Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state

Abstract

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations. Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations. Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, with nearly 80 million cases worldwide each year (1Newman L. Rowley J. Vander Hoorn S. Wijesooriya N.S. Unemo M. Low N. Stevens G. Gottlieb S. Kiarie J. Temmerman M. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting.PLoS One. 2015; 10e0143304Crossref PubMed Scopus (908) Google Scholar). Without antibiotic treatment, infections persist as a chronic disease and can cause serious sequelae, including pelvic inflammatory disease, infertility, arthritis, and disseminated infections (2Hill S.A. Masters T.L. Wachter J. Gonorrhea - an evolving disease of the new millennium.Microb. Cell. 2016; 3: 371-389Crossref PubMed Scopus (41) Google Scholar). For many years, N. gonorrhoeae was treated with a single dose of penicillin, and more recently, ceftriaxone. In 2012, the emergence of several high-level ceftriaxone-resistant strains led the Centers for Disease Control and Prevention to change its recommended treatment for gonorrhea from monotherapy to dual therapy with ceftriaxone and azithromycin (3Workowski K.A. Bolan G.A. Centers for Disease Control and PreventionSexually transmitted diseases treatment guidelines, 2015.MMWR Recomm. Rep. 2015; 64: 1-137Crossref PubMed Scopus (25) Google Scholar, 4Unemo M. Lahra M.M. Cole M. Galarza P. Ndowa F. Martin I. Dillon J.R. Ramon-Pardo P. Bolan G. Wi T. World Health Organization Global Gonococcal Antimicrobial Surveillance Program (WHO GASP): Review of new data and evidence to inform international collaborative actions and research efforts.Sex Health. 2019; 16: 412-425Crossref PubMed Scopus (88) Google Scholar, 5Bignell C. Unemo M. European STI Guidelines Editorial Board2012 European guideline on the diagnosis and treatment of gonorrhoea in adults.Int. J. STD AIDS. 2013; 24: 85-92Crossref PubMed Scopus (293) Google Scholar). However, treatment failures have been reported for and in a strain with high-level resistance to ceftriaxone and azithromycin was N. L. M. Unemo M. G. 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P. penicillin-binding and role in PubMed Scopus Google Scholar). of a with the by the which is by the to a For of the is with its and the is of by is by a which the and the emergence of resistance to and ceftriaxone in N. gonorrhoeae the of of the PBP2 penicillin-binding in strains of Neisseria PubMed Scopus Google Scholar). These to as with by Neisseria PBP2 from the high-level ceftriaxone-resistant H041, mutations with PBP2 from the M. T. S. S. J. Unemo M. Neisseria gonorrhoeae a of of the strain with high-level resistance to PubMed Scopus Google Scholar, M. T. S. S. J. Neisseria gonorrhoeae, PubMed Scopus Google Scholar). how these mutations lower the of ceftriaxone for PBP2 by is for the of antibiotic we have a of these mutations the from of the increase in for ceftriaxone to that of the from J. Unemo M. C. and of of penicillin-binding protein 2 to in Neisseria of PubMed Scopus Google Scholar, J. Unemo M. M. C. of high-level resistance to in the from Neisseria gonorrhoeae strain 2013; PubMed Scopus Google Scholar). We reported the of and PBP2 at and have conformational in and the β3–β4 loop in antibiotic and J. C. of the of Neisseria gonorrhoeae penicillin-binding protein 2019; PubMed Scopus Google Scholar). present in the of the mutations resistance in with ceftriaxone in the of PBP2 J. C. of the of Neisseria gonorrhoeae penicillin-binding protein 2019; PubMed Scopus Google Scholar, J. Unemo M. C. in penicillin-binding protein 2 from Neisseria gonorrhoeae ceftriaxone by protein PubMed Scopus Google Scholar). We have that these mutations the and of PBP2 with ceftriaxone by protein J. Unemo M. C. in penicillin-binding protein 2 from Neisseria gonorrhoeae ceftriaxone by protein PubMed Scopus Google Scholar). the and by which these mutations lower the of we a of solution NMR, X-ray crystallography, and to We report that the β3–β4 loop in the of WT which is to in the J. C. of the of Neisseria gonorrhoeae penicillin-binding protein 2019; PubMed Scopus Google two conformational in of WT PBP2 with mutations in that ceftriaxone resistance the conformational of PBP2 by destabilizing the high-affinity state the inward conformation of the β3–β4 loop and stabilizing a low-affinity conformation an extended β3–β4 loop to the inward conformation for high-affinity drug solution and of PBP2 and its drug the that mutations in PBP2 from ceftriaxone-resistant strains of N. gonorrhoeae antibiotic resistance by conformational to a drug-binding state J. Unemo M. C. in penicillin-binding protein 2 from Neisseria gonorrhoeae ceftriaxone by protein PubMed Scopus Google Scholar). PBP2 is of an a and a J. C. of penicillin-binding protein 2 from and strains of Neisseria gonorrhoeae reveal an mechanism for antibiotic PubMed Scopus Google Scholar). the protein more to by X-ray and NMR, we a of PBP2 the by of the and a loop to to as for as the protein J. 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