Recombinant Staphylococcus aureus Serine protease htrA-like (SAUSA300_0923)
Description
Protein Characteristics
HtrA-like serine proteases in S. aureus are characterized by their dual chaperone and protease activities. These proteins serve crucial roles in stress response and virulence factor regulation. Similar to other HtrA family members, the SAUSA300_0923 protein contains conserved domains that facilitate its proteolytic activity against misfolded proteins.
Based on comparative analysis with other S. aureus HtrA-like proteins, such as MW0903, these proteins typically exhibit high purity levels (>90% as determined by SDS-PAGE) when produced recombinantly . The recombinant forms are often tagged, with His-tags being particularly common to facilitate purification and detection .
Sequence Analysis
The amino acid sequence of S. aureus HtrA-like proteases is characterized by specific functional domains. For comparative reference, the full-length sequence of a similar HtrA-like protease (MW0903) comprises 769 amino acids . The sequence contains regions responsible for both protease activity and substrate recognition. Analysis of these sequences reveals the presence of a catalytic triad characteristic of serine proteases, which is essential for their enzymatic function.
Enzymatic Properties
HtrA-like proteases in Staphylococcus species demonstrate distinctive enzymatic characteristics. Drawing from studies on similar proteins, these proteases typically display maximum proteolytic activity at temperatures above 40°C, consistent with their role in heat stress response . This temperature-dependent activity profile distinguishes them from many other bacterial proteases and aligns with their biological function in stress response.
The enzymatic activity of HtrA-like proteases is typically inhibited by aprotinin, a protease inhibitor with high selectivity for serine proteases, confirming their classification within the serine protease family . Studies on similar HtrA proteins have demonstrated their ability to hydrolyze various synthetic and natural peptides, suggesting versatility in substrate recognition .
Recombinant Expression
The production of recombinant S. aureus HtrA-like proteases typically employs Escherichia coli expression systems. This approach enables the generation of sufficient quantities of purified protein for structural and functional analyses. The recombinant proteins are frequently fused with purification tags, particularly His-tags at the N-terminus, to facilitate efficient isolation and purification .
Table 1: Recombinant Production Specifications for S. aureus HtrA-like Proteases
| Parameter | Specification |
|---|---|
| Expression System | E. coli |
| Tag Type | N-terminal His-tag |
| Protein Length | Full Length (typically 700+ amino acids) |
| Physical Form | Lyophilized powder |
| Purity | >90% (SDS-PAGE verification) |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
Reconstitution Protocols
Proper reconstitution is critical for maintaining the functional integrity of recombinant HtrA-like proteases. The recommended protocol involves briefly centrifuging the vial before opening and reconstituting the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Addition of glycerol (typically to a final concentration of 50%) is recommended for long-term storage to prevent protein degradation and maintain activity .
Stress Response Mechanisms
S. aureus encodes multiple HtrA-like proteases that contribute to bacterial survival under various stress conditions. Research has demonstrated that these proteases are essential for thermal stress survival, particularly in some strains like COL . The inactivation of HtrA1 in the RN6390 strain, for example, results in increased sensitivity to puromycin-induced stress, highlighting their role in protein quality control during cellular stress .
Contribution to Virulence
HtrA-like proteases significantly influence the virulence potential of S. aureus. In strain RN6390, studies have shown that a double mutant lacking both HtrA1 and HtrA2 displayed reduced expression of several secreted virulence factors that comprise the agr regulon . This observation correlated with the disappearance of the agr RNA III transcript in the mutant strain, suggesting that HtrA proteases may influence virulence gene regulation .
The impact of HtrA proteases on virulence has been demonstrated in animal models of infection. The RN6390 strain with mutations in both HtrA1 and HtrA2 showed diminished virulence in a rat model of endocarditis . This finding underscores the importance of these proteases in the pathogenesis of S. aureus infections and highlights their potential as therapeutic targets.
Strain-Specific Differences
An important aspect of HtrA-like proteases in S. aureus is that their roles can vary significantly between different strains. While HtrA1 and HtrA2 are both essential for thermal stress survival in the COL strain, only HtrA1 demonstrated a slight effect on exoprotein expression in this strain . Additionally, unlike in the RN6390 strain, HtrA mutations did not diminish the virulence of the COL strain in the rat model of endocarditis .
These strain-specific differences suggest that the functions of HtrA-like proteases in S. aureus are influenced by the genetic background of the strain, likely depending on specific differences in the regulation of virulence factor and stress protein expression .
Comparison with Mycobacterial HtrA Proteases
Studies on HtrA-like proteases in other bacterial species provide valuable insights into their general properties and functions. For instance, research on ML0176, an HtrA-like protease in Mycobacterium leprae, has shown that this protein can hydrolyze a variety of synthetic and natural peptides . Like S. aureus HtrA-like proteases, the M. leprae enzyme displays maximum proteolytic activity at temperatures above 40°C and is completely inactivated by aprotinin .
Mechanistic Insights
Based on comparative studies, it has been proposed that HtrA proteins in S. aureus act in the agr-dependent regulation pathway by ensuring proper folding and/or maturation of some surface components of the agr system . This suggests a mechanism through which these proteases could influence virulence gene expression and, consequently, bacterial pathogenesis.
Therapeutic Target Potential
The identification of HtrA-like proteases in pathogenic bacteria like S. aureus presents a potential new target for the development of novel prophylactic and/or therapeutic strategies against bacterial infections . Given their role in stress response and virulence, inhibitors targeting these proteases could potentially compromise bacterial survival and reduce pathogenicity.
Research Applications
Recombinant HtrA-like proteases serve as valuable tools for biochemical and structural studies. They enable investigations into protein-protein interactions, substrate specificity, and the mechanisms through which these proteases influence bacterial physiology and pathogenesis.
Future Research Directions
Future research on S. aureus HtrA-like proteases, including SAUSA300_0923, should focus on:
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Determining the precise mechanisms through which these proteases influence virulence gene regulation
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Identifying specific substrates and interaction partners
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Developing selective inhibitors as potential therapeutic agents
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Investigating strain-specific variations in protease function and regulation
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate specific format requests. Please indicate your preferred format in your order notes, and we will do our best to fulfill your needs.
Note: All protein shipments are standardly packaged with blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: saa:SAUSA300_0923
Q&A
What is the fundamental role of HtrA-like serine proteases in S. aureus?
HtrA (high temperature requirement A) surface proteases are involved in virulence of many pathogens, primarily through their role in stress resistance and bacterial survival. S. aureus encodes two putative HtrA-like proteases, referred to as HtrA1 and HtrA2. These proteins contribute to pathogenicity by controlling the production of certain extracellular factors crucial for bacterial dissemination .
In S. aureus, HtrA proteins have strain-specific functions, likely depending on differences in virulence factor regulation and stress protein expression. Research indicates these proteases may act in the agr-dependent regulation pathway by ensuring proper folding and/or maturation of some surface components of the agr system .
How should researchers handle and store recombinant S. aureus HtrA-like proteases for optimal activity?
For optimal handling of recombinant S. aureus Serine protease htrA-like (SAUSA300_0923):
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Storage conditions: Store at -20°C/-80°C upon receipt. For extended storage, maintain at -20°C or -80°C .
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Reconstitution: When supplied as lyophilized powder, briefly centrifuge the vial prior to opening. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .
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Aliquoting: Prepare working aliquots to avoid repeated freeze-thaw cycles, which can significantly reduce protein activity.
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Short-term storage: Store working aliquots at 4°C for up to one week .
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Stabilization: Add glycerol (5-50% final concentration) for long-term storage. This enhances protein stability .
What experimental systems are recommended for studying HtrA protease activity?
Based on research protocols involving similar serine proteases, several experimental approaches are effective:
A. Protease Activity Assays:
Similar to HtrA2/Omi assays, researchers can adapt this method for S. aureus HtrA-like proteases :
| Materials | Specifications |
|---|---|
| Assay Buffer | 50 mM Tris, pH 8.0 |
| Recombinant Protein | S. aureus Serine protease htrA-like |
| Substrate | β-Casein (1.0 mg/mL stock in 25 mM Tris, 0.15 M NaCl, pH 7.5) |
| Analysis | SDS-PAGE and silver staining reagents |
Protocol:
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Dilute substrate to 0.4 mg/mL in Assay Buffer
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Dilute recombinant protein to 0.04 mg/mL in Assay Buffer
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Add 25 μL of substrate and 25 μL of recombinant protein to reaction tube
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Include a control with 25 μL substrate and 25 μL Assay Buffer
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Incubate the reaction and control for 1 hour at 37-45°C
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Stop the reaction by adding SDS-PAGE sample buffer
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Analyze the cleavage by SDS-PAGE followed by silver staining
B. Cell-Based Systems:
For studying the effects on host cells, normal human epidermal keratinocytes (NHEK) have been successfully used to investigate S. aureus protease activity .
How do strain variations affect the function of HtrA proteases in S. aureus?
Research indicates significant strain-dependent differences in HtrA function:
| Strain | HtrA1 Function | HtrA2 Function | Combined Effect |
|---|---|---|---|
| RN6390 | Resistance to puromycin-induced stress | Limited role in stress resistance | Affects expression of secreted virulence factors; diminished virulence in rat endocarditis model |
| COL | Essential for thermal stress survival | Essential for thermal stress survival | Slight effect on exoprotein expression; no decrease in virulence in rat endocarditis model |
These differences likely stem from strain-specific variations in virulence factor regulation and stress protein expression. In the RN6390 strain, mutation of both htrA genes correlated with the disappearance of the agr RNA III transcript, suggesting HtrA proteins may influence the agr regulatory system .
What is the relationship between HtrA proteases and the agr regulatory system?
The agr (accessory gene regulator) system is a key quorum-sensing mechanism that controls virulence gene expression in S. aureus. Research suggests HtrA proteins act in the agr-dependent regulation pathway by:
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Ensuring proper folding and/or maturation of surface components of the agr system
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Controlling the production of extracellular factors crucial for bacterial dissemination
In the RN6390 strain, the htrA1 htrA2 double mutant showed disappearance of the agr RNA III transcript, which correlated with defective expression of several secreted virulence factors comprising the agr regulon. This mechanism appears to be strain-specific, as the COL strain showed different phenotypes with htrA mutations .
How can researchers use HtrA mutants to study S. aureus virulence mechanisms?
To investigate HtrA roles in S. aureus, researchers can construct htrA1, htrA2, and htrA1 htrA2 insertion mutants in different genetic backgrounds. The methodology reported in literature includes:
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Construction of htrA1::cat mutant:
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Construction of htrA2::spc mutant:
These mutants can then be tested for:
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Stress sensitivity (thermal, oxidative, puromycin-induced)
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Virulence factor expression
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In vivo virulence using models such as rat endocarditis
How do HtrA-like proteases interact with host cells during S. aureus infection?
S. aureus and its secreted products can modulate host cell protease activity in a strain-dependent manner. Research has shown:
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S. aureus can induce increased serine protease activity in human keratinocytes, with strain-specific effects:
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This induction of host protease activity shows kinetics:
These findings suggest a complex interplay between bacterial HtrA-like proteases and host protease networks that may contribute to pathogenesis.
What are the technical challenges in expressing and purifying active recombinant HtrA proteases?
Several technical considerations are critical when working with recombinant S. aureus HtrA-like proteases:
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Expression systems: E. coli is commonly used, but proper folding of the active protein may require optimization of expression conditions .
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Protein solubility: The membrane-associated nature of these proteins can present solubility challenges. Expression of truncated forms lacking transmembrane domains may improve solubility.
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Activity preservation: Similar to human HtrA proteins, activity can be affected by:
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Storage stability: Repeated freeze-thaw cycles significantly reduce activity. For improved stability:
How do S. aureus HtrA-like proteases compare to HtrA family proteins in other organisms?
HtrA family proteins are conserved across bacterial and eukaryotic species, with varying functional specializations:
| Organism | HtrA Homolog | Function | Similarity to S. aureus HtrA |
|---|---|---|---|
| Human | HTRA2/Omi | Mitochondrial serine protease involved in apoptosis | Shares serine protease domain and PDZ domain architecture |
| Human | HTRA1 | Secreted protease involved in TGF-β signaling and ECM degradation | Contains PDZ domain and similar catalytic mechanisms |
| E. coli | DegP | Chaperone-protease essential for high temperature survival | Functional analogy in stress response |
| L. lactis | HtrA | Thermal stress protection | Both S. aureus HtrA1 and HtrA2 were tested in L. lactis htrA mutant; only HtrA1 conferred thermal stress protection |
The S. aureus HtrA-like proteases appear functionally closer to bacterial stress response proteases than mammalian HtrAs, though they share structural similarities with both groups .
What are emerging therapeutic applications targeting S. aureus HtrA-like proteases?
Given the growing challenge of antibiotic-resistant S. aureus strains, including MRSA, HtrA-like proteases represent promising therapeutic targets:
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Potential advantages of targeting HtrA-like proteases:
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Essential for stress survival in certain strains
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Involvement in virulence factor expression
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Surface accessibility of these proteins
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Conservation across S. aureus strains
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Therapeutic strategies being explored:
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Development of specific HtrA inhibitors
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Antibody-based approaches targeting surface-exposed HtrA domains
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Vaccine development incorporating HtrA epitopes
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Combination approaches targeting both HtrA and agr systems
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Challenges in therapeutic development:
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Strain-specific functions requiring broad-spectrum approaches
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Potential redundancy in protease functions
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Need for selective targeting to avoid cross-reactivity with human HtrA homologs
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What advanced analytical methods are recommended for characterizing HtrA protease-substrate interactions?
For researchers investigating the specificity and mechanisms of S. aureus HtrA-like proteases, several advanced techniques can be employed:
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Proteomic approaches:
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TAILS (Terminal Amine Isotopic Labeling of Substrates) for identification of protease cleavage sites
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PICS (Proteomic Identification of Cleavage Sites) for mapping substrate specificity
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Differential proteomics comparing wild-type and htrA mutant secretomes
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Structural biology:
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X-ray crystallography of HtrA-substrate complexes
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Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic interactions
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Cryo-EM for visualization of larger HtrA complexes (potentially trimeric arrangements seen in other HtrA family proteins)
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Biochemical characterization:
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Fluorescence resonance energy transfer (FRET)-based assays for real-time monitoring of protease activity
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Surface plasmon resonance (SPR) for measuring binding kinetics with potential substrates and inhibitors
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Isothermal titration calorimetry (ITC) for thermodynamic analysis of interactions
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