Projects & Collaborations 5 foundShow per page10 10 20 50 Influence of phage predation on strain-level diversity in the intestinal microbiota Research Project | 1 Project MembersNo Description available PhagoVax: Combining Vaccination with Phage Therapy Research Project | 2 Project MembersPhagoVax: Proof of concept Oral inactivated vaccines induce secretory Immunoglobulin A (sIgA) with strong affinity for the surface of bacterial strains. sIgA trap growing bacteria in large clumps which neutralizes virulence (enchained growth). In naïve hosts, non-typhoidal Salmonella strains can outgrow the protective intestinal microbiota. In vaccinated hosts, sIgA trapped-pathogens have a reduced fitness. This provides a competitive advantage to the microbiota, which excludes Salmonella from the gut. Oral inactivated vaccines are cheap to produce, safe and simple to administer. We have established a streamlined procedure to construct such vaccines from any cultivable bacterial species. However, pathogens can modify their surface antigens and escape sIgA binding. So we have recently refined the inactivated vaccines and demonstrated that a rationally designed polyvalent oral vaccine reduces escape options. This works in mice exposed to mixtures of all inactivated escape variants that Salmonella Typhimurium is able to generate by mutations or epigenetic changes. The result is a robust production of high affinity sIgA able to neutralize all variants of the strain that we use to prepare the vaccine. We discovered that the sIgA in mice exposed to polyvalent vaccine either lead to exclusion of Salmonella from the gut (problem solved), or select for short O-antigen mutants. These mutants sporadically emerge because the immuno-dominant antigen is the long repetitive O-antigen polysaccharide chain that carpets the surface of the pathogen. The short O-antigen is advantageous in vaccinated mice since sIgA cannot bind efficiently. However, the short O-antigen renders Salmonella sensitive to a large diversity of bacteriophages normally inhibited by long O-antigens. We hypothesize that polyvalent oral vaccines should synergize with bacteriophages to block all evolutionary escape routes and reliably prevent non-typhoidal Salmonellosis. We named this original concept "PhagoVax". The specificity of the phage/vaccine "PhagoVax" approach is a substantial advantage compared to antibiotics which profoundly disrupt the host's microbiota, thus generating a favorable niche for opportunistic resistant pathogens. On the other hand, PhagoVax is designed to target different Salmonella strains and serotypes at once. First, we can easily modify the polyvalent vaccine composition in order to select for short O-antigen mutants in different serotypes. Second, a universal bacteriophage cocktail should be able to target several serotypes. Indeed, evolution of the short O-antigen exposes structures like membrane proteins (e.g. OmpC, BtuB) that are conserved among serotypes and serve as attachment sites for bacteriophages normally inhibited by long O-antigens. Moreover, the short O-antigen phenotype is due to the deletion of the wzyB gene via high frequency site-specific recombination. The unstable configuration of the wzyB locus is conserved among Salmonella enterica prevalent serotypes infecting humans and animals (e.g. Typhimurium, Enteritidis and Javiana). Loss of wzyB occurs at high-frequency and, as such, was already reported in natural isolates of Salmonella Enteritidis from broilers. By selecting for short O-antigen mutants, the polyvalent evolutionary trap vaccine erases what makes Salmonella serotypes difficult to target via one single bacteriophage cocktail i.e., the long, variable and modifiable O-antigen. Using vaccines or phages leads to escapers. Combining the two is synergistic because the respective escapers are more vulnerable to the other treatment: vaccination leads to loss of coating polysaccharides so that phages can attack more easily; bacteria protect themselves against phages by expressing these polysaccharides, which make them a better target for antibodies. In vaccinated animals treated with the right bacteriophage cocktail, there should be no way for Salmonella to escape eradication. Our ability to generate effective vaccines has been proven for non-typhoidal Salmonellosis in mice and is currently developed in pigs. In the murine model, we also have all the tools to follow the exact kinetics of bacteria growth, evolution, clearance and transmission to the next host. This sets the stage for efficient project development, and initial experiments will focus on the synergy between adaptive immunity and bacteriophage cocktails in generating evolutionary robust protection from non-typhoidal salmonellosis. We will challenge the approach by targeting different strains of non-typhoidal Salmonella expressing various serotypes and O-antigen modifying systems. We will determine clearance rates of the targeted bacteria and expansion of bacteriophages in the gut as well as optimal delivery and dosing of the bacteriophage cocktail. EcoStrat: Interrogating the diversity of gut colonization strategies in multidrug-resistant E. coli to deduce robust competitive exclusion approaches Research Project | 1 Project MembersThe rise of multidrug-resistant bacteria limits the options to treat critically ill patients. In 2015, Escherichia coli producing Extended Spectrum β-Lactamases (ESBL) were the leading cause of death attributable to antibiotic resistant bacteria in Europe. The failure to address these infections using standard antibiotherapy calls for better understanding of how these bacteria evolve in order to develop new treatments. Presence of resistance and virulence genes correlates with high prevalence in ESBL clones. Since the intestinal tract of mammals represents a major ecological niche for E. coli, gut colonization ability must have predisposed certain clones to evolutionary success during the antibiotic era. In particular, competition against the gut microbiota should select for colonization factors that predate the acquisition of resistance. To test this hypothesis, we will compare strategies for gut colonization in ESBL clones of different prevalence in absence of antibiotics. Using mice, we will be able to modulate selective pressures exerted by the intestinal microbiota. We will compare gut colonization factors in ESBL clones by performing parallel high-throughput genetic screening in conventional mice (aim 1). We will analyze adaptability and the stability of resistance and virulence genes during long-term colonization (aim 2). Subsequent competitions in gnotobiotic mice harboring permissive microbiota will allow us to deduce functions needed for colonization in the presence of a competitive microbiota. In aim 3, we will measure the impact of competitors transmitted from cohabitant to infected mice on the duration of ESBL E. coli carriage and assess potential synergies with the adaptive immunity in vaccinated hosts. Overall, we will unravel with unprecedented depth the general principles of intestinal colonization in ESBL E. coli, shed new light on the global success of prevalent clones and conceptualize robust antibiotic-free competition-based treatments. Precision Microbiota Engineering for Child Health Research Project | 1 Project MembersIntestinal microbiota composition correlates strongly with disease ranging from cancer to autoimmunity. A growing bank of data now demonstrates causal relationships and reveals mechanisms of host-microbiota crosstalk, indicating a major untapped therapeutic potential. However, a huge gap remains in therapeutic precision engineering of the microbiota. Our key objectives are to develop two complimentary precision microbiota engineering tools to the point of human trial readiness. These complimentary approaches will be tested in gnotobiotic and humanized microbiota murine models for three serious childhood diseases with strong links to microbiota function and urgent need for better therapy/prophylaxis: 1) Urea cycle disorders, 2) Methylmalonic aciduria and 3) necrotizing enterocolitis. REMoVE (Rational Engineering of the Microbiota by Vaccination-Exclusion) employs oral vaccine-induced or recombinant secretory immunoglobulin A (SIgA) to generate a selective pressure in the intestine. This drives replacement of the target bacterium, with a desirable niche competitor. REMoVE-driven compositional changes have proof-of-concept in mice and will here be expanded to human-relevant microbiota. In situ genetic engineering employs broad host-range plasmids to deliver CRISPR interference arrays inhibiting gene expression in the microbiota, directly in the gut lumen. Fundamental insight into microbiota-disease crosstalk, and quantitative preclinical insight of therapy efficacy will be generated with state-of-the art pathophysiology analysis (real-time metabolomics, single cell transcriptomics, novel medical imaging). Toxicity analyses, GMP and scale-up will be carried out for clinical trial readiness. These diseases are prevalent in the developing world. Most of our tools can be produced, distributed and administered in low cost/low tech environments. As correction of pathological microbiota functions is relevant to a broad range of diseases, this represents a disruptive therapeutic approach with major consequences for medicine. Keeping one step ahead: understanding evolution of pathogens to manage their virulence and to stop their transmission Research Project | 1 Project MembersThe current antibiotic resistance crisis testifies that technological progress is lagging behind bacterial evolution. Understanding the general principles underlying the evolution of pathogens will be essential to design strategies shifting away from classical approaches that rely entirely on antimicrobial chemotherapy. I propose to study both the molecular mechanisms supporting virulence in entero-pathogens and to assess their evolutionary dynamics in response to factors limiting the infection (i.e., the colonization resistance, mediated by the intestinal microbiota, and the adaptive immunity in vaccinated hosts, which dampen the fitness of targeted pathogens). This will lay the foundation for preventive treatments based on rational control of pathogens in populations of hosts. My group will focus on the model organism Salmonella enterica Typhimurium, in which the expression of virulence is metabolically costly. This favors the emergence of attenuated mutants (defectors) during infections. Moreover, the expression of virulence is bimodal in S. Typhimurium, i.e., the bacteria switch phenotypically back and forth from virulent to avirulent. Bimodality in virulence expression is a feature of general relevance observed in many pathogens. However, its selective value remains poorly understood. In S. Typhimurium, bimodality seems to offset the cost of virulence and to slowdown the rise of defectors. This suggests that tightly regulated bimodal expression of virulence could represent an unexploited "Achilles' heel". This will be addressed by further developing theoretical and experimental frameworks to quantify the within-host and between-host evolutionary dynamics of S. Typhimurium. This should reveal both the conditions required to drive loss of virulence and pathogen fitness in a population of hosts, and potential drug-able molecular targets that could generate these conditions. 1 1
Influence of phage predation on strain-level diversity in the intestinal microbiota Research Project | 1 Project MembersNo Description available
PhagoVax: Combining Vaccination with Phage Therapy Research Project | 2 Project MembersPhagoVax: Proof of concept Oral inactivated vaccines induce secretory Immunoglobulin A (sIgA) with strong affinity for the surface of bacterial strains. sIgA trap growing bacteria in large clumps which neutralizes virulence (enchained growth). In naïve hosts, non-typhoidal Salmonella strains can outgrow the protective intestinal microbiota. In vaccinated hosts, sIgA trapped-pathogens have a reduced fitness. This provides a competitive advantage to the microbiota, which excludes Salmonella from the gut. Oral inactivated vaccines are cheap to produce, safe and simple to administer. We have established a streamlined procedure to construct such vaccines from any cultivable bacterial species. However, pathogens can modify their surface antigens and escape sIgA binding. So we have recently refined the inactivated vaccines and demonstrated that a rationally designed polyvalent oral vaccine reduces escape options. This works in mice exposed to mixtures of all inactivated escape variants that Salmonella Typhimurium is able to generate by mutations or epigenetic changes. The result is a robust production of high affinity sIgA able to neutralize all variants of the strain that we use to prepare the vaccine. We discovered that the sIgA in mice exposed to polyvalent vaccine either lead to exclusion of Salmonella from the gut (problem solved), or select for short O-antigen mutants. These mutants sporadically emerge because the immuno-dominant antigen is the long repetitive O-antigen polysaccharide chain that carpets the surface of the pathogen. The short O-antigen is advantageous in vaccinated mice since sIgA cannot bind efficiently. However, the short O-antigen renders Salmonella sensitive to a large diversity of bacteriophages normally inhibited by long O-antigens. We hypothesize that polyvalent oral vaccines should synergize with bacteriophages to block all evolutionary escape routes and reliably prevent non-typhoidal Salmonellosis. We named this original concept "PhagoVax". The specificity of the phage/vaccine "PhagoVax" approach is a substantial advantage compared to antibiotics which profoundly disrupt the host's microbiota, thus generating a favorable niche for opportunistic resistant pathogens. On the other hand, PhagoVax is designed to target different Salmonella strains and serotypes at once. First, we can easily modify the polyvalent vaccine composition in order to select for short O-antigen mutants in different serotypes. Second, a universal bacteriophage cocktail should be able to target several serotypes. Indeed, evolution of the short O-antigen exposes structures like membrane proteins (e.g. OmpC, BtuB) that are conserved among serotypes and serve as attachment sites for bacteriophages normally inhibited by long O-antigens. Moreover, the short O-antigen phenotype is due to the deletion of the wzyB gene via high frequency site-specific recombination. The unstable configuration of the wzyB locus is conserved among Salmonella enterica prevalent serotypes infecting humans and animals (e.g. Typhimurium, Enteritidis and Javiana). Loss of wzyB occurs at high-frequency and, as such, was already reported in natural isolates of Salmonella Enteritidis from broilers. By selecting for short O-antigen mutants, the polyvalent evolutionary trap vaccine erases what makes Salmonella serotypes difficult to target via one single bacteriophage cocktail i.e., the long, variable and modifiable O-antigen. Using vaccines or phages leads to escapers. Combining the two is synergistic because the respective escapers are more vulnerable to the other treatment: vaccination leads to loss of coating polysaccharides so that phages can attack more easily; bacteria protect themselves against phages by expressing these polysaccharides, which make them a better target for antibodies. In vaccinated animals treated with the right bacteriophage cocktail, there should be no way for Salmonella to escape eradication. Our ability to generate effective vaccines has been proven for non-typhoidal Salmonellosis in mice and is currently developed in pigs. In the murine model, we also have all the tools to follow the exact kinetics of bacteria growth, evolution, clearance and transmission to the next host. This sets the stage for efficient project development, and initial experiments will focus on the synergy between adaptive immunity and bacteriophage cocktails in generating evolutionary robust protection from non-typhoidal salmonellosis. We will challenge the approach by targeting different strains of non-typhoidal Salmonella expressing various serotypes and O-antigen modifying systems. We will determine clearance rates of the targeted bacteria and expansion of bacteriophages in the gut as well as optimal delivery and dosing of the bacteriophage cocktail.
EcoStrat: Interrogating the diversity of gut colonization strategies in multidrug-resistant E. coli to deduce robust competitive exclusion approaches Research Project | 1 Project MembersThe rise of multidrug-resistant bacteria limits the options to treat critically ill patients. In 2015, Escherichia coli producing Extended Spectrum β-Lactamases (ESBL) were the leading cause of death attributable to antibiotic resistant bacteria in Europe. The failure to address these infections using standard antibiotherapy calls for better understanding of how these bacteria evolve in order to develop new treatments. Presence of resistance and virulence genes correlates with high prevalence in ESBL clones. Since the intestinal tract of mammals represents a major ecological niche for E. coli, gut colonization ability must have predisposed certain clones to evolutionary success during the antibiotic era. In particular, competition against the gut microbiota should select for colonization factors that predate the acquisition of resistance. To test this hypothesis, we will compare strategies for gut colonization in ESBL clones of different prevalence in absence of antibiotics. Using mice, we will be able to modulate selective pressures exerted by the intestinal microbiota. We will compare gut colonization factors in ESBL clones by performing parallel high-throughput genetic screening in conventional mice (aim 1). We will analyze adaptability and the stability of resistance and virulence genes during long-term colonization (aim 2). Subsequent competitions in gnotobiotic mice harboring permissive microbiota will allow us to deduce functions needed for colonization in the presence of a competitive microbiota. In aim 3, we will measure the impact of competitors transmitted from cohabitant to infected mice on the duration of ESBL E. coli carriage and assess potential synergies with the adaptive immunity in vaccinated hosts. Overall, we will unravel with unprecedented depth the general principles of intestinal colonization in ESBL E. coli, shed new light on the global success of prevalent clones and conceptualize robust antibiotic-free competition-based treatments.
Precision Microbiota Engineering for Child Health Research Project | 1 Project MembersIntestinal microbiota composition correlates strongly with disease ranging from cancer to autoimmunity. A growing bank of data now demonstrates causal relationships and reveals mechanisms of host-microbiota crosstalk, indicating a major untapped therapeutic potential. However, a huge gap remains in therapeutic precision engineering of the microbiota. Our key objectives are to develop two complimentary precision microbiota engineering tools to the point of human trial readiness. These complimentary approaches will be tested in gnotobiotic and humanized microbiota murine models for three serious childhood diseases with strong links to microbiota function and urgent need for better therapy/prophylaxis: 1) Urea cycle disorders, 2) Methylmalonic aciduria and 3) necrotizing enterocolitis. REMoVE (Rational Engineering of the Microbiota by Vaccination-Exclusion) employs oral vaccine-induced or recombinant secretory immunoglobulin A (SIgA) to generate a selective pressure in the intestine. This drives replacement of the target bacterium, with a desirable niche competitor. REMoVE-driven compositional changes have proof-of-concept in mice and will here be expanded to human-relevant microbiota. In situ genetic engineering employs broad host-range plasmids to deliver CRISPR interference arrays inhibiting gene expression in the microbiota, directly in the gut lumen. Fundamental insight into microbiota-disease crosstalk, and quantitative preclinical insight of therapy efficacy will be generated with state-of-the art pathophysiology analysis (real-time metabolomics, single cell transcriptomics, novel medical imaging). Toxicity analyses, GMP and scale-up will be carried out for clinical trial readiness. These diseases are prevalent in the developing world. Most of our tools can be produced, distributed and administered in low cost/low tech environments. As correction of pathological microbiota functions is relevant to a broad range of diseases, this represents a disruptive therapeutic approach with major consequences for medicine.
Keeping one step ahead: understanding evolution of pathogens to manage their virulence and to stop their transmission Research Project | 1 Project MembersThe current antibiotic resistance crisis testifies that technological progress is lagging behind bacterial evolution. Understanding the general principles underlying the evolution of pathogens will be essential to design strategies shifting away from classical approaches that rely entirely on antimicrobial chemotherapy. I propose to study both the molecular mechanisms supporting virulence in entero-pathogens and to assess their evolutionary dynamics in response to factors limiting the infection (i.e., the colonization resistance, mediated by the intestinal microbiota, and the adaptive immunity in vaccinated hosts, which dampen the fitness of targeted pathogens). This will lay the foundation for preventive treatments based on rational control of pathogens in populations of hosts. My group will focus on the model organism Salmonella enterica Typhimurium, in which the expression of virulence is metabolically costly. This favors the emergence of attenuated mutants (defectors) during infections. Moreover, the expression of virulence is bimodal in S. Typhimurium, i.e., the bacteria switch phenotypically back and forth from virulent to avirulent. Bimodality in virulence expression is a feature of general relevance observed in many pathogens. However, its selective value remains poorly understood. In S. Typhimurium, bimodality seems to offset the cost of virulence and to slowdown the rise of defectors. This suggests that tightly regulated bimodal expression of virulence could represent an unexploited "Achilles' heel". This will be addressed by further developing theoretical and experimental frameworks to quantify the within-host and between-host evolutionary dynamics of S. Typhimurium. This should reveal both the conditions required to drive loss of virulence and pathogen fitness in a population of hosts, and potential drug-able molecular targets that could generate these conditions.
