Periodic Reporting for period 1 - PERSIST (Antibiotic persisters during infection: a tail of intestinal dominion)
Période du rapport: 2021-11-01 au 2023-10-31
Persisters are transiently non-growing bacteria cells that are able to evade antibiotic treatment and immune response. These pathogenic bacterial persisters, are responsible for the frequent relapses of apparently successfully treated infections (persistent infections) and the spread of mobile genetic elements that encode antibiotic-resistance genes. As a result, persisters contribute to the global threat of antibiotic treatment failure and, therefore, to patient morbidity and mortality and increased medical costs. Novel strategies to eradicate such persistent infections and, in general, the failure of antibiotic treatment that are based on fundamental research address a problem of high clinical, scientific and societal urgency. The overall objective of the work carried out during this fellowship was to study the physiology of persisters in order to aid in the development of experimentally validated strategies to eradicate them.
The work carried out during this fellowship focused on the physiology of Pseudomonas aeruginosa persisters to aid in the development of experimentally validated strategies to eradicate them. Persisters are transiently non-growing bacteria cells that are able to evade antibiotic treatment and immune response. These pathogenic bacterial persisters, are responsible for the frequent relapses of apparently successfully treated infections (persistent infections) and the spread of mobile genetic elements that encode antibiotic-resistance genes. The work carried out here showed for the first time that the human respiratory pathogen Pseudomonas aeruginosa is able to invade lung epithelial cells and macrophages, forming intracellular persisters that allow Pseudomonas to survive treatment with last-resort antibiotics (e.g. meropenem) even when using concentrations 500 times higher than the ones will normally be bactericidal. Most importantly, this work showed that the formation of persisters both in lung epithelial cells and macrophages-like cells depends on the RNA-binding protein ProQ, which has recently been recognized as a major posttranscriptional regulator of gene expression. Given this, the project explored the interactions of ProQ with Pseudomonas mRNAs to determine mechanistically the involvement of ProQ in the formation of Pseudomonas persisters. The results of this analysis indicated that ProQ preferentially regulates genes encoding transporter activity. Further analysis showed that ProQ primarily regulates Pseudomonas efflux pumps that are able to decrease the lethal amount of antibiotics in the bacterial cytoplasm by extruding them. Finally, the result of this project showed that amongst the five efflux pumps encoded in the Pseudomonas genome and regulated by ProQ, mexAB-oprM and mexEF-oprN are involved in the survival of intracellular Pseudomonas persisters during antibiotic treatment. Activities that took place for the dissemination of the results derived from this fellowship include presentations of results as “work-in-progress” during internal scientific, laboratory and department meetings at Harvard Medical School during the outgoing phase, as well as an invited scientific seminar at the Department of Biology of Utrecht University (23rd of January 2023, Utrecht, The Netherlands). A manuscript based on the results derived from this fellowship is under preparation, and it will be submitted to a high-impact and gold open-access journal. This manuscript will include a reference to the EU funding. Apart from scientific publication(s), the results of this fellowship will be communicated through oral presentations at European conferences, a press release to the general media (i.e. EurekAlert!) and social media on which the researcher has already been very active (i.e. Tweets, ResearchGate, departmental/society websites).
Although the formation of bacterial persisters evading antibiotic treatment has been studied in a handful of human pathogens, this is the first time showing the formation and shedding light on the mechanistic details of intracellular persisters in the major human pathogen Pseudomonas aeruginosa. Such persisters are responsible for the frequent relapses of apparently successfully treated infections (persistent infections) and antibiotic treatment failure, increasing patient morbidity and mortality and overall medical costs. These relapses are exacerbated by immunosuppression and are critical for the increasing number of HIV-infected and cancer patients, organ transplant recipients, and patients on immunosuppressive drugs. Hence, the results of this project are of utmost importance for designing treatment strategies that circumvent the formation of Pseudomonas persisters and, therefore, eliminate the frequent relapses documented in Pseudomonas infections.