In Vivo-Selected Compensatory Mutations Restore the Fitness Cost of Mosaic penA Alleles That Confer Ceftriaxone Resistance in Neisseria gonorrhoeae Leah R. Vincent, a * Samuel R. Kerr, b * Yang Tan, b Joshua Tomberg, b Erica L. Raterman, a Julie C. Dunning Hotopp, c Magnus Unemo, d Robert A. Nicholas, b,e Ann E. Jerse a a Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University, Bethesda, Maryland, USA b Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA c Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA d World Health Organization Collaborating Centre for Gonorrhoea and Other Sexually Transmitted Infections, Swedish Reference Laboratory for Sexually Transmitted Infections, Department of Laboratory Medicine, Microbiology, Faculty of Medicine and Health, Örebro University, Örebro, Sweden e Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA ABSTRACT Resistance to ceftriaxone in Neisseria gonorrhoeae is mainly conferred by mosaic penA alleles that encode penicillin-binding protein 2 (PBP2) variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mo- saic penA alleles on gonococcal fitness, we introduced the mosaic penA alleles from two ceftriaxone-resistant (Cro r ) clinical isolates (H041 and F89) into a Cro s strain (FA19) by allelic exchange and showed that the resultant Cro r mutants were signifi- cantly outcompeted by the Cro s parent strain in vitro and in a murine infection model. Four Cro r compensatory mutants of FA19 penA41 were isolated indepen- dently from mice that outcompeted the parent strain both in vitro and in vivo. One of these compensatory mutants (LV41C) displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D) in the acnB gene encod- ing the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduc- tion of the acnB G348D allele into FA19 penA41 conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of ad- ditional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq) analysis of FA19 penA41 acnB G348D revealed a large set of up- regulated genes involved in carbon and energy metabolism. We conclude that com- pensatory mutations can be selected in Cro r gonococcal strains that increase metab- olism to ameliorate their fitness deficit. IMPORTANCE The emergence of ceftriaxone-resistant (Cro r ) Neisseria gonorrhoeae has led to the looming threat of untreatable gonorrhea. Whether Cro resistance is likely to spread can be predicted from studies that compare the relative fitnesses of susceptible and resistant strains that differ only in the penA gene that confers Cro resistance. We showed that mosaic penA alleles found in Cro r clinical isolates are outcompeted by the Cro s parent strain in vitro and in vivo but that compensatory mutations that allow ceftriaxone resistance to be maintained by increasing bacterial fitness are selected during mouse infection. One compensatory mutant that was studied in more detail had a mutation in acnB, which encodes the aconitase that Received 18 October 2017 Accepted 20 February 2018 Published 3 April 2018 Citation Vincent LR, Kerr SR, Tan Y, Tomberg J, Raterman EL, Dunning Hotopp JC, Unemo M, Nicholas RA, Jerse AE. 2018. In vivo-selected compensatory mutations restore the fitness cost of mosaic penA alleles that confer ceftriaxone resistance in Neisseria gonorrhoeae. mBio 9:e01905-17. https://doi.org/10.1128/ mBio.01905-17. Editor Michael S. Gilmore, Harvard Medical School This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply. Address correspondence to Robert A. Nicholas, nicholas@med.unc.edu, or Ann E. Jerse, ann.jerse1@usuhs.edu. * Present address: Leah R. Vincent, NIAID, Bethesda, Maryland, USA; Samuel R. Kerr, Yale University, New Haven, Connecticut, USA. RESEARCH ARTICLE crossm March/April 2018 Volume 9 Issue 2 e01905-17 ® mbio.asm.org 1 mbio.asm.org on April 5, 2018 - Published by mbio.asm.org Downloaded from