Deciphering the underlying mechanisms of a novel peptidoglycan hydrolase-dependent protein secretion system in Salmonella enterica serovar Typhi

• Applicant: Dr. Tobias Geiger
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 523077943

Bacterial pathogens have evolved sophisticated protein secretion systems that play a pivotal role in the pathogenicity of bacteria. They facilitate attachment to various surfaces, defense against host antimicrobial systems, delivery of virulence factors, and effector proteins to the extracellular environment or directly in eukaryotic cells, nutrient acquisition, and communication with the surrounding environment. The diversity of these secretion systems and their various functions underline their great importance in the establishment of bacteria within different ecological niches. Protein transport across the bacterial envelope is particularly challenging in Gram-negative bacteria, as proteins must move through at least three physical barriers: the inner membrane, the peptidoglycan layer, and the outer membrane. Studies on typhoid toxin, a major virulence factor of the human pathogen Salmonella enterica serovar Typhi, discovered a novel protein secretion system that differs significantly from other known secretion systems. Interestingly, phylogenetic analyses revealed major components of this system in other bacterial genomes, which indicates a high prevalence of this protein secretion system in many bacteria. The studies characterized TtsA (typhoid toxin secretion protein A), a specialized bacterial cell wall hydrolyzing enzyme, as the essential component for the secretion process. Mainly localized at the bacterial poles, TtsA locally degrades specifically modified peptidoglycan of the bacterial cell wall, which subsequently facilitates the secretion of periplasmic localized typhoid toxin at the bacterial poles. Nevertheless, many aspects and components of the secretion mechanism are still unknown. The first goal of the project is to verify preliminary results that show that TtsA facilitates active secretion of typhoid toxin from viable and intact bacteria, despite targeting the peptidoglycan of the bacterial cell wall. Here, comprehensive expression and secretion studies with fluorescence reporter strains will be realized. Secondly, our research will also focus on identifying additional components associated with typhoid toxin secretion. An intriguing question is how per se cytoplasmic localized TtsA can reach the peptidoglycan layer, its target in the bacterial periplasm. This is even more astonishing since TtsA lacks a discernible signal peptide that would explain its translocation across the inner membrane via the canonical Sec machinery. Here, transposon mutant screens will reveal components responsible for TtsA translocation to the periplasm. Furthermore, additional transposon mutant screens and specifically deployed electron microscopy analyses will elucidate how typhoid toxin finally gets released from the bacterial cell through the bacterial outer membrane layer. Overall our studies will shed light on the mechanisms behind a novel peptidoglycan hydrolase-dependent protein secretion system in Salmonella Typhi.

• DFG Programme: Research Grants