Deletion of cyp125 Confers Increased Sensitivity to Azoles in Mycobacterium tuberculosis

RESEARCH ARTICLE Deletion of cyp125 Confers Increased Sensitivity to Azoles in Mycobacterium tuberculosis Paul Carroll1, Tanya Parish1,2* 1 Queen Mary University of London, Barts & The London School of Medicine and Dentistry, Centre for Infectious Disease, London, United Kingdom, 2 TB Discovery Research, Infectious Disease Research Institute, Seattle, Washington, United States of America * a11111 OPEN ACCESS Citation: Carroll P, Parish T (2015) Deletion of cyp125 Confers Increased Sensitivity to Azoles in Mycobacterium tuberculosis. PLoS ONE 10(7): e0133129. doi:10.1371/journal.pone.0133129 Editor: Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, FRANCE Abstract Mycobacterium tuberculosis is able to utilize cholesterol as a carbon source, and this ability is linked to its virulence in macrophages and in the mouse model of infection. The M. tuberculosis cytochrome P450 Cyp125 plays a key role in cholesterol metabolism being involved in the first steps of its degradation. Cyp125 is a cholesterol hydroxylase which is essential for cholesterol catabolism in M. bovis BCG and some strains of M. tuberculosis. We generated an unmarked, in-frame deletion of Cyp125 in M. tuberculosis H37Rv. The deletion strain was able to grow as well as wild-type in medium containing glucose as the carbon source. The Cyp125 deletion strain was more sensitive to growth inhibition by clotrimazole consistent with the ability of Cyp125 to bind azoles with high affinity. The deletion strain showed no difference in sensitivity to nitric oxide or hydrogen peroxide and was not attenuated for growth inside THP-1 human macrophage-like cells. These data suggest that the attenuation of virulence seen in operon deletion strains is not linked to the lack of Cyp125 alone. Received: May 6, 2015 Accepted: May 19, 2015 Published: July 21, 2015 Copyright: © 2015 Carroll, Parish. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by the European Union Project LSHP-CT-2005-018923. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for the death of over 1 million people and 9 million new cases per year [1]. The current treatment of tuberculosis is time consuming and with the current treatment procedures there is an increase in the number of both multidrug resistant (MDR-TB) and extensively drug resistant (XDR-TB) M. tuberculosis strains [1]. This indicates a need for an increased understanding of the biology and pathogenic mechanisms of the bacterium in order to develop new therapeutic strategies. Unusually, the M. tuberculosis genome encodes 20 cytochrome P450 enzymes (Cyps) [2,3]. Among the mycobacteria, the closely related species M. bovis has 18, and the non-pathogenic species M. smegmatis has 39 [4]. In relation to other bacteria this is a relatively high number, since very few prokaryotes encode any Cyp homologues; for example, Escherichia coli has none [4,5]. In contrast, M. leprae has only one, which may represent the minimal requirement for a mycobacterium, since it has undergone substantial reductive evolution of its genome [6]. PLOS ONE | DOI:10.1371/journal.pone.0133129 July 21, 2015 1/8 Deletion of cyp125 Confers Increased Sensitivity to Azoles in Mtb P450s are of interest as drug targets, for example the polycyclic azoles are P450 inhibitors which have been used topically to combat fungal pathogens. Azoles inhibit Cyp51, an essential lanosterol 14α–demethylase, involved in the synthesis of ergosterol—an integral component of the cell membrane [7]. Azoles also have anti-mycobacterial properties, with activity against M. tuberculosis both in vitro and in vivo in the mouse model of infection [8–12]. Unfortunately, azoles have hepatotoxic and teratogenic properties, which precludes their use in long term treatment for tuberculosis [13]. The main mechanism of resistance to azoles is increased drug efflux, mediated in M. tuberculosis by the MmpS5-MmpL5 system [14–16]. Cyp125 plays a role in cholesterol metabolism in M. tuberculosis [17,18]. Cholesterol degradation is required for both the spread and persistence of the bacteria in vivo [19–21]. In addition, nitric oxide has been shown to bind to Cyp125, and other P450 enzymes, and a role in removing dangerous free radicals has been suggested [22]. Cyp125 catalyses the C27 hydroxylation of both cholesterol and choles-4-ten-3-one, although its deletion results in accumulation of the latter metabolite which is presumed to be toxic to the cells [23–25]. Cyp125 forms part of the igr locus [24,25] and is found in a region of the genome with other genes encoding enzymes also involved in cholesterol degradation [26]. In M. bovis BCG, Rhodococcus jostii RHA1 and M. tuberculosis CDC1551, Cyp125 is essential for growth on cholesterol [18,23,27]. In contrast in M. tuberculosis H37Rv, a Cyp125 deletion mutation was able to grow on cholesterol as the only carbon source [18] and this was linked to the ability of Cyp142 to provide similar C27 hydroxylation capability [28]. Materials and Methods Bacterial Strains, Growth Media and Antibiotics M. tuberculosis H37Rv (ATCC25618) was grown in Middlebrook 7H9 medium plus 10% v/v OADC supplement (Becton Dickinson) and 0.05% w/v Tween 80 or on Middlebrook 7H10 agar plus 10% v/v OADC. Hygromycin was added at 100 μg/ml, kanamycin at 20 μg/ml, gentamicin at 10 μg/ml, X-gal at 50 μg/ml. Generation of a M. tuberculosis CYP125 Deletion Strain A deletion delivery vector was generated by amplifying the upstream and downstream regions of Rv3545c, encoding Cyp125, using primer pairs F1 5’ AAG CTT ACG AAG ATC TGC TGC TCG AT 3’ and R1 5’ GGA TCC CAC TGG CAG GTC GAC TAC ACC 3’, and F2 5’ GGA TCC CTC CAC TGA CTG GTG ATT CCA3 ‘ and R2 5’ GCG GCC GCT CGT TGA TCT CGA CGA TGT 3’ and cloned into p2NIL [29] as a HindIII-NotI fragment to generate an unmarked inframe deletion. Restriction sites used for cloning are underlined. The gene cassette from pGOAL19 [29] was cloned in as a PacI fragment to generate the final delivery vector pTACK125. The deletion delivery vector pTACK125 was electroporated into M. tuberculosis and single cross-overs (SCOs) were isolated. Double cross-overs (DCOs) were isolated from the SCO strain as previously described [29]. DCOs were screened for the presence of the wildtype or deletion allele using primers CYP125D1 5’ CGT CTG AAC CAT TCG ATG TG 3’ and CYP125D2 5’ TTC AAC GAT GAC CGG GTA AC, which amplify a product of 2.0 kb from (...truncated)