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Staphylokinase has distinct modes of interaction with antimicrobial peptides, modulating its plasminogen-activation properties.

ABSTRACT: Staphylokinase (Sak) is a plasminogen activator protein that is secreted by many Staphylococcus aureus strains. Sak also offers protection by binding and inhibiting specific antimicrobial peptides (AMPs). Here, we evaluate Sak as a more general interaction partner for AMPs. Studies with melittin, mCRAMP, tritrpticin and bovine lactoferricin indicate that the truncation of the first ten residues of Sak (Sak?N10), which occurs in vivo and uncovers important residues in a bulge region, improves its affinity for AMPs. Melittin and mCRAMP have a lower affinity for Sak?N10, and in docking studies, they bind to the N-terminal segment and bulge region of Sak?N10. By comparison, lactoferricin and tritrpticin form moderately high affinity 1:1 complexes with Sak?N10 and their cationic residues form several electrostatic interactions with the protein's ?-helix. Overall, our work identifies two distinct AMP binding surfaces on Sak?N10 whose occupation would lead to either inhibition or promotion of its plasminogen activating properties.

SUBMITTER: Nguyen LT

PROVIDER: S-EPMC4995489 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Staphylokinase has distinct modes of interaction with antimicrobial peptides, modulating its plasminogen-activation properties.

Nguyen Leonard T LT Vogel Hans J HJ

Scientific reports 20160824

Staphylokinase (Sak) is a plasminogen activator protein that is secreted by many Staphylococcus aureus strains. Sak also offers protection by binding and inhibiting specific antimicrobial peptides (AMPs). Here, we evaluate Sak as a more general interaction partner for AMPs. Studies with melittin, mCRAMP, tritrpticin and bovine lactoferricin indicate that the truncation of the first ten residues of Sak (SakΔN10), which occurs in vivo and uncovers important residues in a bulge region, improves its ...[more]

PMID: 27554435

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Project description:With the growing problem of the emergence of antibiotic-resistant bacteria, the search for alternative ways to combat bacterial infections is extremely urgent. While analyzing the effect of antimicrobial peptides (AMPs) on immunocompetent cells, their effect on all parts of the immune system, and on humoral and cellular immunity, is revealed. AMPs have direct effects on neutrophils, monocytes, dendritic cells, T-lymphocytes, and mast cells, participating in innate immunity. They act on B-lymphocytes indirectly, enhancing the induction of antigen-specific immunity, which ultimately leads to the activation of adaptive immunity. The adjuvant activity of AMPs in relation to bacterial and viral antigens was the reason for their inclusion in vaccines and made it possible to formulate the concept of a "defensin vaccine" as an innovative basis for constructing vaccines. The immunomodulatory function of AMPs involves their influence on cells in the nearest microenvironment, recruitment and activation of other cells, supporting the response to pathogenic microorganisms and completing the inflammatory process, thus exhibiting a systemic effect. For the successful use of AMPs in medical practice, it is necessary to study their immunomodulatory activity in detail, taking into account their pleiotropy. The degree of maturity of the immune system and microenvironment can contribute to the prevention of complications and increase the effectiveness of therapy, since AMPs can suppress inflammation in some circumstances, but aggravate the response and damage of organism in others. It should also be taken into account that the real functions of one or another AMP depend on the types of total regulatory effects on the target cell, and not only on properties of an individual peptide. A wide spectrum of biological activity, including direct effects on pathogens, inactivation of bacterial toxins and influence on immunocompetent cells, has attracted the attention of researchers, however, the cytostatic activity of AMPs against normal cells, as well as their allergenic properties and low stability to host proteases, are serious limitations for the medical use of AMPs. In this connection, the tasks of searching for compounds that selectively affect the target and development of an appropriate method of application become critically important. The scope of this review is to summarize the current concepts and newest advances in research of the immunomodulatory activity of natural and synthetic AMPs, and to examine the prospects and limitations of their medical use.

Project description:Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

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Project description:Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr(90)-Glu(100)) for the docking of the substrate PG to the SAK-Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr(92)-Glu(100)) from the SAK 90-loop, resulted in approximately 60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKDelta90) and Pm. The preformed activator complex of SAKDelta90 with Pm, however, displayed a 50-60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK-Pm complex displayed approximately 50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKDelta90. Lysine residues (Lys(94), Lys(96), Lys(97) and Lys(98)) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAK(K97A) and SAK(K98A) exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the K(m) values of PG towards the activator complex of these two SAK mutants were 4-6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys(97) and Lys(98) residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK-Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.

Dataset Information

Staphylokinase has distinct modes of interaction with antimicrobial peptides, modulating its plasminogen-activation properties.

Publications

Staphylokinase has distinct modes of interaction with antimicrobial peptides, modulating its plasminogen-activation properties.

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