Staphylokinase Research Articles

Engineered Microrobots for Targeted Delivery of Bacterial Outer Membrane Vesicles (OMV) in Thrombus Therapy.

In the realm of thrombosis treatment, bioengineered outer membrane vesicles (OMVs) offer a novel and promising approach, as they have rich content of bacterial-derived components. This study centers on OMVs derived from Escherichia coli BL21 cells, innovatively engineered to encapsulate the staphylokinase-hirudin fusion protein (SFH). SFH synergizes the properties of staphylokinase (SAK) and hirudin (HV) to enhance thrombolytic efficiency while reducing the risks associated with re-embolization and bleeding. Building on this foundation, this study introduces two cutting-edge microrobotic platforms: SFH-OMV@H for venous thromboembolism (VTE) treatment, and SFH-OMV@MΦ, designed specifically for cerebral venous sinus thrombosis (CVST) therapy. These platforms have demonstrated significant efficacy in dissolving thrombi, with SFH-OMV@H showcasing precise vascular navigation and SFH-OMV@MΦ effectively targeting cerebral thrombi. The study shows that the integration of these bioengineered OMVs and microrobotic systems marks a significant advancement in thrombosis treatment, underlining their potential to revolutionize personalized medical approaches to complex health conditions.

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2–2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex.

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Commercial proteases, such as Nattokinase (NK), Staphylokinase (SAK), and Streptokinase (SK) play an important role in the destruction of thrombus, the main cause of death in cardiovascular disease. The latest technology combining enzymes with certain drugs is the target of new research in the thrombolytic area. The first step is to develop protease from Bacillus thuringiensis HSFI-12 bacteria as an antithrombotic agent, characterization of the bacterial enzyme is necessary. This study aims to determine the specificity of protease from Bacillus thuringiensis HSFI-12 to explore its potential as an antithrombotic agent in terms of anticoagulant and fibrinolytic activities. The molecular weight and specificity of bacterial protease were determined with a zymographic method with casein as substrate. Bacillus thuringiensis HSFI-12 was first cultured on Nutrient Agar (NA) media and then on Skim Milk Agar (SMA) media. The obtained crude protease from Skim Milk Broth (SMB) was then concentrated as dialysate. Both crude and dialysate proteases were tested for their specific activity, as well as anticoagulant and fibrinolytic activities. Next, the dialysate’s molecular weight and specificity on the casein substrate were investigated using the zymographic method. As result, protease activity in crude form is lower than that in dialysate, which was 0.5570 ± 0,004 U/mL and 2.1767 ± 0,005 U/mL, respectively. The molecular weight of the obtained bacterial protease was between 117 – 133 kDa and the enzyme is capable of degrading casein as shown on the zymogram. Overall, both crude and dialysate proteases of Bacillus thuringiensis HSFI-12 show potential as an antithrombotic agent for exhibiting anticoagulant and antiplatelet activities. Yet, it could not exhibit direct fibrinolytic activity implying the possibility that the enzyme plays a role as a plasminogen activator, which can dissolve fibrin by activating plasmin.

Indirect optimization of staphylokinase expression level in dicistronic auto-inducible system

Design of experiment (DOE) is a statistical approach for designing, performing, and interpreting a large set of data with the minimum number of tests. In our previous study, we developed a novel Hsp27 SILEX system for production of recombinant proteins. In the present study, we optimized indirectly the most effective factors including inoculation load, self-induction temperature, and culture media on autoinduction of staphylokinase (SAK) expression using RSM methodology and fluorometry. The expression level of SAK was assayed at different runs after 6 h incubation at 90 rpm. The results indicated all parameters significantly affect the SAK expression level (p < 0.05). The optimum expression condition was obtained with an inoculation load of 0.05, a temperature of 25 °C, and TB culture medium. The analysis of variance with a R2 value of 0.91 showed that a quadratic model well described this prediction (p < 0.05). Applying the optimized condition led to an approximately fourfold increase in the SAK expression level (from 1.3 to 5.2 µg/ml). Moreover, the recombinant protein was purified using immobilized metal affinity chromatography and the activity was also confirmed by semi-quantitative caseinolytic method.

Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin.

Staphylococcus aureus (S. aureus) is an invasive and life-threatening pathogen that has undergone extensive coevolution with its mammalian hosts. Its molecular adaptations include elaborate mechanisms for immune escape and hijacking of the coagulation and fibrinolytic pathways. These capabilities are enacted by virulence factors including microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the plasminogen-activating enzyme staphylokinase (SAK). Despite the ability of S. aureus to modulate coagulation, until now the sensitivity of S. aureus virulence factors to digestion by proteases of the coagulation system was unknown. Here, we used protein engineering, biophysical assays, and mass spectrometry to study the susceptibility of S. aureus MSCRAMMs to proteolytic digestion by human thrombin, plasmin, and plasmin/SAK complexes. We found that MSCRAMMs were highly resistant to proteolysis, and that SAK binding to plasmin enhanced this resistance. We mapped thrombin, plasmin, and plasmin/SAK cleavage sites of nine MSCRAMMs and performed biophysical, bioinformatic, and stability analysis to understand structural and sequence features common to protease-susceptible sites. Overall, our study offers comprehensive digestion patterns of S. aureus MSCRAMMs by thrombin, plasmin, and plasmin/SAK complexes and paves the way for new studies into this resistance and virulence mechanism.

The pro-inflammatory effect of Staphylokinase contributes to community-associated Staphylococcus aureus pneumonia

Pneumonia caused by community-associated Staphylococcus aureus (CA-SA) has high morbidity and mortality, but its pathogenic mechanism remains to be further investigated. Herein, we identify that staphylokinase (SAK) is significantly induced in CA-SA and inhibits biofilm formation in a plasminogen-dependent manner. Importantly, SAK can enhance CA-SA-mediated pneumonia in both wild-type and cathelicidins-related antimicrobial peptide knockout (CRAMP−/−) mice, suggesting that SAK exacerbates pneumonia in a CRAMP-independent manner. Mechanistically, SAK induces pro-inflammatory effects, especially in the priming step of NLRP3 inflammasome activation. Moreover, we demonstrate that SAK can increase K+ efflux, production of reactive oxygen species production, and activation of NF-κB signaling. Furthermore, the NLRP3 inflammasome inhibitor can counteract the effective of SAK induced CA-SA lung infection in mice. Taken together, we speculate that SAK exacerbates CA-SA-induced pneumonia by promoting NLRP3 inflammasome activation, providing new insights into the pathogenesis of highly virulent CA-SA and emphasizes the importance of controlling inflammation in acute pneumonia.

Cloning & expression of SAK enzyme from Staphylococcus aureus in E. coli BL21-CodonPlus.

Staphylokinase (SAK), also known as staphylococcal fibrinolysin, is a protein with a molecular mass of about 15 kDa produced by Staphylococcus aureus. Staphylokinase is synthesized in the late exponential phase, similar to streptokinase. The current study identified and predicted the protein SAK from Staphylococcus aureus. SAK is a fibrinolytic enzyme of the third generation that acts as an indirect activator of plasminogen. The current study cloned and expressed SAK protein isolated from Staphylococcus aureus and used in the form of a grid for enhancement of SAK Catalyst with PCR, disengagement, and change into articulation vector PET24b(+). The recombinant plasmid was changed into E. coli strain BL21 (codon additionally to 440) acceptance with isopropyl β-D-1-thiogalactopyranoside (IPTG).

Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin

Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.

PURIFICATION, CHARACTERIZATION, AND EVALUATION OF FIBRINOLYTIC ACTIVITY OF STAPHYLOKINASE FROM LOCALLY ISOLATED STAPHYLOCOCCUS AUREUS GH38

This study was aimed to purify, characteristic, and fibrinolytic activity of staphylokinase (SAK), is an enzyme activates plasminogen to form plasmin, which digest fibrin clots that cause thrombosis clot. Staphylokinase was purified from local isolate Staphylococcus aureus GH38 by ammonium sulfate precipitation at 70% saturation followed by ion exchange chromatography (CM-Cellulose) with a purification fold 2.73, and 1.53, and recovery 72.1, and 33.11% in wash and elution steps respectively. The partially purified enzyme was high activity at 40°C with pH 7 and the enzyme retained 100% of its activity at 35°C with pH 7. The activity of an enzyme increased by its treatment with calcium and sodium chloride, while the activity affected when incubated with mercury, silver, and iron chloride. The enzyme have high effective against thrombus (blood clot), which encourage the use of enzyme in the treatment as therapeutic agent to remove clots formed in the human body.

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat /Km , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.

Purification and in vivo stability and half-life of recombinant lipid modified staphylokinase

Staphylokinase (SAK), the thrombolytic protein holds a significant position in treating cardiovascular diseases. However, the rapid clearance of this protein from blood circulation reduces its effective usage and as a strategy to increase the half-life of SAK, initial work focussed on lipid modification of SAK (LMSAK) in E. coli GJ1158. Effective purification of the modified protein achieved using the two step method of hydrophobic interaction chromatography in succession with size exclusion chromatography, indicated a better yield. The thrombolytic activity of purified LMSAK analysed in heated plasma agar plate assay confirmed an enhanced activity. In vivo pharmacokinetic studies carried out for determining the half-life of LMSAK in blood circulation of mice presented that it has a half-life of 43.3 ± 3.4 min which is much higher than 21.6 ± 2.1 min that of the unmodified version of SAK. The studies confirmed the role of lipid modification as a crucial factor in confirming in vivo stability of LMSAK and proves to be beneficial in therapeutic usage.

Construction of a novel Staphylokinase (SAK) mutant with low immunogenicity and its evaluation in rhesus monkey

The heterologous nature of SAK, a thrombolytic drug, elicits high titers of neutralizing antibodies, which limits its clinical use. Here, we aim to establish a SAK mutant with equivalent activity to the wild type but reduced antigenicity, which may allow for multiple injections. Biosun software was used to predict SAK antigenic epitopes, and several main epitopes were modified by gene deletion and mutation. Ten SAK mutants were constructed, and their thrombolytic activity and immunogenicity were analyzed in vitro. SAK6, with a high expression level (45%), similar thrombolytic activity, and lower antibody reaction, was chosen for in vivo analysis in rhesus monkey. In the nearly 8-month experimental period, the antibody level of the SAK6 group was significantly lower than that of the SAK group. Moreover, only 5% of SAK activity was retained, whereas 75.6% of SAK6 activity was retained after incubating with respective antiserum. Overall, these results demonstrated that SAK6, established through comprehensive site-directed mutagenesis program, had identical thrombolytic activity to SAK, low immunogenicity, and less side effects, demonstrating its efficient clinical potential for thrombus disease.

Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris

There has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more “life-style”factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke etc. but re-occlusion and bleeding that are co-incident to their clinical usage are not addressed. Hence, there is need to develop thrombolytics having properties like increased fibrin clot specificity and thrombin inhibition capability to prevent re-occlusion. In the present work, a fusion protein construct containing two components i.e. Staphylokinase (SAK) and Epidermal Growth Factor (EGF) 4, 5, 6-like domains of human thrombomodulin (THBD) was expressed in Pichia pastoris after genetic optimization. SAK isolated from Staphylococcus aureus is a fibrin-specific plasminogen activator while EGF 4, 5, 6-like domains are reported to be responsible for imparting thrombin inhibition to human thrombomodulin, and therefore, expected could help prevent re-occlusion in the novel construct - SAK_EGF, which is a 43 kDa protein. After expression, it was purified (approx. 13-fold) using two-step purification protocol involving ion-exchange followed by Gel Filtration Chromatography (GFC). The functional characterization including plasminogen activation and thrombin inhibition showed that both the fusion partners viz. SAK and 4,5,6 EGF-like domains retained their respective activities after fusion, confirming it to be a bio-active construct. Thus, this engineered protein could be clinically promising due to the combinatorial effect of fibrin-specific thrombus lysis and prevention of re-occulusion.

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production.

The methylotrophic yeast Hansenula polymorpha, known as a non-conventional yeast, is used for the last 30 years for the production of recombinant proteins, including enzymes, vaccines, and biopharmaceuticals. Although a large number of reviews have been published elucidating the applications of this yeast as a cell factory, the latest was released about 10 years ago. Therefore, this review aimed at summarizing available information on the use of H. polymorpha as a host for recombinant protein production in the last decade. Examples of chemicals and virus-like particles produced using this yeast also are discussed. Firstly, the aspects that feature this yeast as a host for recombinant protein production are highlighted including the techniques available for its genetic manipulation as well as strategies for cultivation in bioreactors. Special attention is given to the novel genomic editing tools, mainly CRISPR/Cas9 that was recently established in this yeast. Finally, recent examples of using H. polymorpha as an expression platform are presented and discussed. The production of human Parathyroid Hormone (PTH) and Staphylokinase (SAK) in H. polymorpha are described as case studies for process establishment in this yeast. Altogether, this review is a guideline for this yeast utilization as an expression platform bringing a thorough analysis of the genetic aspects and fermentation protocols used up to date, thus encouraging the production of novel biomolecules in H. polymorpha.

Conjugation of staphylokinase with the arabinogalactan-PEG conjugate: Study on the immunogenicity, in vitro bioactivity and pharmacokinetics.

Staphylokinase (SAK) is a bacterial protein with profibrinolytic activity. However, SAK suffers from short serum half-life and high immunogenicity. PEGylation with high Mw (20 kDa or 40 kDa) could decrease the immunogenicity and prolong the serum half-life of the proteins. However, the PEGylated protein could induce the anti-PEG antibodies and its bioactivity was significantly decreased. Arabinogalactan (AG) is a health-promoting substance with numerous biological activities. Conjugation of AG is an alternative strategy to solve the above-mentioned problems. However, conjugation with AG significantly decreased the bioactivity of a protein by shielding the bioactive domain. Here, AG conjugation and PEGylation were combined to improve the therapeutic efficacy of SAK. PEG with low Mw (2 kDa or 5 kDa) acted as a linker to conjugate AG from Larix. As compared with SAK-AG (22.3%), the conjugates (SAK-P2K-AG and SAK-P5K-AG) largely maintained the bioactivity of SAK (73.8% and 62.9%). The two conjugates both showed an 8-fold decrease in the SAK-specific IgG titers and a prolonged serum half-life. Moreover, the conjugates did not render any apparent toxicity to the heart, liver and renal functions of mice. Thus, our conjugation strategy is promising for the development of an effective long-acting therapeutic protein.

Screening and optimization of staphylokinase from Staphylococcus aureus isolated from nasal swab of healthy students in Himachal Pradesh University, India

Background: One of virulence factors produced by Staphylococcus aureus is staphylokinase (SAK), which enhances their proteolytic activity leading to tissue damage and improving bacterial invasiveness. In the present study, we estimated the ability to produce SAK by S. aureus isolates from nasal carriers. We would like to verify relationship between SAK production and different S. aureus isolates. Methods: In this study, all nasal swab samples from healthy nasal carriers were collected and further processed in the Department of Biotechnology, Himachal Pradesh University. All S. aureus isolates were screened for SAK and optimization was done. Results: Out of all S. aureus isolates 20% isolates were positive for SAK production. Isolate SAK-24 shown increase in SAK production after optimization and response surface methodology (RSM). Conclusion: Production of SAK varies from strain to strains. SAK production can be increased by optimization and RSM.

Plasminogen-Dependent Collagenolytic Properties of Staphylococcus aureus in Collagen Gel Cultures of Human Corneal Fibroblasts.

Staphylococcus aureus is a common cause of corneal ulceration, and staphylokinase (SAK) produced by this bacterium is a plasminogen activator. To investigate the pathogenesis of corneal ulceration induced by S. aureus, we examined the effects of bacterial culture broth and SAK on collagen degradation in a culture model in which human corneal fibroblasts are embedded in a collagen gel. Corneal fibroblasts embedded in collagen were exposed to S. aureus culture broth or SAK. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Expression of pro-matrix metalloproteinase-1 (pro-MMP-1) was detected by immunoblot analysis as well as reverse transcription and real-time polymerase chain reaction analysis. Both S. aureus culture broth and SAK markedly increased collagen degradation in the presence of corneal fibroblasts and plasminogen. This effect of the culture broth was dependent on cell number to a greater extent than was that of SAK. Whereas the culture broth also increased the expression of pro-MMP-1 in corneal fibroblasts at both mRNA and protein levels, SAK did not. Our results suggest that S. aureus may promote collagen degradation both by upregulating pro-MMP1 expression in corneal fibroblasts, with pro-MMP-1 then being converted to active MMP-1 by plasmin, and by directing plasmin activity toward collagen in a SAK-dependent manner.

Lipid modification of staphylokinase and its implications on stability and activity

Thrombolytic agents are routinely used to dissolve blood clot by activating fibrinolytic system. Among different thrombolytic agents available, staphylokinase (SAK) is gaining much attention because of their fibrin specificity and reduced inhibition by α2 antiplasmin. Though SAK had exhibited less circulatory half life, they are equipotent to tissue plasminogen activator and streptokinase and had shown more potency for clot dissolution during retracted thrombi. In this study, SAK was lipid modified at the N-terminal by a protein engineering approach to enhance its stability and activity. Native SAK as well as the gene encoding SAK with lipobox was cloned into E. coli GJ1158 using pRSET-B expression vector for higher expression. The lipid modification of SAK was confirmed by a mobility shift of 1.3 kDa against the 15.5 kDa of native SAK using tricine SDS-PAGE. Lipid modification of SAK was confirmed by LC MS/MS. The secondary structure analysis was carried out using circular dichroism and deconvoluted fourier transform infrared spectroscopy. LMSAK was found to have a slightly higher denaturation temperature compared to SAK. The improved stablility and activity of lipid modified SAK was studied by heated plasma agar plate assay and mouse tail bleeding test.

Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from Slaughter Poultry, Their Toxin Gene Profiles and Prophage Patterns

In this work, 85 strains of Staphylococcus aureus were isolated from samples taken from slaughter poultry in Poland. Attempts were made to determine the prophage profile of the strains and to investigate the presence in their genome of genes responsible for the production of five classical enterotoxins (A–E), toxic shock syndrome toxin (TSST-1), exfoliative toxins (ETA and ETB) and staphylokinase (SAK). For this purpose, multiplex PCR was performed using primer-specific pairs for targeted genes. The presence of the mecA gene was found in 26 strains (30.6%). The genomes of one of the methicillin-resistant S. aureus (MRSA) strains and two methicillin-sensitive S. aureus (MSSA) strains contained the gene responsible for the production of enterotoxin A. Only one MRSA strain and two MSSA strains showed the presence of the toxic shock syndrome toxin (tst) gene. Only one of the MSSA strains had the gene (eta) responsible for the production of exfoliative toxins A. The presence of the staphylokinase gene (sak) was confirmed in 13 MRSA strains and in 5 MSSA strains. The study results indicated a high prevalence of prophages among the test isolates of Staphylococcus aureus. In all, 15 prophage patterns were observed among the isolates. The presence of 77-like prophages incorporated into bacterial genome was especially often demonstrated. Various authors emphasize the special role of these prophages in the spread of virulence factors (staphylokinase, enterotoxin A) not only within strains of the same species but also between species and even types of bacteria.

Detection of Sak Gene and Expression of Staphylokinase in Different Clinical Isolates of Staphyloccocus spp.

This study had investigated the presence of Staphylokinase (SAK) phenotypically and genetically. A total of 100 Staphylococcus spp. samples were isolated from different clinical cases. Morphological, biochemical tests and for further conformation; Viteck2 technique were adopted to isolate and diagnose the staphylococcal samples. Phenotypic assay tests including hydrolysis of casein and plasma agar were performed to check for the production of Staphylokinase from producing Staphylococcus spp. Out of 100, only 32 isolates had exhibited (SAK) activity whereby 29 of them represented the species S. aureus while 3 belonged to others. All isolates were subjected to Polymerase Chain Reaction (PCR) to determine the possessed sak gene. This investigation certainly had shown that not all isolates of S. aureus has the sak gene, only the lysogenic one has it and that it is not species committed but also other staphylococcus species could have it.