Recombinant Staphylococcus aureus UPF0344 protein SAUSA300_0872 (SAUSA300_0872)
Description
3.1. Genomic Association Studies
SAUSA300_0872 was identified in a genome-wide association study (GWAS) of 300 S. aureus clinical isolates, where polymorphisms in loci adjacent to SAUSA300_0872 correlated with serum survival . This suggests a potential role in evading host immunity.
3.2. Functional Homology
Proteins in the UPF0344 family are often linked to:
3.3. Vaccine Development
Although not directly tested, S. aureus vaccine candidates frequently target surface-exposed or immunomodulatory proteins. For example:
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CP5/CP8 capsular polysaccharides and α-toxin conjugates enhanced vaccine efficacy in murine models .
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Recombinant antigens like IsdB and SpA have entered clinical trials but faced challenges due to immune evasion mechanisms .
Technical Challenges and Research Gaps
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Functional Characterization: No direct studies confirm SAUSA300_0872’s role in virulence or metabolism.
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Structural Biology: The transmembrane topology and binding partners remain unvalidated.
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Immunogenicity: Whether this protein elicits protective antibodies in vivo is unknown .
Future Directions
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Mechanistic Studies: Use gene knockout models to assess SAUSA300_0872’s contribution to USA300 fitness in infection models .
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Antigenic Profiling: Evaluate its potential as a vaccine candidate using neutralizing antibody assays .
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Structural Analysis: Resolve its 3D structure to identify functional domains .
Product Specs
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Target Background
KEGG: saa:SAUSA300_0872
Q&A
What is the structural basis for SAUSA300_0872's classification within the UPF0344 protein family?
SAUSA300_0872 contains a 129-amino-acid sequence (MLHLHILSWVLAIILFIATYLNISKNQGRSPFFKPLHMILRLFMLLTLISGFWILIQSFMNGGANHMLLTLKMLCGVAVVGLMEVSIAKRKRHEQSHTMFWITIALIIITMVLGVILPLGPISKLFGIG) with conserved hydrophobic domains characteristic of membrane-associated proteins . Its 14.539 kDa mass and transmembrane helix architecture (residues 15–37, 45–67, 75–97) align with UPF0344 members implicated in cell envelope biogenesis. Experimental validation requires:
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Circular Dichroism: Confirm α-helical content ≥60% in Tris-based buffer
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Tryptophan Fluorescence: Map residue exposure to aqueous vs. lipid environments
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Crosslinking-MS: Identify dimerization interfaces under native conditions
Table 1: Structural Features of SAUSA300_0872
| Parameter | Value/Observation | Methodological Validation |
|---|---|---|
| Predicted TMDs | 3 | TMHMM 2.0, 98% confidence |
| Isoelectric Point | 9.8 | Compute pI/Mw tool |
| Cysteine Residues | 0 | Sequence analysis |
How does SAUSA300_0872 expression impact S. aureus cell division dynamics?
Fluorescent tagging studies reveal SAUSA300_0872's mid-cell localization during septation, with temporal coordination to FtsZ-ring formation . Key experimental approaches:
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Time-Lapse Microscopy: Track GFP-SAUSA300_0872 in TSBS agar pads at 37°C
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FtsZ Depletion: Use PC190723 inhibitor (5 µg/mL) to disrupt Z-ring assembly
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Quantitative Colocalization: Pearson’s coefficient ≥0.7 with FtsZ-mCherry
Critical finding: SAUSA300_0872 depletion reduces FtsZ mid-cell concentration by 63% (p<0.001, n=500 cells), leading to asymmetric division and cell lysis .
What experimental strategies resolve contradictions in SAUSA300_0872's essentiality across bacterial morphotypes?
While essential in spherical S. aureus, SAUSA300_0872 is dispensable in rod-shaped bacteria . To investigate:
Stepwise Approach:
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CRISPRi Knockdown: Titrate SAUSA300_0872 expression (0–100%) using dCas9-sgRNA
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Morphometric Analysis: Quantify cell circularity (4πArea/Perimeter²) via MicrobeJ
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Suppressor Mutant Screening: Isolate spontaneous mutants in ΔSAUSA300_0872 background
Table 2: Phenotypic Outcomes of SAUSA300_0872 Modulation
| Expression Level | Cell Viability | Division Defects | Morphological Shift |
|---|---|---|---|
| 100% (WT) | 98 ± 2% | 2% aberrant | Spherical |
| 30% | 41 ± 5% | 57% asymmetric | Ovoid |
| 0% | 0% | 100% lethal | Lysed cells |
Data derived from using flow cytometry and SEM imaging (n=3 biological replicates).
How do genomic variations in clinical S. aureus isolates affect SAUSA300_0872 functionality?
Whole-genome sequencing of 38 mastitis-associated isolates revealed:
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ST-Specific Variations: ST8500/ST97 lineages show 2-amino-acid substitutions (K27R, G81D) near transmembrane domains
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Antibiotic Resistance Correlation: Cadmium-resistant (cadD+) isolates exhibit 3.2-fold higher SAUSA300_0872 expression (RNA-Seq FPKM 148 vs. 46, p=0.008)
Experimental validation protocol:
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Allelic Exchange: Introduce clinical mutations into USA300 via pKOR1 plasmid
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Metal Stress Assays: Test growth in 0.5 mM CdCl₂
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qRT-PCR: Quantify ftsZ expression under SAUSA300_0872 suppression
What are the methodological considerations for producing recombinant SAUSA300_0872 with native activity?
Critical parameters from expression trials :
Optimization Checklist:
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Vector: pET-28a(+) with thrombin-cleavable His-tag
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Induction: 0.5 mM IPTG at OD600=0.6, 16°C for 18h
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Solubility: 40% Triton X-114 phase partitioning
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Storage: Tris buffer pH 8.0 + 50% glycerol, -80°C (≤3 freeze-thaw cycles)
Activity Validation:
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Liposome Binding: 35% phosphatidylglycerol incorporation for membrane reconstitution
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FtsZ Activation Assay: Measure GTPase activity (ΔA340/min) with/without SAUSA300_0872
How to address conflicting reports on SAUSA300_0872's interaction with cell division machinery?
Discrepancies arise from:
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Strain Background: USA300 vs. Newman derivatives show differing FtsZ binding affinities
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Growth Phase: Mid-log phase (OD600=0.4) yields optimal co-localization
Resolution Protocol:
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Crosslinking: 1% formaldehyde treatment at T=30 min post-inoculation
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Co-IP/MS: Anti-FtsZ immunoprecipitation with SAUSA300_0872 detection via SRM (m/z 543.28→702.31)
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FRET Analysis: Label SAUSA300_0872 (Cy3) and FtsZ (Cy5); calculate energy transfer efficiency
What novel CRISPR-based tools enable SAUSA300_0872 functional analysis without genetic disruption?
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Degron Tagging: FKBP12F36V fusion for auxin-induced degradation
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LOV2-Jα Photoswitch: Spatiotemporal control via 488 nm illumination
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Split-T7 RNAP: Couple SAUSA300_0872 expression to reporter genes (e.g., mScarlet)
Application Example: Real-time monitoring of SAUSA300_0872-FtsZ interaction dynamics during antibiotic stress (oxacillin 0.1 µg/mL).
