Unmasking the spread barrier of a major driver of antibiotic resistance: the Integron.
Name: Bacterial Genomes Plasticity
Affiliation: Institut Pasteur
Name: Didier Mazel
Name: Egill Richard
Summary of lab’s interests:
We are looking for an M2 intern interested in bacterial genetics and issues regarding antibiotics resistances; and more fundamentally by genome fluidity. Our models are Escherichia coli and Vibrio cholerae, both who possess a key genome region used for horizontal gene transfers: the integron (see Mazel et al. 2006 or Escudero et al. 2015). We are trying to understand the mechanisms of gene capture in the integrons (molecular biology) as well as its impact on bacterial evolution (genomics). You’ll find enclosed one of the available proposals in the lab. Keep in mind that the projet can/will be tuned according to your interest, and that other projects are available.
Antibiotic resistance (AMR) is a major public health concern, now regarded as a threat to antibiotic chemotherapy and, consequently, to modern medicine. Antibiotic resistance genes (ARG) can disseminate between human and animal bacterial pathogens, and their associated commensal populations. Integrons are bacterial genetic elements able to stockpile, express and disseminate ARG (1). They are considered to be the principal agent in the continuous emergence of AMR in Gram-negative bacteria (GNB). This project is based on the observation that integrons successfully invaded the Gram-negative bacterial world while failing to disseminate in Gram-positive bacteria (GPB). The project proposes to determine the origin of this disparity, by identifying and studying the possible barriers impeding the integron dissemination and adaptive success. We think that the low dissemination in GPB may rather reflect a specific and strong counter selection after transfer such as integrase cell toxicity, when its expression is not properly regulated. Indeed, in GNB, it was already shown that the SOS response prevents the expression of the costly IntI1 integrase. Therefore, we will study integrase toxicity and expression regulation in GPB.
We propose to establish the fitness cost of integrase in GPB. The fitness cost and/or toxicity can constitute a barrier impeding integron dissemination in these strains. We will express the IntI1 integrase in Corynebacterium glutamicum using the well-described pCLTON1 plasmid (2). This is an expression vector which allows a gradual induction of a reporter protein and a tightly repressed basal expression under non-induced conditions. We will measure cell and rate growth in presence of increasing concentrations of the inducer (anhydrotetracycline) and several versions of the integrase (wild type and catalytic mutant integrases). We already obtained preliminaries results which seem to show a very high integrase toxicity in C. glutamicum. The integrase toxicity could be the result of extensive anarchic cleavages altering the chromosome integrity. We will perform pulse-field electrophoresis assay in order to visualize if there is an increase in double strand breaks specifically in GPB compare to GNP.
The SOS response, by increasing the expression of the integrase, accelerates the dynamics of integron shuffling and dissemination and ensures a rapid and “on demand” adaptation to novel environmental contexts. In addition, SOS regulation also limits the pleiotropic effects and genomic instability apparently brought by integrase expression in the host bacterium. Due to its toxicity, the expression of the integrase has to be finely regulated. In this way, it was already shown that the SOS response prevents the expression of the costly IntI1 integrase in GNB (3). We hypothesize that, due to an absence of repression of the integrase expression by the LexA protein, the integrase toxicity in GPB could be too high. Indeed, we know that the LexA box consensus sequence varies among bacterial species (4). Especially, the LexA box sequence of class 1 integrons completely match with the consensus sequence of β and γ-proteobacteria, while considerably differs from the consensus sequences of GPB such as Bacillus subtilis, Actinobacteria and Mycobacterium. Thus, it is likely that the GPB LexA protein will not able to repress the Pint1 promoter of class 1 integrons. Therefore, we strongly suspect that the Pint1 promoter would not be SOS regulated in GPB, rendering the integrase expression constitutive and potentially toxic for the cell (see above). To demonstrate it, a transcriptional fusion coupling a green fluorescent protein (GFP) gene with the PintI1 promoter will be done using the pCLTON1 vector. We will compare the degree of green fluorescence (GFP expression), reflecting the PintI1 activity, in absence and in presence of SOS inducer such as mitomycin C. If the PintI1 promoter is regulated by the SOS response, we expect an induction of integrase expression increasing the fluorescence in presence of mitomycin C. The PintI1 promoter will be cloned with its native intI RBS and with a GPB optimized RBS. We also performed gel shift assays using GPB LexA purified protein.
This project, by providing new precious information on the conditions required for a bacterium to develop AMR, will help predictive modeling of resistance spread. We will understand why, paradoxically, are integrons so little prevalent in GPB while the selective pressure due to the massive antibiotic use is high for these bacteria. On top of it, it will provide a better understanding of the integron system as a whole and therefore will help to explore new approaches to cope with the worldwide problem of AMR.
1. J. A. Escudero, C. Loot, A. Nivina, D. Mazel. Microbiology spectrum 3, MDNA3-0019-2014 (2015).
2. F. Lausberg, A. R. Chattopadhyay, A. Heyer, L. Eggeling, R. Freudl. Plasmid 68, 142-147 (2012).
3. Y. Lacotte, M. C. Ploy, S. Raherison. The ISME journal 11, 1535-1544 (2017).
4. Z. Baharoglu, D. Mazel. FEMS Microbiol Rev 38, 1126-1145 (2014).