Novel insights regarding the sigmoidal pattern of resistance to neomycin conferred by the aphII gene, in Streptomyces lividans
© Seghezzi et al.; licensee Springer. 2013
Received: 30 January 2013
Accepted: 4 February 2013
Published: 8 February 2013
A library of synthetic promoters of various strengths, specifically constructed for Streptomyces species, was cloned in the promoter-probe plasmid pIJ487, upstream of the promoter-less aphII gene that confers resistance to neomycin. The survival rates conferred by promoters were assessed in the presence of 100 μg.ml−1 neomycin. The correlation between the transcriptional activity of the aphII gene (estimated by RT-PCR) and the resistance to neomycin (expressed as survival rate) indicated a sigmoid rather than a linear correlation. In this issue, we propose a tentative explanation for this sigmoidal pattern of resistance in relation with the level of aph II gene expression. Beyond this specific example, our model might constitute a sound explanation for the generally observed but never explained sigmoidal shape of classical inhibition curves obtained in the presence of linearly increasing antibiotic concentrations.
Antibiotics have been the most useful therapeutic agents of the twentieth century (Levy 2002). However, more and more bacteria have developed resistance to all existing antibiotics and antimicrobial resistance was recently recognized as one of the greatest threats to human health (Gyssens 2011). Indeed an increasing number of patients suffer from serious life-threatening antimicrobial-resistant infections against which only very few, if any, effective antibiotics are available (Levy 1998). Alarmingly, as the number of patients dying from antibiotic-resistant infections rises, the number of new antibiotics in development is plummeting (Butler and Cooper 2012; Mahajan and Balachandran 2012). The antibiotic resistance genes acquired by human pathogens are thought to originate from micro-organisms of the environment including Streptomyces (Forsberg et al. 2012). These bacteria are antibiotic producers and thus contain the corresponding antibiotic resistance genes for self protection (Allen et al. 2010; Davies and Davies 2010; Nikaido 2009). The genes conferring resistance to antibiotics are spreading by horizontal transfer in the microbial population and the release of antimicrobials in the environment likely selects micro-organisms carrying these genes (Alonso et al. 2001; Martinez 2008). The mechanisms conferring antibiotic resistance in micro-organisms include enzymatic inactivation or modification of the antibiotic, modification of host targets to prevent antibiotic binding, efflux pumps. A major challenge to counteract the development of resistance to antibiotic treatment is to get a better understanding of how bacteria react to antibiotics.
The efficiency of many antibiotics is known to be impaired by the existence of resistance mechanisms. In this study, an antibiotic resistance gene, aph II, was used to assess the impact of the level of expression of this gene on survival to an antibiotic selective pressure. aph II encodes a phosphotransferase, phosphorylating the aminoglycoside antibiotic, neomycin, impairing its ability to interact with the ribosome and thus preventing inhibition of mRMA translation by neomycin (Beck et al. 1982; Hermann 2007). The expression of this gene present on the multicopy plasmid pIJ487 (Ward et al. 1986) was put under the control of 18 promoters of varying strengths originating from a previous study (Seghezzi et al. 2011). These different constructs were introduced into Streptomyces lividans and survival of these different clones, exposed to a constant and rather high concentration of neomycin (100 μg/ml), was assessed.
Our results revealed that the relation between promoter strength (as determined by RT-PCR) and survival rate was not linear but indicated a sigmoidal correlation. A model, consistent with this behaviour, based on the well-known mechanism by which aminoglycoside antibiotics are lethal to bacteria, and how AphII counteracts this poisoning activity, was designed and discussed. This model might have a more general scope to rationalize the currently observed but never explained sigmoidal shape of classical inhibition curve obtained with linearly increasing antibiotic concentrations (Baudoux et al. 2007).
Materials and methods
Bacterial strains, plasmid and media
Streptomyces lividans TK24 strains transformed with 38 pIJ487-derived plasmids, each carrying a 300 bp DNA fragment with promoter activity of various strength were used in this study (Ward et al. 1986). The strength of these promoters was previously roughly estimated, using the replica-plating technique as described in ( Lederberg and Lederberg 1952). These promoters were classified as weak, medium or strong based on their ability to allow growth of the different transformants in the presence of up to 20, 50 or 100 μg.ml−1 of neomycin in HT medium (Seghezzi et al. 2011). Media as well as Streptomyces manipulations were carried out according to Practical Streptomyces Genetics manual (Kieser et al. 2000). SFM was used to grow up transformants to prepare spores suspensions for quantitative estimation of survival rates.
Estimation of survival rates
Estimation of aphII transcriptional activity using RT-PCR
RNA was extracted, using RNeasy Mini Kit from Qiagen, from selected transformants representative of each strength class and grown for 48h on the surface of cellophane disks laid on solid HT medium containing only 50 μg.ml−1 thiostrepton.
RT PCR was performed using the OneStep RT-PCR Kit from Qiagen and the following conditions: initial denaturation at 97°C, 5 min followed by 25 cycles of denaturation (97°C, 30 s), annealing (50°C, 30 s) and extension (72°C, 30 s). The absence of DNA in RNA samples was systematically checked by running a control PCR reaction made in absence of reverse transcription. Quantification of the RT-PCR signals was made using ImageQuant pixel counts in non-saturated conditions. Values were normalised on pIJ487 signals. The normalisation was done with the negative control that is the plasmid pIJ487 with no promoter cloned upstream of aph II. Some weak transcription of aph II was detected in this context.
Assessment of promoter strength using viability assays and RT-PCR
An accurate quantification of promoter strength, expressed as survival rate, was carried out in the presence of 100 μg.ml-1 neomycin, for 38 selected clones belonging to a previously constructed bank of synthetic promoter designed for Streptomyces species and fused to the reporter gene aphII conferring resistance to neomycin. These promoters were previously roughly classified as weak, medium and strong by replicate plating (Seghezzi et al. 2011). A transformant containing pIJ487ermE* carrying the strong ermE* promoter was used as a positive control (Bibb et al. 1985). It should be stressed that in a genetically homogenous bacterial population, all the bacteria are not in the same physiological state and the expression of aphII (as that of any other genes) varies stochastically around a mean value (Elowitz et al. 2002; Losick and Desplan 2008). This variability explains why, even when a weak promoter is driving aphII expression, a small fraction of the bacterial population is able to resist to a high level of neomycin.
Eighteen of these transformants, representative of each class of promoter strength (weak, medium and strong) were precisely ranked according to the survival rate they conferred in the presence of 100 μg.ml-1 neomycin. All colonies had approximately the same size. Each promoter was plotted against the log of the survival rate it conferred in the presence of 100 μg.ml-1 neomycin. Interestingly, the resulting curve appears to be sigmoidal (Figure 1). The first and third parts represent the low and high survival rates and the central part of the curve shows an abrupt transition between these two states. We thus wondered whether the transcriptional activity of these promoters followed a similar sigmoidal pattern. To answer this question, we assessed the transcriptional activity of promoters corresponding to the three parts of the curve, using RT-PCR.
The aphII gene is the most extensively used reporter system in Streptomyces, however, this useful system was sometimes blamed for some non-understood paradoxical behaviour. The dynamics of the system, as revealed by our study, can explain these paradoxes. Consequently, the aphII gene should thus be used with caution to accurately assess promoter strength, since a small variation of gene expression around a specific threshold might lead to a huge change in the pattern of resistance to neomycin.
Furthermore, our study suggests that it might be sufficient to reduce (and not totally preclude) the expression of a resistance gene just under a certain threshold to greatly enhance the killing efficiency of the corresponding antibiotic. That is why nowadays even imperfect inhibitors of transcription and/or translation are sometimes associated in prescription to overcome some reluctant antibiotic resistant strains.
This work was supported by the European program ACTINOGEN (http://www.swan.ac.uk/ils/Research/BioMed/ActinoGen/), the Centre National de la Recherche Scientifique (http://www.cnrs.fr/), the University Paris Sud (http://www.u-psud.fr) and the Pôle de Recherche et d’Enseignement supérieur UniverSud Paris (http://www.universud-paris.fr).
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