Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

Scott A. Lujan, Laura M. Guogas, Heather Ragonese, Steven W. Matson, Matthew R. Redinbo,
PNAS 2007 vol. 104 no. 30 12282-12287

Since mid sixties when so-called “R factors” were found to spread antibiotic resistance between different bacterial strains, lots of effort has been put on finding the simplest and universal method to stop horizontal plasmid transfer. Many papers described the effect of various substances on conjugation. Many substrates have a detrimental effect on plasmid transfer just by influencing the bacterial propagation and general life functions. Sometimes the results could be quite surprising and unexpected, but some ordinary everyday products like green tea (epigallocatechin gallate), coffee (caffeine), and papaya seed macerate can inhibit conjugation and ipso facto spreading of an antibiotic resistance. The most common approach to this kind of study is just to add the material under investigation to the conjugation mixture and evaluate plasmid transfer efficiency over a time. Conjugation is a very complicated, multistep process. The functions necessary for conjugation are encoded by plasmids and do not depend on the host. Many studies have been done to establish the function of particular genes. One of the most important proteins involved in plasmid transfer is a DNA relaxase. The conjugative relaxase initiates DNA transfer with a site- and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5′-phosphotyrosine intermediate. The nicked T-strand moves from the donor cell to the recipient cell via an intercellular junction mediated by a type IV secretion system. The relaxase completes DNA transfer by reversing the covalent phosphotyrosine linkage and releasing the T-strand. In the F plasmid, this relaxase is located in the N-terminal domain of a large multifunctional protein, TraI (DNA helicase I). The relaxase active site contains one or several tyrosine residues: F-like relaxases (found in IncF, IncN, IncP9 and IncW plasmids) contain 2 to 5 tyrosines while relaxases of IncQ, IncP, IncI plasmids and Ti plasmid of Agrobacterium sp. possess only one. F-like relaxases encoded by the traI gene shares significant sequence identity with relaxases of many R plasmids (e.g., 98% with R100 TraI); thus, the F plasmid serves as a model system for examining conjugative plasmids and the inhibition of conjugative transfer.
In this study the authors first describe the role that the relaxase enzyme plays in the initiation and termination of DNA conjugation and then use that information to identify potent relaxase-specific inhibitors. This is the first paper which described a bottom-up approach to identify the first small molecule inhibitors of conjugative DNA transfer.
The authors determined the 2.4-Å crystal structure of the 300-residue N-terminal relaxase domain of F plasmid TraI and found it similar to other, previously described relaxase domains. They found that the tyrosine at the active site is responsible for binding the oriT thymidine. Based on electron density they found presence of a divalent cation in the active site and identified it as Mg2+. A survey of magnesium-binding proteins in the Protein Data Bank revealed that the chelation of Mg2+ by neutral amino acid residues is diagnostic of a site that simultaneously binds to multiple phosphate groups. Mutation of the metal-chelating residue histidine-159 to glutamic acid eliminated relaxase activity. These data indicated that the 2+ charge on the bound metal ion is critical to relaxase function. The proposed models for relaxase role in binding DNA strands during conjugation suggested that relaxase binds two phosphate groups. To prove this theory authors used a simple and relatively stable bisphosphonate – imidobisphosphate (PNP) molecule and found that at nanomolar concentration PNP inhibited relaxase activity in vitro. Further studies established that relaxase can be effectively inhibited by substrates where two phosphonate residues are separated by three or fewer atoms and have no additional negative charge at pH 7.4. Five additional inhibitors were found: methylenediphosphonic acid (PCP), iminobis(methylphosphonic acid) (PCNCP), etidronic acid (ETIDRO), clodronic acid (CLODRO), and 1,2-bis(dimethoxyphosphoryl)benzene (PBENP). ETIDRO and CLODRO are bisphosphonates clinically approved as drugs used to treat bone loss by inhibiting farnesyl diphosphate synthase Two other inhibitors identified, PCP and PNP, have been used as radioisotope carriers in humans. The simplest inhibitors, PCP, ETIDRO, and CLODRO, were then characterized further by using a kinetic assay and exhibited purely competitive inhibition, with Kic,app values ranging from 3 to 145 nM. Taken together with the PNP results, these data validate the prediction that F-like conjugative relaxases can accommodate two phosphotyrosine intermediates simultaneously within their active sites. Significantly, these data also establish that bisphosphonates (including clinically approved compounds) potently inhibit the in vitro relaxase activity of F TraI with Ki values in the nanomolar range.
The in vivo tests confirmed the results obtained in vitro. In addition to conjugation inhibition, micromolar concentrations of PNP caused death of plasmid-containing, but not plasmid free cells by blocking relaxase activity.
The presented results show that the clinically approved bisphosphonates etidronate (Didronel) and clodronate (Bonefos) are potently effective at killing F+ cells and preventing conjugative DNA transfer. These particular compounds could also be combined with existing antibiotics to create potent antimicrobial cocktails. Etidronate and clodronate exhibit low absorption and can be administered at high oral doses. According to the authors, extrapolating from the results, approved doses of etidronate and clodronate would be expected kill >90% of plasmid + cells and to stop >80% of conjugative transfer within the gastrointestinal tract. Such results are relatively mild, given the large bacterial populations present in the gastrointestinal tract or at wound sites, but may be enough shift the balance toward success in a variety of recalcitrant clinical infections, especially given the prevalence of conjugative plasmids within multidrug-resistant bacterial strains. The treatment of skin infections, primary sites of nosocomial antibiotic resistance transfer, using topical applications of bisphosphonates may also be effective. In summary, this study establishes conjugative relaxases as a unique antimicrobial target. The results suggest that approved therapeutics could have an immediate impact, alone or in combination with existing antibiotics, in the prevention of resistance propagation during clinical treatment of bacterial infections, thus extending the lifetime of our antibiotic arsenal.
In conclusion this paper shows us a very important thing: basic research on the conjugation process in plasmid transfer that showed the crucial role of relaxase protein led to more detailed application studies that give us a potential weapon to fight bacterial conjugation and the spread of antibiotic resistance in the world of microorganisms.

Additional papers.

Zhao, W.-H. , Z.-Q. Hu, Y. Hara and T. Shimamura 2001 Inhibition by epigallocatechin gallate (EGCg) of conjugative R plasmid transfer in Escherichia coli. J.Infect. Chemotherapy 7: 195-197

Tiagunenko IuV, Glatman LI, Antsiferova NG., 1975. Caffeine as an inhibitor of the conjugation transfer of R-factors. A study of certain aspects of the mechanism of action of caffeine on the conjugation transfer of R-factors], Antibiotiki 20: 253-257.

Leite A.A.M., Nardi R.M.D., Nicoli J.R., Chartone-Souza E. and Nascimento A.M.A., 2005. Carica papaya seed macerate as inhibitor of conjugative R plasmid transfer from Salmonella typhimurium to Escherichia coli in vitro and in the digestive tract of gnotobiotic mice. Gen. Appl. Microbiol. 51: 21-26

Fernandez-Lopez R., Machón C., Longshaw C.M., Martin S., Molin S., Zechner E.L., Espinosa M., Lanka E. and de la Cruz F. 2005. Unsaturated fatty acids are inhibitors of bacterial conjugation. Microbiology 151: 3517–3526


dr Jaroslaw E. Krol
UofI