Recombinant Staphylococcus aureus Uncharacterized leukocidin-like protein 1 (SAR2107)

What is SAR2107 and how does it relate to characterized S. aureus leukocidins?

SAR2107 belongs to the family of Staphylococcus aureus leukocidins, which are pore-forming toxins that target and kill human immune cells. These proteins are critical virulence factors in S. aureus pathogenesis. Similar to characterized leukocidins like LukAB, SAR2107 likely contributes to immune evasion by targeting and killing phagocytes such as human monocytes. LukAB specifically has been demonstrated to be a major contributor to human monocyte death during S. aureus infection . As an uncharacterized leukocidin-like protein, SAR2107 may share structural and functional similarities with better-characterized family members while potentially possessing unique target specificity or mechanism of action.

What are the recommended storage conditions for recombinant SAR2107?

Based on protocols for similar leukocidin-like proteins such as SAR2108, optimal storage of recombinant SAR2107 likely depends on its formulation. For lyophilized preparations, storage at -20°C/-80°C typically provides a shelf life of approximately 12 months. For proteins in liquid form, the shelf life is generally around 6 months at -20°C/-80°C . To maintain protein stability, it is advisable to:

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for no more than one week

• Add glycerol (5-50% final concentration) for long-term storage

• Aliquot reconstituted protein to minimize freeze-thaw damage

What expression systems are most effective for producing recombinant SAR2107?

E. coli expression systems have been successfully used to produce recombinant leukocidin-like proteins from S. aureus, as demonstrated with SAR2108 . When designing expression constructs for SAR2107, researchers should consider:

• Codon optimization for the host expression system

• Inclusion of appropriate affinity tags for purification

• Expression of the mature protein without the signal peptide

• Use of tightly controlled inducible promoters to manage potential toxicity to the expression host

For optimal protein folding and solubility, expression conditions such as temperature, induction time, and inducer concentration should be systematically optimized.

How does SAR2107 interact with host immune cell receptors?

While specific receptor interactions for SAR2107 have not been fully characterized, research on related leukocidins provides a framework for investigation. For instance, LukAB targets CD11b receptors on human monocytes . Research approaches to identify SAR2107 receptors should include:

Researchers should pay particular attention to integrins like CD11b, which has been demonstrated as the receptor for LukAB .

What role does the inflammasome play in SAR2107-mediated cell death?

Based on studies of LukAB, the NLRP3 inflammasome likely plays a significant role in the cellular response to SAR2107. LukAB has been shown to activate Caspase 1, promote IL-1β secretion, and induce necrosis in human monocytes through the NLRP3-ASC inflammasome . To investigate SAR2107's interaction with inflammasome components, researchers should consider:

• Measuring activation of Caspase 1 following SAR2107 exposure

• Quantifying IL-1β secretion in response to SAR2107

• Evaluating the requirement for NLRP3 and ASC using knockout cell lines

• Comparing intracellular versus extracellular exposure models

It's important to note that the cellular context may significantly influence these interactions. With LukAB, extracellular toxin-mediated killing requires ASC, NLRP3, and CD11b, while killing by phagocytosed bacteria expressing LukAB is independent of ASC and NLRP3 but still dependent on CD11b .

How do extracellular versus intracellular infection models affect SAR2107 activity?

The study of LukAB has revealed that the cellular context significantly impacts toxin-mediated killing mechanisms. Research approaches should address both scenarios:

When designing these experiments, researchers should establish appropriate controls including heat-inactivated toxin, receptor-blocking antibodies, and inflammasome inhibitors to dissect the precise mechanisms involved .

What purification strategies yield functional recombinant SAR2107?

Based on protocols used for related leukocidin proteins, a systematic purification approach should include:

• Expression optimization in E. coli or other suitable hosts

• Initial capture using affinity chromatography (His-tag or other fusion tags)

• Tag removal using specific proteases if the tag affects function

• Further purification using ion exchange and size exclusion chromatography

• Quality control assessment using SDS-PAGE (target purity >85%)

For SAR2107 specifically, researchers should validate protein functionality after each purification step using cytotoxicity assays with human monocytes or other target cells.

How can protein-protein interactions between SAR2107 and host factors be characterized?

Understanding the interactions between SAR2107 and host proteins is crucial for elucidating its mechanism of action. Recommended approaches include:

When conducting these studies, researchers should consider the oligomeric state of SAR2107, as many leukocidins function as bi-component toxins requiring partner proteins for full activity.

What cell-based assays are most appropriate for measuring SAR2107 cytotoxicity?

To evaluate the cytotoxic activity of SAR2107, researchers should employ multiple complementary assays:

When establishing these assays, it's essential to include appropriate controls such as LukAB (positive control) and non-pore-forming proteins (negative control). Cell types should include THP1 monocytic cells as a model system and primary human monocytes for physiological relevance .

How does genetic diversity in S. aureus strains affect SAR2107 expression and activity?

To address strain variation, a comprehensive approach should include:

• Genomic analysis across clinical isolates to identify SAR2107 sequence variants

• Quantitative PCR and proteomics to measure expression levels under various conditions

• Functional comparison of recombinant SAR2107 variants

• Correlation of SAR2107 sequence/expression with strain virulence in infection models

This approach will help identify whether SAR2107 contributes to strain-specific virulence patterns and could reveal naturally occurring functional variants with distinct activities or target specificities.

What strategies can neutralize SAR2107 activity in experimental or therapeutic contexts?

Based on approaches used with other leukocidins, potential neutralization strategies include:

• Monoclonal antibodies targeting critical epitopes required for pore formation

• Soluble receptor decoys that compete for toxin binding

• Small molecule inhibitors that prevent oligomerization or pore formation

• Peptide inhibitors designed to disrupt protein-protein interactions

When developing neutralization approaches, researchers should consider:

• Specificity for SAR2107 versus cross-reactivity with other leukocidins

• Potential for complementary targeting of multiple toxins simultaneously

• Effects on both extracellular and intracellular toxin activity

• Compatibility with existing antimicrobial approaches

How can SAR2107 research contribute to vaccine development against S. aureus?

Investigating SAR2107 as a potential vaccine component requires:

This research should be conducted in the context of developing multi-component vaccines that target multiple virulence factors, as S. aureus pathogenesis involves numerous redundant mechanisms .