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Pepe, M. V.

Publications and source records attributed to Pepe, M. V..

2 recordsLinked to original sources
bioRxiv2022

The SLAPTAG: A new molecular tag adapted for the development of a high-performance, low-cost, affinity chromatography system

The SLAPTAG is a novel molecular TAG derived from a protein domain present in the sequence of Lactobacillus acidophilus SlpA (SlpA284-444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAG and efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). Different binding and elution conditions were evaluated to set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC). The binding equilibrium between SLAPTAG and BM was reached after a few minutes at 4{degrees}C, being the apparent dissociation constant (KD) of 4.3 {micro}M, a value which is similar to different Kd determined for other S-layer proteins and their respective bacterial cell walls. A reporter protein was generated (H6-GFP-SLAPTAG) to compare the efficiency of the SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAG purification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE (a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmag was successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.

biochemistry↗
bioRxiv2022

Reversible adhesion by type IV pili leads to formation of irreversible localized clusters

Despite the fact a fundamental first step in the physiopathology of many disease-causing bacteria is the formation of long-lived, localized, multicellular clusters, the spatio-temporal dynamics of the cluster formation process, particularly on host tissues, remains poorly understood. Experiments on abiotic surfaces suggest that the colonization of a surface by swimming bacteria requires i) irreversible adhesion to the surface, ii) cell proliferation, and iii) a phenotypic transition from an initial planktonic state. Here, we investigate how Pseudomonas aeruginosa (PA) infects a polarized MDCK epithelium and show that contrary to what has been reported on the colonization of abiotic surfaces, PA forms irreversible bacterial clusters on apoptotic epithelial cell without requiring irreversible adhesion, cell proliferation, or a phenotypic transition. By combining experiments and a mathematical model, we reveal that the cluster formation process is regulated by type IV pili (T4P). Furthermore, we unveil how T4P quantitatively operate during adhesion on the biotic surface, finding that it is a stochastic process that involves an activation time, requires the retraction of pili, and results in reversible adhesion with a characteristic attachment time. Using a simple kinetic model, we explain how such reversible adhesion process leads to the formation of irreversible bacterial clusters and quantify the cluster growth dynamics.

biophysics↗