Cambios en la fisiología de Pseudomonas aeruginosa causados por la sobre-expresión del sistema múltiple de bombeo MexEF-OprN

Resumen

One paradigm on antibiotic resistance is that the acquisition of resistance may cause a fitness cost in the organism. In this doctoral thesis we have tested the alternative hypothesis that the acquisition of resistance not always produces a general metabolic burden in the microorganisms. We propose that this acquisition lead changes in the bacterial physiology, which can affect positively or negatively the fitness of bacteria, depending on environmental conditions. To investigate this possibility we use a multidrug efflux pump (MexEF-OprN) over-producer on Pseudomonas aeruginosa as a model. The over-expression of this system is caused by the mutation in mexT, the transcriptional activator of the mexEF-OprN genes. In order to analyze the physiological changes associated with the MexEF-OprN over-expression we considered five different aspects: (i) effects over the bacterial fitness, measured by classical experiments; (ii) changes in the P. aeruginosa transcriptomic profile; (iii) effects over the quorum sensing system; (iv) virulence and; (v) effects in the energy generation metabolism. In this work we found that the over-expression of MexEF-OprN does not cause a decrease in P. aeruginosa fitness in classical competition tests performed in LB medium, indicating the absence of an important, non-specific, metabolic burden. The transcriptomic analysis revealed that the overexpression of MexEF-OprN results in reduced expression of several quorum sensing regulated genes and the reduction in the expression of the Type III and Type VI Secretion Systems is caused by the activation of MexT. Virulence factors regulated by quorum sensing like pyocyanin, elastase, caseinase and hemolysin decrease their production in the MexEF-OprN over-producer also. Previous works have described that RND-efflux pumps can extrude quorum sensing related molecules. We determine that the malfunctioning of the quorum sensing system is caused by the delay in PQS production due to over-extrusion of kinurenine, a PQS precursor, through the efflux pump. Experiments in the Caenorhabdities elegans model demonstrated that over-production of MexEF-OprN impairs virulence in P. aerginosa. This effect was mainly due to the decrease in the expression of quorum sensing virulence factors and not by the reduction in the expression of Type III and Type VI secretion systems, both regulated by MexT. RND efflux pumps need the energy of the proton flux to work. Over-expression of these systems could cause an acidification of the cytoplasm. Increasing the oxygen respiration rate is the fastest way to eliminate the excess of protons. In this work we found that RND-efflux over-producers consume more oxygen compared with the wild type strain. The possible decrease in the oxygen concentration in the cytoplasm may activate other mechanisms of energy production normally activated under anaerobic condition like the nitrate respiratory chain. The data obtained in this doctoral thesis indicate that the acquisition of antibiotic resistance can produce specific changes in the bacterial physiology and not necessarily imply a severe, nonspecific cost in the fitness of organisms.