Research project: Persistence of Pseudomonas aeruginosa in blood
Supervisor: Dr. François Cretin & Dr. Ina Attrée
Lab: IBS (Institute de Biologie Structurale), PBRC team
Year: 2021-2022
Bacterial bloodstream infections (BSIs) are an important burden for human health system. Pseudomonas aeruginosa (Pa) is one of the most common nosocomial pathogens with a mortality rate between 33 to 61% in BSIs. This opportunistic bacterium possesses a great panoply of virulence factors allowing the infection of different organs and BSIs. Pa reaches the bloodstream where it faces the complement system (CS) which is the main innate immune actor responsible for its elimination (1). The CS involves more than 50 proteins and consists of a cascade resulting in the deposition and polymerization of the CS components (2). This leads to the formation of the membrane attack complex (MAC) in the outer membrane (OM) causing the lysis of the bacterial pathogen2. Despite the CS activity in the human plasma, many Pa isolates persist by forming a sub–population called evaders that corresponds to less than 1% of the initial bacterial load1. The survival in plasma greatly varies between different Pa strains and it ranges from total resistance to persistence1. A recent work by the IBS–PBRC team, using Tn–seq methodology, indicated that the ATP levels may modulate Pa plasma resilience (3).
The goal of Serena’s M2 internship was to further characterize the evaders and explore the link between ATP and Pa persistence in plasma. Getting more evaders, monitored by colony forming unit (CFU) count, is critical for further analyses. Therefore, I optimized the conditions of bacteria/plasma interactions and yielded 4 times more evaders. The second step was to isolate the evaders by osmotic shock in order to perform a variety of molecular characterizations. First, the deposition and polymerization of the CS components was evaluated by flow cytometry. My results show that evaders indeed have CS molecules on their surface meaning that they have been recognized by the CS, but somehow resisted the MAC–dependent lysis. To determine whether evaders survive despite MAC insertion in their OM, I will use nisin, an antimicrobial peptide able to kill Gram–negative bacteria only if the OM is damaged. I also directly investigated the ATP levels in evaders from different strains. However, there was no significant correlation between the formation of evaders in plasma and ATP levels, probably due to a variability in the mechanisms of resilience between strains and possible
heterogeneity within each evaders’ populations. Altogether, this work allowed a better molecular characterization of the evaders and calls for single–cell approaches to investigate the ATP levels in the evaders’ populations in BSI clinical strains.
Serena is now pursuing a PhD with Dr. Ina Attrée and Dr. Eric Faudry at Institut de Biologie Structurale.
(1) Pont, S., Fraikin, N., Caspar, Y., Van Melderen, L., Attrée, I. and Cretin, F., 2020. Bacterial behavior in human blood reveals complement evaders with some persister–like features. PLoS pathogens, 16(12), p.e1008893.
(2) Bjanes, E. and Nizet, V., 2021. More than a pore: Nonlytic antimicrobial functions of complement and bacterial strategies for evasion. Microbiology and Molecular Biology Reviews, 85(1), pp.e00177–20.
(3) Janet–Maitre, M., Pont, S., Masson, F., Trouillon, J., Robert–Genthon, M., Dumestre–Perard, C., Elsen, S., Moriscot, C., Bardoel, B., Rooijakkers, S. and Cretin, F., 2021. Molecular features underlying Pseudomonas aeruginosa persistence in human plasma. bioRxiv.