Ongoing projects
Legionella pneumophila is a facultative intracellular human pathogen directly transmitted from the environment. Legionella is responsible for the life-threatening pneumonia termed Legionnaires’ disease (LD)(ref). Worrisomely, inventoried cases have doubled since 2013(ECDPC). LD is fatal for 5-15% of the patients (up to 50% among the elderly), even when promptly diagnosed and treated(ref). LD may progress to (i) complicated presentations such as abscess formation, relapse of an uncured disease despite appropriate treatment, or (ii) persistent and progressive primary LD that is poorly responsive to drug therapy In both cases, persistence of pulmonary infiltrates are observed beyond 30 days after the onset of disease(ref). A nation-wide retrospective study describing those cases of slowly or non-resolving LD proposed that recalcitrance to antibiotic treatments or relapsing infections were likely caused by bacterial persisters(ref). In agreement, we recently found that Legionella developed intracellular subpopulation of nonreplicating persisters(ref). The ability of a pathogen to produce persisters that survive both the antibiotic and their interaction with the host is an important yet under-investigated field.
Our objectives are to decipher the molecular interplay between the persisters and their host that fine-tunes persistence, resuscitation and evolution in mammalian niches. To achieve an unbiased characterisation, we combine methods in microbiology, cell biology, molecular biology to innovative infections assays and cutting-edge high-throughput single-cell acquisition systems, multi-parametric high-resolution microscopy, omics analysis and loss of function approaches.
RECENT ACHIEVEMENTS
1. Single-cell fluorescent reporter to track, collect and analyse within-host persisters
Antibiotic therapy often fails to eliminate a fraction of transiently refractory nonreplicating bacteria, causing relapses and chronic infections. Multiple mechanisms can induce such persisters with high antimicrobial tolerance
in vitro, but
in vivo their relevance remained unclear due to the lack of suitable methodologies that could resolve variation between pathogen subpopulations in infected organs. To track the nonreplicating persisters hidden deep in the infected tissues, we devised a plasmid-based growth rate reporter suitable for both flow-cytometry and microscopy analysis. We took advantages of the DsRed S197T variant called Timer, a stable fluorescent protein that slowly maturates from a green to a red fluorescent protein. In growing bacteria, constitutively producing Timer, green fluorescent Timer dominates over red fluorescent Timer, which is diluted by cell division before maturation, and the individual bacteria show a high [500 nm (green)/600 nm (red)] fluorescence (color) ratio (i.e., “green” fluorescence). By contrast, growth-arrested bacteria accumulate both green and red fluorescent Timer, and the individual bacteria show a low green/red color ratio (i.e., “red” fluorescence,
A). In a murine model for human typhoid fever, Timer producing
Salmonella enterica Typhimurium showed extensive division rate variations in infected spleen (
B). This included fast-growing subsets driving disease progression as well as nongrowing individuals.
Salmonella growth in spleen leads to drastic changes of the host environment with the development of structured pathological lesions in which nongrowers were however exclusively found within red pulp resident macrophages. Monitoring the impact of growth rate heterogeneity on antimicrobial therapies, under clinically relevant conditions (natural oral route, disease signs before starting treatment, realistic antibiotic regime) revealed that antibiotic killing correlated with single-bacterium division rates (
C). Fast-growing
Salmonella, that drives disease progression, survived poorly, whereas nongrowing
Salmonella subsets survived best. Unexpectedly, initially, most survivors originated from the abundant moderately growing, and then partially tolerant, subpopulation. Throughout therapy, repeating cycles of extensive killing/partial tolerance and resumption of growth resulted in
slow Salmonella clearance. If the initial dominance of moderately growing
Salmonella subsets delays the chemotherapy, the dominance of the nongrowers, at the later stage of the antibio-therapy, may be responsible for therapeutic failure and relapsing infection. The single cell approaches developed in this study demonstrate that host tissues diversify pathogen physiology, with major consequences for disease progression and control and that, nonreplicating persistence is a relevant phenomenon during the infection.
Online access.
2. Intracellular persistence of Legionella.
Research on intracellular persisters has gained heightened attention in an effort to tackle antibiotic tolerance and relapsing infections. However, the physiological state of the persisters during the infection was elusive. Using the Timer growth rate reporter and the natural interaction of
Legionella pneumophila, a facultative intravacuolar pathogen, with amoebae and macrophages, we reveal extended intracellular
Legionella division rates variations ranging from 0 h
-1 (growth-arrest) to a maximum of about 0.4 h
-1, 24 h p.i. (
A). Contrary to the growing individuals, the nongrowers survived the exposure to antibiotic better defining them as nonreplicating persisters. To characterize the intracellular nonreplicating persisters on a biochemical level, FACS-sorted growers and persisters were subjected to proteomics analysis. The persister’s specific proteome, that we termed
persistome (
B), revealed a unique physiology with an active, yet distinct, metabolism and the upregulation of drug tolerance mechanisms. Hence, the persisters are not dormant bacteria. The persistome also unveiled the features of the micro-environment in which the persisters nested (
e.g., carbon sources, cues encountered...). Remarkably, the persisters produced components of the Icm/Dot Type 4 Secretion System (T4SS) and upregulated
a specific portfolio of Icm/Dot translocated effector proteins. A functional T4SS significantly increased the survival of the intracellular persisters that echoed with the capability of persisters to build a protective phosphatidylinositol-4-phosphate (Pi(4)
P) positive vacuole (Persister containing vacuole, PCV,
C, micrographs) avoiding the fusion with bactericidal lysosomes. Hence, persisters are non-dormant and preestablished subpopulation of nonreplicating individuals that emerge in evolutionarily distant phagocytes. The intracellular persisters not only have the capacity to survive antibiotic treatments but actively subvert the host cell functions, through a specific virulence program
. Online access 
