic activity from microbial sources can contribute to this degradation. Candida albicans, an opportunistic yeast that is susceptible to the histatins, was used as target organism. The most potent histatin (histatin 5: sequence: DSHAKRHHGYKRKFHEKHHSHRGY), two histatin 5 fragments (dh-5: sequence: KRKFHEKHHSHRGY; P-113: sequence: AKRHHGYKRKFH) and an all-D isomer of the latter (P-113D) were used as model peptides. All L-peptides were susceptible to degradation by C. albicans. Cleavage was established at Lys5 and His19 of histatin 5, Lys11, Arg12, Phe14, Glu16, Lys17, His18 and Ser20 of dh-5 and Ala4 and Lys11 of P-113. In addition, it was found that secreted C. albicans enzymes are not involved in the degradation process and that blocking cell entry of the peptides greatly impedes degradation. Moreover, P-113D, which is biologically as active as P-113, was hardly susceptible to proteolysis. These data imply that proteolysis occurs mainly intracellularly and is not used as a protective mechanism against histatin activity. Together, our results suggest that, besides host proteinases, microbial enzymes play an important role in histatin degradation.

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily.

Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.