RESEARCH SUMMARY

Infectious diseases remain among the major causes of human death in the world. Several infections at hospitals are due to opportunistic pathogens, microorganisms that rarely infect healthy people, but are a frequent cause of infection in people with basal diseases, who are immunodepressed or debilitated. Environmental bacteria, frequently antibiotic resistant, constitute a large percentage of those pathogens. Our work focuses on understanding the mechanisms of virulence and resistance, as well as possible crosstalk, of these pathogens.

Within this scope, in the last two years, we have been defining those genes whose mutation changes the phenotype of antibiotic susceptibility of Pseudomonas aeruginosa. As a result, we have selected nearly three hundred genes for future analysis and are currently studying whether those mutations that challenge intrinsic resistance also alter the virulence of P. aeruginosa. We found that mutations in several genes encoding proteins from different categories that include multidrug efflux pumps, two component systems, metabolic enzymes or global regulators, simultaneously alter the antibiotic susceptibility and the virulence of P. aeruginosa. One of these is Crc, a global regulator involved in Pseudomonas carbon metabolism. We found that a crc-defective mutant is more susceptible to several antibiotics and expresses several virulence determinants at lower levels than the wild-type strain. These results indicate that the resistance to antibiotics and the virulence of P. aeruginosa are intrinsically linked to bacterial metabolism.

Another opportunistic pathogen we are working with is Stenotrophomonas maltophilia, which is characterized by its intrinsic low susceptibility to several antibiotics. Part of this low susceptibility relies on the expression of chromosomally-encoded multidrug efflux pumps, of which SmeDEF is the most relevant antibiotic resistance efflux pump so far studied in this bacterial species. Expression of smeDEF is downregulated by the SmeT repressor, encoded upstream of smeDEF in its complementary DNA strand. We determined the crystal structure of SmeT and analysed its interactions with its cognate operator. SmeT behaves as a dimer and presents some common structural features with other TetR regulators. At difference from other TetR proteins for which the structure is available, SmeT turned out to have two extensions at the N and C termini that might be relevant to its function. In vitro studies showed that SmeT binds to a 28 bp pseudopalindromic region, forming two complexes. This operator region overlaps the promoters of smeT and smeDEF, a finding consistent with a role for SmeT in simultaneously downregulating smeT and smeDEF transcription.