Periodic Reporting for period 5 - INVADIS (Microbial invasion and dissemination within the host, mechanisms and effects)
Reporting period: 2022-11-01 to 2024-04-30
An infection is defined by the deleterious consequences of the interactions between a pathogen and a host. Thus, studying the biology of infection reveals critical properties of hosts and pathogens, and is a way forward to address basic biological questions and improve health. We study listeriosis, a systemic infection caused by Listeria monocytogenes (Lm). Lm is a human foodborne pathogen that crosses the intestinal barrier, disseminates systemically, replicates in liver and spleen and reaches the central nervous system (CNS) and fetoplacental unit. Given the remarkable journey Lm makes in its host, studying listeriosis offers unprecedented opportunities to understand host cell biology, physiology and immune responses, guided by Lm. The mucosal, CNS and fetal-placental tropisms of Lm are shared by other microbes which pathogenesis is far less understood. Lm therefore stands as a unique model microorganism of general biological and medical significance. The major challenge of this project is to go beyond reductionist approaches and embrace the complexity of actual infections. We will use stem cell-derived organoids, live imaging, genetically engineered mouse models, the clinical and biological data from a unique cohort of 900 patients and the corresponding causative Lm strains, to investigate the molecular mechanisms of Lm tissue invasion, dissemination and host responses. Specifically, we will (i) decipher the cell biology of microbial translocation across the intestinal epithelium; (ii) study the impact of microbial portal of entry on microbial fate, dissemination and host responses; (iii) harness Lm biodiversity to identify novel virulence factors and (iv) discover new host factors predisposing to invasive infections. Building on the unique combination of advanced experimental systems and exclusive clinical data, this integrative and innovative project will reveal novel, physiologically relevant mechanisms of infection, with scientific and biomedical implications.
During the reporting period we have acquired following results regarding the objectives of the project:
Objective 1: Decipher the cell biology of microbial translocation across intestinal epithelium
We have generated an ex vivo model of intestinal organoid to study the mechanism by which Lm translocates through goblet cells. Using the light-sheet microscope purchased in the context of INVADIS, we have shown by live imaging that Listeria transcytoses through goblet cells within a membrane vacuole. This phenotype is InlA- and microtubule-dependent. We have determined in inducible genetically modified organoids that Listeria, upon docking onto its luminally accessible receptor Ecad, hijacks its recycling pathway to be transferred by transcytosis across goblet cells. These results have been published in Current Biology (Kim et al, Curr Biol 2021) and STAR Protocols (Kim et al, STAR Protoc 2024).
Objective 2: Study the impact of microbial portal of entry on microbial fate, dissemination and host responses
We have shown that InlA-independent entry of Lm in Peyer's patch induces an IL-23 and IFN-response leading to an increase of epithelial proliferation and a depletion of mature goblet cells in the villi. This pathway therefore blocks Lm subsequent entry via goblet cells, while sensitizing host to colitis due to mucus depletion (Disson et al, J Exp Med 2018).
Objective 3: Decipher the molecular bases of Listeria monocytogenes hypervirulence
We have identified clonal complexes (CCs) of Lm that are associated with clinical isolates and are hypervirulent (Maury et al, Nature Genetics 2016). We have shown that these hypervirulent clones are adapted to mammalian gut environment, which accounts for their association with dairy products (Maury et al, Nature Communications 2019). We have also uncovered a mechanism involved in Lm infection and neurolisteriosis. We have shown that hypervirulent strains of Lm overexpress InlB which, upon activation of its receptor c-Met, protects infected monocytes from Fas-mediated cell death induced by CD8+ T-cells. Therefore, infected monocytes remain longer in the host, increasing the probability for Lm to infect the brain, but also to be released in the environment. This work has been published in Nature (Maudet al, Nature 2022), and led to a patent application (Lecuit et al, n°63/115,779 (United States)).
We have also set up a new method of core genome multi locus sequence typing (cgMLST) based on whole-genome sequencing (WGS) for surveillance of Lm as well as to determine candidate factors involved in Lm virulence (Moura et al, Nature Microbiology 2016; Emerg Infect Dis 2017). Based on this methodology, we determined how the main hypervirulent clonal complex from Lm (Lm-CC1) emerged and spread (Moura et al, Science Advances 2021). We analyzed isolates collected from 40 countries, and showed that Lm-CC1 spread worldwide from North America, which correlates with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century.
We have also studied hypovirulence and described a mechanism underlying loss of virulence in natural populations of Lm (Maury et al, Infection and Immunity 2017).
Objective 4: Identify novel host factors predisposing to invasive infections
We studied Lm carriage in non symptomatic hosts (Hafner et al, Nature Communications 2021). We showed that Lm is present in 5 to 10% of human stools, more than expected. We discovered a specific microbiota signature associated with Lm fecal carriage, both in humans and experimentally inoculated mice, in which it precedes Lm fecal carriage. We therefore showed that Lm fecal carriage is common and depends on the gut microbiota
We recently reported a human case of horizontal transmission of Lm in neonates, independently of placental infection. We demonstrated in a mouse model of infection that the neonatal susceptibility to listeriosis is due to the immaturity of the gut microbiota (Charlier et al, Cell Rep Med 2023).
Objective 1: Decipher the cell biology of microbial translocation across intestinal epithelium
We have generated an ex vivo model of intestinal organoid to study the mechanism by which Lm translocates through goblet cells. Using the light-sheet microscope purchased in the context of INVADIS, we have shown by live imaging that Listeria transcytoses through goblet cells within a membrane vacuole. This phenotype is InlA- and microtubule-dependent. We have determined in inducible genetically modified organoids that Listeria, upon docking onto its luminally accessible receptor Ecad, hijacks its recycling pathway to be transferred by transcytosis across goblet cells. These results have been published in Current Biology (Kim et al, Curr Biol 2021) and STAR Protocols (Kim et al, STAR Protoc 2024).
Objective 2: Study the impact of microbial portal of entry on microbial fate, dissemination and host responses
We have shown that InlA-independent entry of Lm in Peyer's patch induces an IL-23 and IFN-response leading to an increase of epithelial proliferation and a depletion of mature goblet cells in the villi. This pathway therefore blocks Lm subsequent entry via goblet cells, while sensitizing host to colitis due to mucus depletion (Disson et al, J Exp Med 2018).
Objective 3: Decipher the molecular bases of Listeria monocytogenes hypervirulence
We have identified clonal complexes (CCs) of Lm that are associated with clinical isolates and are hypervirulent (Maury et al, Nature Genetics 2016). We have shown that these hypervirulent clones are adapted to mammalian gut environment, which accounts for their association with dairy products (Maury et al, Nature Communications 2019). We have also uncovered a mechanism involved in Lm infection and neurolisteriosis. We have shown that hypervirulent strains of Lm overexpress InlB which, upon activation of its receptor c-Met, protects infected monocytes from Fas-mediated cell death induced by CD8+ T-cells. Therefore, infected monocytes remain longer in the host, increasing the probability for Lm to infect the brain, but also to be released in the environment. This work has been published in Nature (Maudet al, Nature 2022), and led to a patent application (Lecuit et al, n°63/115,779 (United States)).
We have also set up a new method of core genome multi locus sequence typing (cgMLST) based on whole-genome sequencing (WGS) for surveillance of Lm as well as to determine candidate factors involved in Lm virulence (Moura et al, Nature Microbiology 2016; Emerg Infect Dis 2017). Based on this methodology, we determined how the main hypervirulent clonal complex from Lm (Lm-CC1) emerged and spread (Moura et al, Science Advances 2021). We analyzed isolates collected from 40 countries, and showed that Lm-CC1 spread worldwide from North America, which correlates with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century.
We have also studied hypovirulence and described a mechanism underlying loss of virulence in natural populations of Lm (Maury et al, Infection and Immunity 2017).
Objective 4: Identify novel host factors predisposing to invasive infections
We studied Lm carriage in non symptomatic hosts (Hafner et al, Nature Communications 2021). We showed that Lm is present in 5 to 10% of human stools, more than expected. We discovered a specific microbiota signature associated with Lm fecal carriage, both in humans and experimentally inoculated mice, in which it precedes Lm fecal carriage. We therefore showed that Lm fecal carriage is common and depends on the gut microbiota
We recently reported a human case of horizontal transmission of Lm in neonates, independently of placental infection. We demonstrated in a mouse model of infection that the neonatal susceptibility to listeriosis is due to the immaturity of the gut microbiota (Charlier et al, Cell Rep Med 2023).
Our study published in Nature (Maudet et al, Nature 2022) significantly advanced our field beyond the state of the art. It was not expected, as we were looking for a bacterial factor specifically involved in brain invasion, and uncovered an unexpected mechanism that mediates Lm evasion of the host immune response, ultimately leading to neuroinvasion.
We have shown that hypervirulent strains of Lm overexpress InlB, which, upon activation of its receptor c-Met, protects infected monocytes from Fas-mediated cell death induced by CD8+ T cells. As a result, infected monocytes remain in the host longer, increasing the likelihood that Lm will infect the brain but also be released from the intestinal lumen back into the environment. This work has led to a patent application (n°63/115,779 (United States)) and several press releases (altmetric 182).
Bacterial inhibition of Fas-mediated killing promotes neuroinvasion and persistence.
Maudet C, Kheloufi M, Levallois S, Gaillard J, Huang L, Gaultier C, Tsai YH, Disson O, Lecuit M*
Nature 2022 Mar;603(7903):900-906. doi: 10.1038/s41586-022-04505-7
We have shown that hypervirulent strains of Lm overexpress InlB, which, upon activation of its receptor c-Met, protects infected monocytes from Fas-mediated cell death induced by CD8+ T cells. As a result, infected monocytes remain in the host longer, increasing the likelihood that Lm will infect the brain but also be released from the intestinal lumen back into the environment. This work has led to a patent application (n°63/115,779 (United States)) and several press releases (altmetric 182).
Bacterial inhibition of Fas-mediated killing promotes neuroinvasion and persistence.
Maudet C, Kheloufi M, Levallois S, Gaillard J, Huang L, Gaultier C, Tsai YH, Disson O, Lecuit M*
Nature 2022 Mar;603(7903):900-906. doi: 10.1038/s41586-022-04505-7