Ongoing projects
We have more than 10 years of expertise on studying the pathogen phenotypic variations to decipher & control the infection process and the treatment failures. Our pionnering work at the single-cell level led to (i) the development of unprecedented methodological approaches and (ii) new concepts in the fields of microbiology, infection biology and antibiotic resistance / tolerance / persistence.
Phenotypic heterogeneity and antibiotic persisters: Infection success and treatment failure: ESKAPE: Klebsiella pneumoniae. TBA. Legionnaire's disease: 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). 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. 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. 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. In agreement, we recently found that Legionella developed intracellular subpopulation of nonreplicating persisters. 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. Ongoing research aim at understanding the molecular bases of intracellular Legionella persister formation, survival and growth resumption.
One Heath: Bacterivorous protists are fascinating autonomous microorganisms that play a pivotal yet overlooked role in diverse ecosystems. Numerous human bacterial pathogens have evolved as facultative endosymbionts within these protists. Yet, impact of the protists on the life cycle of human bacterial pathogens remains elusive. By delving into the natural interplay between the protozoan predators and the human bacterial pathogens, we have uncovered an unforeseen function of the protists as catalysts for diversifying the pathogen physiology. We have already unveiled various clonal bacterial subsets with deep physiological realignments and functional specialization. This sustains an elaborated intracellular bacterial community behavior that spans antibiotic persistence, cooperative virulence or cooperative colonization.
Methods developments: Studying the the biology of the bacterial phenotypic variants is challenging, as they represent a reversible physiological state occurring in a fraction of the pathogen population. To track and analyse individual bacterium and bacterial subpopulation, we developped (and keep developping) innovative single-cell and Omics based pipeline. We notably engineered the Timer single-cell bacterial growth rate fluorescent reporter to track and analyse the variants based on their growth rate. To achieve an quantitative and unbiased analysis, we combine methods in microbiology, cell biology, molecular biology to infections assays and cutting-edge high-throughput single-cell acquisition systems, multi-parametric high-resolution automated microscopy, omics analysis and genome-wide loss of function approaches. We are also coordinating an intra-mural research project at @CIRI_Lyon, focused on establishing a human lung organoid model. Learn more here: https://ciri.ens-lyon.fr/ciri/trans-projects/funded-for-the-year-2021
Service/Collaboration: By coupling infection models, genome-wide loss of functions, single cell technologies, and Omics analysis, we have establish a powerful pipeline (i) to better understand the mechanisms underlying cell-to-cell variations during the infection, and (ii) to identify new molecular determinants based on which new medicines may be developed or evaluated. Our plateform is suitable for a wide range of pathogens. For collaboration (industrial partners, academic research) contact Nicolas Personnic.
SOME 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
3. Bistable quorum sensing activity governs persisters formation and resuscitation
The
Legionella quorum sensing (QS) system of
Legionella centers on the production and the detection of the autoinducer molecule LAI-1 produced by the autoinducer synthase LqsA. A regulatory node bridges the QS to the stringent response, a stress response in reaction to starvation that is commonly associated to persistence. We monitored the activation of the QS at the single-cell level by designing a plasmid-based biosensor reporting the promoter activity of
6SRNA, found strongly upregulated by synthetic LAI-1 (P
tac-
mCherry-P
6SRNA-
gfp). This way, we unveiled that the QS was heterogeneously activated among infectious
Legionella grown in broth as well as by bacteria within microcolonies (
A). Remarkably, activation of the QS was dependent on the temperature (i.e., triggered above 30°C) as well as the microcolony topology since the LAI-1 activated
Legionella localized preferentially to the colonies boundaries (
A). Remarkably, the frequency of bacterial growth initiation positively correlated with the activation of the LAI-1 signaling pathway (
A), indicating a possible role of QS in nonreplicating persister formation. Using deletion mutants inactivating the autoinducer LAI-1 synthesis, sensing or signal transduction, we confirmed that the QS regulated the formation of intracellular
Legionella virulent nonreplicating persisters (
B).The persisters resuscitation has long been overlooked, despite its functional importance, and the underlying mechanisms has remained elusive.
We monitored the growth resumption of Legionella persisters produced by growing the bacteria to stationary phase. Upon nutrients supply, stationary phase grown bacteria resumed growth heterogeneously, leading to the coexistence of growers (“green” microcolonies) and a substantial subset of nonreplicating persisters (“red” individuals). The growth resumption frequency was determined by the LAI-1 signaling pathway (
C) and contributed to the pathogen bet-hedging strategy by maintaining a subpopulation of persisters even in favorable growth conditions. Here, we broke with the central assumption that the quorum sensing primarily represents a regulatory mechanism to uniform and to coordinate bacterial behavior as it also determines functionally distinct subpopulations of
Legionella and governs persister formation and resuscitation. Persisters are thus much more versatile than commonly appreciated to fine tune persistence and growth resumption.
Online access
4. Intravacuolar phenotypic specialization of Legionella.
In the late stage of the host cell infection, within Pi4P-positive vacuoles, the proliferative subpopulation of Legionella formed a densely populated microcolony of isogenic individuals (A) that may exhibit community traits, a hallmark of which is the bi-stability in gene expression. Legionella assembles a single flagellum prior to its release into the environment. We monitored the induction of the bacterial flagellum during the course of an infection, by engineering a dual fluorescence reporter plasmid (Ptac-mCherry-PflaA-gfp) to visualize and quantify the promoter activity of flagellin (flaA). We discovered, in the late stage of the host cell infection, a spatially regulated bi-stable expression of the flagellin, the expression of which was restricted to the bacteria located at the periphery of the intravacuolar microcolony (B). Inactivation of the quorum sensing system (QS), one of the major regulator of the flagellum machinery abolished the bistable expression of flagellin within the vacuole. Proteomics analysis of the subpopulations showed that the interior flaA-negative population preferentially produced the proteins promoting cell division. In contrast, the replication inhibitors were produced by the peripherical flaA-positive population along with proteins implicated in the production, and assembly of the flagellum (C). Both subpopulations produced the Icm/Dot T4SS and, for each, a specific set of translocated effectors. Bi-stability in the flagellum assembly led an optimal cooperative colonization of the extracellular environment with the preferential exit and long-distant spreading of the flagellated individuals whilst the interior non-motile and proliferative bacteria remained clustered within the dead host cell (D). Type IV- and type II-secreted Legionella phospholipases promoted the release of the flagellated individual. Hence, the intravacuolar Legionella perform a “division of labor” to maximize the colonization of the local and distant environment. While the peripheral flagellated bacteria spread and can readily infect other host cells in a distance, the interior individuals might be better suited to infect “grazing” phagocytes, and/or might be physiologically better equipped to adhere to and thrive in a comparably rich (biofilm) environment. Our proteomics analysis of the intracellular subpopulation revealed that Legionella organises and deploys its record number of >300 effectors according to the functional needs of each intracellular subpopulations.
