Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

Abstract

Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs) form conserved protein interaction “hubs” that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

“…We have previously shown that RecA-GFP focus formation is an in vivo marker for replicative stress in B. subtilis (27,30,60,61).…”

“…The coefficient describing the severity of the curve determined by the rate of replication fork collapse is defined here as the fork collapse factor (FCF) (see Materials and Methods). Upon Ϫ19 a Cells were grown in S7 50 defined minimal medium as described previously (27,29,30,60,61). After cells reached an OD 600 of 0.4, cultures were split, with 50 ng/ml phleomycin added to one culture, while the other culture was left untreated.…”

DNA replication proteins as potential targets for antimicrobials in drug-resistant bacterial pathogens

“…These proteins are also an integral part of nucleoprotein complexes involved in recombination and repair 65 , 66 . Although present in all domains of life, the amino acid sequence, subunit composition and oligomeric state of these proteins differ substantially between organisms 65 .…”

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

“…Many such targets exist within the mechanisms of DNA replication. 4,5 Notably, DNA topoisomerases and nucleotide biosynthesis are the only antibiotic targets within this pathway used clinically. 6 Single-stranded DNA (ssDNA)-binding proteins (SSBs) are essential for DNA replication and are well studied in eubacteria, particularly in E. coli (extensively reviewed by Meyer and Laine 7 ).…”