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Antimicrobial Agents and Chemotherapy, March 2007, p. 1004-1010, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.01103-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Reduced Susceptibility of Haemophilus influenzae to the Peptide Deformylase Inhibitor LBM415 Can Result from Target Protein Overexpression Due to Amplified Chromosomal def Gene Copy Number

Charles R. Dean,^1* Shubha Narayan,¹ Joel Richards,^1, Denis M. Daigle,¹ Stacy Esterow,¹ Jennifer A. Leeds,¹ Heather Kamp,^1, Xiaoling Puyang,¹ Brigitte Wiedmann,¹ Dieter Mueller,² Hans Voshol,² Jan van Oostrum,² Daniel Wall,³ James Koehn,³ JoAnn Dzink-Fox,¹ and Neil S. Ryder¹

Infectious Diseases, Novartis Institute for Biomedical Research, Cambridge, Massachusetts 02139,¹ Genome and Proteome Sciences, Novartis Institute for Biomedical Research, Basel, Switzerland 4002,² Discovery Technologies, Novartis Institute for Biomedical Research, Cambridge, Massachusetts 02139³

Received 31 August 2006/ Returned for modification 6 October 2006/ Accepted 21 December 2006

Previous genetic analysis of Haemophilus influenzae revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene acrR and in the fmt gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 µg/ml versus 4 µg/ml against the parent strain NB65044) that lacks mutations in the acrR or fmt structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in def transcript titer. No mutations were found in the region upstream of def that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing def had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the def gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.

Published ahead of print on 12 January 2007.

Present address: Massachusetts Department of Public Health, Boston MA 02108.

Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston MA 02115.