RESUMO
Translocator protein (TSPO) is an 18 kDa transmembrane protein, localized primarily on the outer mitochondrial membrane. It has been found to be involved in various physiological processes and pathophysiological conditions. Though studies on its structure have been performed only recently, there is little information on the nature of dynamics and doubts about some structures referenced in the literature, especially the NMR structure of mouse TSPO. In the present work, we thoroughly study the dynamics of mouse TSPO protein by means of atomistic molecular dynamics simulations, in presence as well as in absence of the diagnostic ligand PKA. We considered two starting structures: the NMR structure and a homology model (HM) generated on the basis of X-ray structures from bacterial TSPO. We examine the conformational landscape in both the modes for both starting points, in presence and absence of the ligand, in order to measure its impact for both structures. The analysis highlights high flexibility of the protein globally, but NMR simulations show a surprisingly flexibility even in the presence of the ligand. Interestingly, this is not the case for HM calculations, to the point that the ligand seems not so stable as in the NMR system and an unbinding event process is partially sampled. All those results tend to show that the NMR structure of mTSPO seems not deficient but is just in another portion of the global conformation space of TSPO.
RESUMO
Background: Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. Methods: In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Results: Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. Conclusions: We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.