An Inhibitory Effect of Dryocrassin ABBA on Staphylococcus aureus vWbp That Protects Mice From Pneumonia.
ABSTRACT: Von Willebrand factor-binding protein (vWbp), secreted by Staphylococcus aureus (S. aureus), can activate host prothrombin, convert fibrinogen to fibrin clots, induce blood clotting, and contribute to pathophysiology of S. aureus-related diseases, including infective endocarditis, staphylococcal sepsis and pneumonia. Therefore, vWbp is an promising drug target in the treatment of S. aureus-related infections. Here, we report that dryocrassin ABBA (ABBA), a natural compound derived from Dryopteris crassirhizoma, can significantly inhibit the coagulase activity of vWbp in vitro by directly interacting with vWbp without killing the bacteria or inhibiting the expression of the vWbp. Using molecular dynamics simulations, we demonstrate that ABBA binds to the "central cavity" in the elbow of vWbp by interacting with Arg-70, His-71, Ala-72, Gly-73, Tyr-74, Glu-75, Tyr-83, and Gln-87 in vWbp, thus interfering with the binding of vWbp to prothrombin. Furthermore, in vivo studies demonstrated that ABBA can attenuate injury and inflammation of mouse lung tissues caused by S. aureus and increase survival of mice. Together these findings indicate that ABBA is a promising lead drug for the treatment of S. aureus-related infections. This is the first report of potential inhibitor which inhibit the coagulase activity of vWbp by directly interacting with vWbp.
Project description:For screening the active phloroglucinols on influenza virus (H5N1) from Dryopteris crassirhizoma NaKai, a database was established including twenty-three phloroglucinols that had been isolated from Dryopteris crassirhizoma. Their inhibitory effect on the neuraminidase (NA) of influenza virus H5N1 was screened by molecular docking. As a result, three candidates were selected. The rhizomes of D. crassirhizoma were subjected to isolation and purification processes to obtain the inhibitor candidates. Thirteen phloroglucinols were obtained, including three selected candidates and two new phloroglucinols. The five phloroglucinols were investigated for their inhibitory activity on NA in vitro. The results showed that dryocrassin ABBA and filixic acid ABA exhibited inhibitory effects on NA with IC50 as 18.59 ± 4.53 and 29.57 ± 2.48 ?M, respectively, and the other three phloroglucinols showed moderate inhibitory activity. Moreover, the anti-influenza virus (H5N1) activity and cytotoxicity of dryocrassin ABBA and filixic acid ABA were tested on Madin-Darby canine kidney (MDCK) cells with the cell counting kit-8 (CCK8) method. The results confirmed that dryocrassin ABBA exhibited an inhibitory activity with low cytotoxicity (TC50 > 400 ?M) against influenza virus (H5N1) which will have to be investigated in further detail. In conclusion, phloroglucinols from D. crassirhizoma were shown to have anti-influenza virus activity, and especially dryocrassin ABBA, one of the phloroglucinols, may have the potential to control influenza virus (H5N1) infection.
Project description:During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.
Project description:The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine.
Project description:Staphylococcus aureus produces numerous factors that facilitate survival in the human host. S. aureus coagulase (Coa) and von Willebrand factor-binding protein (vWbp) are known to clot plasma through activation of prothrombin and conversion of fibrinogen to fibrin. In addition, S. aureus clumping factor A (ClfA) binds fibrinogen and contributes to platelet aggregation via a fibrinogen- or complement-dependent mechanism. Here, we evaluated the contribution of Coa, vWbp and ClfA to S. aureus pathogenesis in a rabbit model of skin and soft tissue infection. Compared to skin abscesses caused by the Newman wild-type strain, those caused by isogenic coa, vwb, or clfA deletion strains, or a strain deficient in coa and vwb, were significantly smaller following subcutaneous inoculation in rabbits. Unexpectedly, we found that fibrin deposition and abscess capsule formation appear to be independent of S. aureus coagulase activity in the rabbit infection model. Similarities notwithstanding, S. aureus strains deficient in coa and vwb elicited reduced levels of several proinflammatory molecules in human blood in vitro. Although a specific mechanism remains to be determined, we conclude that S. aureus Coa, vWbp and ClfA contribute to abscess formation in rabbits.
Project description:The two coagulases, von Willebrand factor binding protein (vWbp) and Coagulase (Coa), are critical virulence factors in several animal models of invasive Staphylococcus aureus (S. aureus) infections. These proteins are part of an intricate system of proteins that S. aureus uses to assemble a fibrinogen (Fg)/fibrin protective shield surrounding itself. This shield allows the microorganism to evade clearance by the host phagocytic cells. The coagulases can non-proteolytically activate the zymogen prothrombin to convert Fg to fibrin and promote the Fg/fibrin shield formation. The coagulases also bind directly to Fg and the interaction between Coa and Fg has been previously characterized in some detail. However, the mechanism(s) by which vWbp interacts with Fg remains unclear. Here, we show that vWbp and Coa have distinct interactions with Fg, despite being structurally similar. Coa binds with a significantly higher affinity to soluble Fg than to Fg coated on a plastic surface, whereas vWbp demonstrates no preference between the two forms of Fg. The two coagulases appear to target different sites on Fg, as they do not compete with each other in binding to Fg. Similar to Coa, both the N- and C-terminal halves of vWbp (vWbp-N, vWbp-C, respectively) harbor Fg-binding activities. The higher affinity Fg-binding activity resides in vWbp-N; whereas, the C-terminal region of Coa encompasses the major Fg-binding activity. Peptides constituting the previously identified Coa/Efb1 Fg-binding motif fail to inhibit vWbp-C from binding to Fg, indicating that vWbp-C lacks a functional homolog to this motif. Interestingly, the N-terminal prothrombin-binding domains of both coagulases recognize the Fg ?-chain, but they appear to interact with different sequence motifs in the host protein. Collectively, our data provide insight into the complex interactions between Fg and the S. aureus coagulases.
Project description:Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.
Project description:The intractability of bacterial resistance presents a dilemma for therapies against Staphylococcus aureus (S. aureus) infection. Effective anti-virulence strategies are urgently needed, reflecting the proliferation of resistant strains. Inhibitors of sortase A (SrtA), enzymes that anchor virulence-related surface proteins, are regarded as promising candidates for countermeasures against bacterial infections. In the present study, the inhibitory effect of dryocrassin ABBA (ABBA) against SrtA and its molecular basis has been examined. Fluorescence resonance energy transfer (FRET) assays were used to determine the inhibitory activity of ABBA against SrtA. To identify the mechanism underlying this activity, molecular dynamics simulations and mutagenesis assays were applied, and the results revealed that the direct engagement of SrtA via ABBA through binding to V166 and V168 significantly attenuated the catalytic activity of SrtA. Taken together, these findings indicated that ABBA is a potential novel antimicrobial agent for S. aureus infection via targeting SrtA.
Project description:Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options.
Project description:The blood-clotting protein fibrinogen has been implicated in host defense following <i>Staphylococcus aureus</i> infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared <i>S. aureus</i> following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator <i>vWbp</i> was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate <i>S. aureus</i> deficient in <i>vWbp</i>, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.
Project description:<i>Dryopteris crassirhizoma</i> Nakai is a fern plant with important evolutionary and medicinal values. Herein, we assembled the complete chloroplast genome of <i>D. crassirhizoma</i> by next-generation sequencing technology. The complete chloroplast genome of <i>D. crassirhizoma</i> was 153,355 bp in length, and the GC content was 42.86%; the genome consisted of a pair of inverted repeats (IRs, 23,470 bp), a small single copy region (SSC, 21,570 bp) and a large single copy region (LSC, 84,854 bp). The genome contained 111 genes, namely, 73 protein-coding genes, 34 tRNA genes and four rRNA genes. The phylogenetic analysis suggested that both <i>D. crassirhizoma</i> and <i>D. decipiens</i> from Dryopteridaceae were most closely related to <i>Lepisorus clathratus</i> from Polypodiaceae.