Silver is known to possess anti-microbial properties that are of origin. It is believed that either Ag atoms bind to thiol groups in bacterial enzymes or Ag ions enter bacterial cells and denature the DNA molecule to kill bacteria. Silver , however, may kill bacteria by another mechanism: it is possible that the sharp tips of the nanorods puncture bacterial cells and kill bacteria via impalement-a mechanism. To test if this can indeed happen, we have compared the anti-microbial properties of silver and CdS nanorods. No significant difference is found between the two even though CdS does not possess the chemical properties of silver. This indicates that the physical kill mechanism is indeed likely and therefore nanorods of material may possess anti-microbial properties. In that case, it is possible to overcome serious short- and long-term health hazard issues which have been posed by silver nanoparticles by replacing them with nanorods of innocuous elements or compounds. A surface containing nanorods of varying heights presents an undulating bed of spikes to microbes and is most inhospitable to bacteria.

In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell Penetrating Peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable “cargo” , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and PVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice Structured Illumination Microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

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