Development of bacterial autolysis strains to detect innate immune responses triggered by production of local mediators active on mucosal surfaces

• Applicant: Dr. Mareike Lembke
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 465465835

Vibrio cholerae is a Gram-negative bacterium and the causative agent of cholera, an enteric disease with an estimated burden of 100,000 deaths worldwide each year. Upon ingestion by the human host, V. cholerae colonizes the small intestine and is then detected by the host innate immune system. The resulting inflammatory response seems more robust in the presence of commensals and V. cholerae strains that express the Type VI Secretion System (T6SS), a syringe-like nanomachine that injects toxic effectors into prey cells by piercing their cellular envelope. The killing of sensitive commensal bacteria in the gut of infected animals presumably leads to the release of their contents including DNA, lipopolysaccharides (LPS), peptidoglycan (PG) or lipoproteins. Such bacterial debris are commonly rich in molecules that display pathogen-associated molecular patterns (PAMPs), bacterial-specific conserved structures that are known to be potent inducers of the innate immune system; these include molecules such as LPS, PG and others. For V. cholerae it has been postulated that the inflammatory milieu shaped by microbial antagonistic interactions improves its fitness by upregulating V. cholerae virulence factors, including toxin coregulated pilus and cholera toxin but by a mechanism that has yet to be defined. One way to address the role of PAMPs, e.g. derived from bacterial cell wall components such as PG, in communication with the host innate immune system is to create novel systems (“biological signal amplifiers”) that will generate these signalling molecules at the mucosal surface after colonization. In this study I propose two approaches for the controlled lysis of bacteria in the suckling mouse model, one using CRISPR-interference (CRISPRi) for the knockdown of V. cholerae genes essential to cell wall integrity and other using heterologous murein hydrolases as a life-death switch in commensal and pathogenic E. coli strains as well as V. cholerae to create a temporally controllable imbalance in cell wall biosynthesis/repair. These orthogonal strategies will recreate a controlled scenario that mimics V. cholerae’s antagonistic behavior towards commensal bacteria and allows the quantification of its pathogenicity and host innate immune responses driven by cell wall debris. Advances in synthetic biology as described in this proposal also have the potential to develop innovative therapeutic strategies that can be applied to diseases that affect mucosal surfaces such as Crohn’s disease or colorectal cancer. Investigating which set of PAMPs sent off from our novel ‘biological signal amplifiers’ is related to which specific immune response may positively impact cancer treatment in the future by stimulating suppressed immune cells in ways analogous to immune checkpoint inhibitors used in immunotherapy.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor John J. Mekalanos, Ph.D.