PATRICE COURVALIN

CONTRIBUTIONS TO SCIENCE

The goal of my scientific work has been to understand the major public health problem of how bacteria pathogenic for humans become resistant to antibiotics. Basing my research on genetic and biochemical approaches, I pioneered the application of molecular biology techniques to the field of medical bacteriology and the use of such approaches on species other than Escherichia coli. Using contemporary state-of-the-art technology, I have described numerous new types of resistance, elucidated their genetic basis and biochemical mechanisms, and developed molecular tools for their detection. In addition, I investigated the origin, evolution, and dissemination of resistance genes, and these findings had significant implications for the understanding of the biology of bacteria and the evolution of their genomes.

Together with colleagues, we have demonstrated that strains of Methicillin-Resistant Staphylococcus aureus (MRSA) were in fact resistant to all ß-lactams, an observation of major therapeutic importance.

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I also provided the first evidence for the presence of plasmids conferring multiple resistance in Enterococcus, a genus that has since become one of the major nosocomial pathogens.

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I contributed to show that some resistance genes found in human pathogens originate from antibiotic producers, the latter having to protect themselves against suicide by the product of their secondary metabolism (1). This finding was further illustrated by the observation in 2002 that aminoglycoside resistance can be conferred in enterobacteria by methylation of their 16S rRNA, a mechanism known at that time only in antibiotic-producing bacteria (12,13,15).

In 1985, my laboratory demonstrated the first transfer of DNA in a natural environment from Gram-positive cocci (staphylococci, streptococci, pneumococci, and enterococci) to Gram-negative bacteria related to Escherichia coli (2). Further studies on the heterologous expression of resistance genes enabled them to explain the polarity of this genetic transfer between the two groups of bacteria that diverged more than a billion years ago: genes from Gram-positive bacteria can be expressed in Gram-negatives whereas the reverse is not true. They succeeded in reproducing this DNA transfer by conjugation under laboratory conditions and in vivo in an animal model. These observations brought an end to the dogma that the exchange of genetic information could only occur between closely-related bacterial species.

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With my collaborators, we elucidated in 1989 the molecular mechanism responsible for the mobility (excision and insertion) of conjugative transposons, which confer multiple resistance in Gram-positive cocci (4). As a consequence, we developed integrative vectors for mutagenesis of Gram-positive bacteria and for cloning of genes from these organisms into E. coli as well as trans-Gram conjugative shuttle vectors (6). These tools have since then been widely used for the genetic study of bacteria pathogenic for humans.

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I demonstrated in 1988 that extended-spectrum b-lactamases, enzymes that confer resistance to the most recent cephalosporins and carbapenems in a large variety of Gram-negative bacteria, are in fact ‘old’ penicillinases with substitutions at single amino acids.

With my group, I studied the origin, and elucidated the genetics (7), biochemistry (17,20), regulation (19), mechanisms for the dissemination of vancomycin resistance in enterococci, and were the first to describe this in 1988 (3). This is a unique and highly sophisticated dual mechanism that involves synthesis of a new ‘resistant’ target and elimination of the ‘susceptible’ target (8). Moreover, the four genes required for resistance are expressed only when required, i.e. in the presence of the antibiotic. This is achieved by means of a two-component regulatory system upstream of the resistance operon. These findings put an end to the dogma that synthesis of the bacterial cell wall could only proceed from peptidoglycan precursors terminating in the dipeptide D-alanine-D-alanine, and have formed the basis for the rational design of new antibiotics that might escape the vancomycin resistance mechanism. In 2010, we evaluated the biological cost for an Enterococcus host when it acquires inducible vancomycin resistance. We showed that, despite the predicted high fitness cost of this dual mechanism, there was no burden on the host in the absence of vancomycin due to the tightly regulated expression of resistance (14). These findings are in agreement with the observation that regulation of expression is common in horizontally acquired resistance and represents an efficient evolutionary pathway for resistance determinants to become selectively neutral in the absence of drugs and favors their persistence in the environment.

We have described and studied acquisition of multiple drug resistance in Listeria monocytogenes (5), Yersinia pestis (9), and chloramphenicol resistance in Neisseria meningitidis (10), which are genetic events with important consequences for the therapy of these major human pathogens.

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Our laboratory has detected and elucidated, starting in 1983, the vast majority of resistance mechanisms found in Acinetobacter baumannii, a bacterial species responsible for epidemics of nosocomial infections. Since 2001, we have shown that multidrug resistance in this species was also due to overexpression of efflux systems and have elucidated their expression regulation mechanisms (21), a work that we are pursuing in close collaboration with the Broad Institute.

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In 1995, my group reported the first direct transfer of DNA from bacteria to mammalian cells. The bacterial vector allows efficient transfer of functional genetic material to a large number of mammalian cells and uses the ability of certain bacteria to invade the host cell to deliver DNA into the cytosol. This represents the ultimate, trans-kingdom, transfer of genetic information when considering the evolutionary distance between the donor and the recipient cells. The bacterial vectors that we engineered are used in numerous laboratories worldwide as tools for the delivery and expression of large genomic DNA constructs, both in vitro and in vivo in animal models (11). Transfer of functional genes from wild type intracellular bacteria was also achieved recently.

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In summary, my research has provided new concepts for horizontal gene transfer and has described numerous new biochemical pathways in bacteria involved in drug resistance. This was made possible by the very early application of the most up-to-date molecular biology techniques to the study of bacterial species pathogenic for humans. This work has provided a solid foundation for the rational design of new antibiotics that can escape clinically relevant resistance mechanisms.

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1. Courvalin, P., B. Weisblum, and J. Davies. 1977. Aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in Escherichia coli. Proc. Natl. Acad. Sci. USA 74:999-1003.

2. Trieu-Cuot, P., G. Gerbaud, T. Lambert, and P. Courvalin. 1985. In vivo transfer of genetic information between Gram-positive and Gram-negative bacteria. EMBO J. 4:3583-3587.

3. Leclercq, R., E. Derlot, J. Duval, and P. Courvalin. 1988. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium.  N. Engl. J. Med. 319:157-161.

4. Poyart-Salmeron, C., P. Trieu-Cuot, C. Carlier, and P. Courvalin. 1989. Molecular characterization of two proteins involved in the excision of the pneumococcal transposon Tn1545: homologies with other site-specific recombinases. EMBO J. 8:2425-2433.

5. Poyart-Salmeron, C., C. Carlier, P. Trieu-Cuot, A. Courtieu, and P. Courvalin. 1990. Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes. Lancet 335:1422-1426.

6. Trieu-Cuot, P., C. Carlier, C. Poyart-Salmeron, and P. Courvalin. 1991. Shuttle vectors containing a multiple cloning site and a lacZ𝑥 gene for conjugal transfer of DNA from Escherichia coli to Gram-positive bacteria. Gene 102:99-104.

7. Arthur, M., C. Molinas, F. Depardieu, and P. Courvalin. 1993. Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J. Bacteriol. 175:117-127.

8. Reynolds, P.E., F. Depardieu, S. Dutka-Malen, M. Arthur, and P. Courvalin. 1994. Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine. Mol. Microbiol. 13:1065-1070.

9. Galimand, M., A. Guiyoule, G. Gerbaud, B. Rasoamanana, S. Chanteau, E. Carniel, and P. Courvalin. 1997. Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. N. Engl. J. Med. 337:677-680. [Editorial of the Journal. 1997, 337:702-704]

10. Galimand, M., G. Gerbaud, M. Guibourdenche, J.-Y. Riou, and P. Courvalin. 1998. High-level chloramphenicol resistance in Neisseria meningitidis. N. Engl. J. Med. 339:868-874. [Editorial of the Journal. 1998, 339:917-918]

11. Grillot-Courvalin, C., S. Goussard, F. Huetz, D.M. Ojcius, and P. Courvalin. 1998. Functional gene transfer from intracellular bacteria to mammalian cells. Nat. Biotechnol. 16:862-866.

12. Galimand, M., P. Courvalin, and T. Lambert. 2003. Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob. Agents Chemother. 47:2565-2571. [Journal Highlights. ASM News. 2003, 69:457]

13. Liou, G.F., S. Yoshizawa, P. Courvalin, and M. Galimand. 2006. Aminoglycoside resistance by ArmA-mediated ribosomal 16S methylation in human bacterial pathogens. J. Mol. Biol. 359:358-364.

14. Foucault, M.-L., F. Depardieu, P. Courvalin, and C. Grillot-Courvalin. 2010. Inducible expression eliminates the fitness cost of vancomycin resistance in enterococci. Proc. Natl. Acad. Sci. U.S.A. 107:16964-16969.

15. Galimand, M., E. Schmitt, M. Panvert, S. Douthwaite, Y. Mechulam, and P. Courvalin. 2011. Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m⁵C1404-specific methyltransferase EfmM. RNA. 17:251-262.

16. McGann*, P., P. Courvalin*, E. Snesrud, R.J. Clifford, E.-J. Yoon, F. Onmus-Leone, A.C. Ong, Y.I. Kwak, C. Grillot-Courvalin, E. Lesho, and P.E. Waterman. 2014. Amplification of aminoglycoside resistance gene aphA1 in Acinetobacter baumannii results in tobramycin therapy failure. mBio 5:e00915-14. (* co-first authors)

17. Meziane-Cherif, D., P.J. Stogios, E. Evdokimova, A. Savchenko, and P. Courvalin. 2014. Structural basis for the evolution of vancomycin resistance D, D-peptidases. Proc. Natl. Acad. Sci. USA. 111:5872-5877.18.

18. Yoon, E.-J., L. Krizova, S. Goussard, M. Touchon, C. Murphy, C. Grillot-Courvalin, T. Lambert, A. Nemec, and P. Courvalin. 2014. Aminoglycoside modifying enzyme Aph(3’)-VI originates in Acinetobacter guillouiae. mBio 5:e01972-14.

19. Depardieu, F., V. Méjean, and P. Courvalin. 2015. Competition between VanUG repressor and VanRG activator leads to rheostatic control of vanG vancomycin resistance operon expression. PLoS Genet. 11(4):e1005170.

20. Meziane-Cherif, D., P. J. Stogios, E. Evdokimova, A. Savchenko, and P. Courvalin. 2014. Structural basis for the evolution of vancomycin resistance D, D-peptidases. Proc. Natl. Acad. Sci. USA. 111:5872-5877.

21. Yoon, E.-J., V. Balloy, L. Fiette, M. Chignard, P. Courvalin, and C. Grillot-Courvalin. 2016. Contribution of the Ade RND-type efflux pumps to fitness and pathogenesis in Acinetobacter baumannii. mBio 3:e00697-16.

22. Cecchini, T., E.-J. Yoon, Y. Charretier, C., Bardet, C., Beaulieu, X., Lacoux, J.-D., Docquier, J., Lemoine, P., Courvalin, C., Grillot-Courvalin, and J.-P., Charrier. 2018. Deciphering multifactorial resistance phenotypes in Acinetobacter baumannii by genomics and targeted label-free proteomics. Mol. Cell. Proteomics 17.3: 442-451.

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