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High-throughput sequencing enhanced phage display identifies peptides that bind mycobacteria.

ABSTRACT: Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections.

SUBMITTER: Ngubane NA

PROVIDER: S-EPMC3827053 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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Project description:PURPOSE:Osteosarcoma represents the most common malignant primary bone tumor in childhood; however, the survival rate has remained unchanged for the past 20 years. To improve existing diagnosis and treatment methods and broaden the spectrum of imaging agents that can be used for early detection and assessment of tumor response to therapy, we performed a phage display-based screening for peptide sequences that bind specifically to osteosarcoma cells. EXPERIMENTAL DESIGN:From the Ph.D.-12 phage display peptide library composed of 2.7 x 10(9) different displayed peptides, one peptide was enriched after four rounds of in vitro selection in 143B osteosarcoma tumor cells with 293T human embryonic kidney cells as a control. Both the peptide and the phage clone displaying the peptide were conjugated with fluorescent dyes for in vitro cell and ex vivo tumor tissue stainings. The peptide was further labeled with (18)F for positron emission tomography imaging studies. Cell uptake and efflux and ex vivo biodistribution were also done with (18)F-labeled osteosarcoma specific peptide. RESULTS:ASGALSPSRLDT was the dominant sequence isolated from biopanning and named as OSP-1. OSP-1 shares a significant homology with heparinase II/III family protein, which binds and reacts with heparan sulfate proteoglycans. The fluorescence staining showed that FITC-OSP-1-phage or Cy5.5-OSP-1 had high binding with a panel of osteosarcoma cell lines, much less binding with UM-SCC1 human head and neck squamous cell carcinoma cells, and almost no binding with 293T cells, whereas the scrambled peptide OSP-S had virtually no binding to all the cell lines. (18)F-OSP-1 had significantly higher accumulation in 143B tumor cells both in vitro and in vivo than (18)F-OSP-S. (18)F-OSP-1 also had higher uptake in 143B tumors than in UM-SCC-1 tumors. CONCLUSIONS:Our data suggest that OSP-1 peptide is osteosarcoma specific, and the binding site of OSP-1 might be related to heparan sulfate proteoglycans. Appropriately labeled OSP-1 peptide has the potential to serve as a novel probe for osteosarcoma imaging.

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High-throughput sequencing enhanced phage display identifies peptides that bind mycobacteria.

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