Fabien J. Fuche

Abstract Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health 1 . Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells 2 , we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a β-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.

Nontyphoidal Salmonella (NTS; i.e., Salmonella enterica organisms that do not cause typhoid or paratyphoid) are responsible for 94 million infections and 155,000 deaths worldwide annually, 86% of which are estimated to be foodborne. Although more than 50 serogroups and 2,600 serovars have been described, not all Salmonella serovars cause disease in humans and animals. Efforts are being made to develop NTS vaccines, with most approaches eliciting protection against serovars Typhimurium and Enteritidis (serogroups B [O:4] and D [O:9], respectively), as they are widely considered the most prevalent. Here, we show that serogroup C (O:6,7, O:6,8, or O:8 epitopes) is the most common serogroup in the United States, and the prevalence of serovars from this serogroup has been increasing in Europe and the United States over the last decade. They are also the most commonly isolated serovars from healthy cattle and poultry, indicating the underlying importance of surveillance in animals. Four out of the 10 most lethal serovars in the United States are serogroup C, and reports from African countries suggest that strains within this serogroup are highly antibiotic resistant. Serogroup C consists of highly diverse organisms among which 37 serovars account for the majority of human cases, compared to 17 and 11 serovars for serogroups B and D, respectively. Despite these concerning data, no human vaccines targeting serogroup C NTS are available, and animal vaccines are in limited use. Here, we describe the underestimated burden represented by serogroup C NTS, as well as a discussion of vaccines that target these pathogens.

Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells.

Non-typhoidal Salmonella (NTS) are a leading cause of foodborne infections worldwide, and serogroups B, C1, C2-C3 and D are the most common serogroups associated with human disease. While live vaccine candidates that protect against S. Typhimurium (serogroup B) and S. Enteritidis (serogroup D) have been described by us and others, far less effort has been directed towards vaccines that target either serogroup C1 or C2-C3 Salmonella. Here we describe a Salmonella Newport-based live-attenuated vaccine (serogroup C2-C3). Deletion of the genes clpX or rfaL, previously used in live vaccines to attenuate S. Typhimurium and/or S. Enteritidis, failed to attenuate S. Newport. However, we found that deletion of either guaBA or htrA raised the 50% lethal dose of S. Newport in an intraperitoneal infection model in BALB/c mice. Our live-attenuated vaccine candidate CVD 1966 (S. Newport ΔguaBA ΔhtrA) elicited strong antibody responses against COPS, flagellin and outer membrane proteins when administered intraperitoneally or orally. Following lethal challenge with the parental virulent strain of S. Newport, we observed vaccine efficacies of 53% for immunization via the intraperitoneal route and 47% for immunization via the oral route. Following intraperiteonal immunization, the vaccine also significantly reduced the bacterial burden of challenge organisms in the liver and spleen. Interestingly, reducing the LPS chain length by deleting rfaL did not induce a stronger immune response towards surface antigens, and failed to elicit any protection against lethal homologous challenge. In conclusion, we have developed a live-attenuated Salmonella serogroup C2-C3 vaccine that we are further evaluating.