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Stéphane Pont

Strategies employed by Pseudomonas aeruginosa to withstand the innate immune system in human whole blood

Published on 17 November 2020
Thesis presented November 17, 2020

Abstract:
Pseudomonas aeruginosa (Pa) is a Gram-negative opportunistic pathogen primarily affecting immunocompromised and cystic fibrosis patients. In addition to lung and urinary tract infections, Pa is often isolated from blood of bacteremic patients. Blood stream infections caused by this particular pathogen register with higher mortality rates compared to septicemia associated with other bacteria. Once in the circulation, Pa faces the innate immune system of the blood, however the strategies employed by this pathogen to escape killing by the complement system and circulating phagocytes have not been investigated in whole blood. In this work, we assessed the survival of a collection of six Pa strains in human whole blood. We found that rather than cytotoxicity toward circulating leukocytes, complement resistance is the main driver of survival in the blood. The origin, serotype of the isolate, as well as the nature of the toxins expressed by the strains did not affect their survival. We also discovered that complement-sensitive strains prevent their eradication through the generation of a minor subpopulation of "evaders", able to withstand complement-mediated lysis. Depending on the strain, between 0.0001 and 0.01% of the initial population survived in human plasma. The characterization of evaders showed that they harbor a transient persister-like phenotype, and are also observed in Acinetobacter baumannii, Burkholderia multivorans, enteroaggregative Escherichia coli, Klebsiella pneumoniae, and Yersinia enterocolitica. Formation of evaders likely represent an efficient strategy for complement-sensitive bacteria to persist within the blood and disseminate through the host. Using a genome-wide screen of a transposon mutant library in human plasma (Tn-Seq), we identified new bacterial factors influencing the interplay between Pa and the complement system. We observed that bacteria negatively affected in de novo biotin and purine synthesis survive better in plasma than the parental strain. In addition, Tn-Seq identified an operon of three genes, that we named "srg" for "serum resistant genes" whose products are predicted to be membrane bound. SrgABC overexpression confers tolerance to plasma (1000 fold-increased survival compared to wt) and triggers an overproduction of alginates, suggesting complex interplay between bacterial membrane, exopolysaccharides and complement-mediated lysis.

Keywords:
Pseudomonas aeruginosa, whole blood, innate immunity, complement, evaders