β-lactamases are the most common agents of antimicrobial resistance in bacteria (Bush et al., 1995). These enzymes translocate to the periplasm via the Sec or Tat pathway (Pradel et al., 2008) depending on their signal sequence at the N terminal (Berks et al., 2003). Most well-known β-lactamases are known to translocate via the Sec pathway, which transports unfolded proteins across the inner membrane while a few are known to proceed via the Tat pathway which transports folded proteins. In this project, we aimed to investigate the basis of this differential β-lactamase translocation by studying the translocation of BKC-1, a plasmid-borne carbapenemase recently reported in Brazilian clinical strains of Klebsiella pneumoniae (Nicoletti et al., 2015).
Signal peptides of various β-lactamases from BLDB were analysed using TatP and sequence alignment was carried out using Clustal Omega. Antibiotic sensitivity assays were carried out using broth dilution and protein expression profiles in whole cell lysates and periplasmic fractions were determined using Western blots.
Among the 1089 β-lactamase sequences that were fed into TatP, only 14, including BKC-1, were predicted to be Tat translocated. BKC-1 from K.pneumoniae revealed to possess a repeating h region in the signal peptide when aligned with BKC-1 from Shinella zoogloeoides. Strains lacking TatC and expressing BKC-1 continued to depict beta-lactam resistant phenotypes similar to those of the wild type with the native signal sequence but yielded relatively sensitive phenotypes when this sequence was modified with a shorter hydrophobic chain. This was consistent with BKC-1 protein expression levels.
We thus suggest that translocation of BKC-1 is not only dependant on the presence of the twin arginine motif, but also on the hydrophobicity and length of the signal peptide and the type of organism. This study also suggests that a β-lactamase that may be originally Tat translocated can potentially modify its signal sequence or be dual-targeting when acquired by another organism depending on the similarity of the Tat complex between the organisms.