Establishment of a comprehensive pathogen protein array for basic and applied research

Staphylococcus aureus represents a major gram-positive human pathogen showing an increasing resistance rate towards many latest-generation antibiotics. In light of this development, there is not only an urgent requirement to in-depth characterize basic S. aureus physiology, but also to accelerate the advancement and concomitant merging of high-throughput technologies focusing on innovative anti-staphylococcal therapy plus prevention strategies. Starting from our in-house established genome-wide collection of full-length S. aureus genes available in recombinational cloning compatible entry format, we thus propose to produce a systematic S. aureus expression ORFeome containing 2562 unique protein-encoding GST-tagged fusion constructs. This flexible resource creates the basis for subsequent innovative S. aureus whole-proteome chip fabrication. Although protein chips (arrays) have in general already been widely acknowledged for assessing biochemical activities or binding specificities in massively parallel fashion, numerous technical challenges related to array fabrication and limited content have, however, so far hampered their ubiquitous adoption. At this juncture, our meanwhile in part already optimized shotgun format in brief directly combines transformed E. coli colony spotting, protein expression, screening for interactors plus subsequent detection on a single nitrocellulose membrane. Applying a mixture of four different inducible E. coli expression strains - each with its own advantageous features -, the problem of bacterial inclusion body formation resulting in denatured protein conformation can be largely overcome. Therefore, the unbiased screening of such a comprehensive protein collection yields many unexpected associations that might otherwise be missed in a more limited design. Further substantial aim of our application will be to fabricate a bona fide focused protein array encompassing 351 recombinantly expressed and natively purified gene products selected for their essential participation in S. aureus growth and survival. Due to enclosed controls, both kinds of arrays will, despite differences in composition and quality, enable the standardization, signal-to-background quantification, quality estimation as well as inter-blot normalization of digitized images. We hence envision both products to play an important role in future S. aureus proteomics reasearch deciphering the molecular subnetworks governing medically relevant phenoma, plus in the discovery of diagnostic markers, vaccine candidates, and therapeutics.

Staphylococcus aureus represents a major gram-positive bacterium temporarily or persistently carried within the nasal mucosa by a significant percentage of the human population. Under particular circumstances that permit or even promote its invasion - like disruption of epithelial barriers or compromised immune status - it can provoke life-threatening catheter-associated (or in general indwelling medical device) bacteremia with concomitant septicaemia, distinct toxin-mediated disorders, etc. Both the increasing emergence and pandemic spreading of S. aureus isolates developing multi-drug resistances to last-barrier antibiotics (such as penicillin, vancomycin or methicillin) thus constitute a significant threat to global human health. For that reason, novel alternatives to the currently fairly limited arsenal of prevention and treatment strategies are required, and such an endeavour can solely be achieved by advancing and merging innovative high-throughput compatible technologies. Because of their intrinsic propensity towards parallelism, low sample consumption, automation, and miniaturisation, protein chips (that consist of hundreds of proteins printed on defined, traceable position on a membrane support) could constitute a valuable screening platform. At this juncture, topic of our granted project (T648-B22) was to assemble two different kinds of protein chip prototypes that differ in underlying manufacturing process, composition, quality as well as comprehensiveness, and are now after three years of successful research ready to be subjected to forthcoming applications. Starting from our in-house established genome-wide collection of full-length genes, we first of all produced a systematic S. aureus expression ORFeome consisting of 2562 unique constructs. The latter were after manifold optimizations exploited for proteome-wide chip production in crude shotgun format, combining transformed E. coli colony spotting, protein expression, mild on-membrane lysis, screening, plus final detection on a single nitrocellulose substratum. In parallel, 505 S. aureus gene products selected for their essential participation in bacterial growth and survival were recombinantly expressed as 6xHIS-tagged fusions, purified one-by-one via IMAC, and used to assemble a bona fide focused protein chip version (90% ready). A first miniature proof-of-concept chip has already successfully reproduced the specific interaction between spotted penicillin-binding protein 2 and a biotinylated beta-lactam compound, demonstrating the practical feasibility for analogous procedures on proteome-scale. Besides, several appealing backup strategies (double-tagged vector system for production of secreted protein chips applying a double-membrane configuration; collection of S. aureus RN4220 strains constitutively overexpressing respective essentials) have been developed, and protein functionality / activity in-depth validated on non-chip (assay) level. Continuous upgrade of our product in terms of quality, dimension or detection sensitivity will ultimately transform our S. aureus protein chip prototype(s) into practicable, robust, reliable tools for interaction screening (deciphering the molecular subnetworks governing medically relevant phenomena); for prioritization of diagnostic markers or vaccine candidates; and for discovery of novel therapeutics (lead compound characterization, enhanced target selectivity, etc.).