Evaluation of the Seca Inhibitors as Novel Anti-Microbial Agents

The misuse of conventional antibiotics and natural selection of the infectious bacterial population has produced drug resistance. Thus, novel effective antibiotic compounds that treat bacterial infections resistant to available therapies are needed. SecA is an indispensable ATPase of the protein translocation machinery present in all bacteria. SecA is responsible for the secretion of many essential proteins, some toxins and virulence factors, and is essential for bacterial survival. SecA has no counterpart in mammalian cells, thus provides an ideal target for developing antimicrobial agents. SCA-13 (HO) is a pyrimidine analog derived from virtual screening; it exerts the ability to inhibit SecA translocation ATPase activity with an IC50 of 75 µM. HO showed promising bacteriostatic activities against a vacomycin resistant strain of S. aureus Mu50 and B. anthracis Sterne. No significant difference in antimicrobial activity of HO was observed among efflux pump strains of S. aureus, suggesting that compound HO is not a substrate of NorA or MepA efflux pumps. Resistant mutants of E. coli NR698 selected from HO need to be characterized to gain a better understanding of the resistance mechanisms and subsequently will allow for the identification of the drug target

Dimeric SecA Couples the Preprotein Translocation in an Asymmetric Manner

The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles

Nucleotide-Dependent Dimerization of the C-Terminal Domain of the ABC Transporter CvaB in Colicin V Secretion

The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer