Chitinase A and Cytolysin A: important virulence factors of Salmonella enterica, secreted by type 10 secretion systems

Krone, Lena (2025): Chitinase A and Cytolysin A: important virulence factors of Salmonella enterica, secreted by type 10 secretion systems. Dissertation, LMU München: Medizinische Fakultät

Abstract

Salmonella enterica is a foodborne pathogen which is spread by contaminated food or water. The infection by Salmonella leads to millions of illnesses and death cases per year. It is a facultative intracellular pathogen, which is able to invade into host cells and to survive and proliferate within the cells by translocation of effector proteins and secretion of toxins. During invasion, a membrane compartment emerges, which is restructured to a Salmonella-containing vacuole (SCV). While non-typhoidal serovars typically result in self-limiting gastroenteritis, typhoidal serovars have the capability to spread from the initial intestinal infection site to secondary sites within the human body. This leads to systemic disease and long-term bacterial persistence. S. enterica harbors several virulence factors crucial for colonizing the host and infiltrating diverse host target cells. We first focused on the pathogenicity factor chitinase A (ChiA). It was known that ChiA functions as an enzyme, breaking down chitin by cleaving its β1-4 glycosidic bonds. Our hypothesis was that ChiA also plays an important role for S. enterica virulence in a chitin-free environment such as the human host. It was our aim to elucidate the effect of ChiA on Salmonella invasion into human cells and further to clarify the effect of transcriptional regulators and other proteins encoded in the chiA operon on ChiA regulation and secretion. We found that the deletion of chiA leads to reduced invasion into human host target cells, comparable with data of other studies, published at about the same time. Intriguingly, our studies revealed that ChiA-mediated invasion occurs specifically in polarized human intestinal epithelial cells, whereas non-polarized or non-intestinal epithelial cells did not show reduced invasion phenotypes. In addition, we found that both cell-associated and free mucus lead to ChiA expression and secretion. This finding aligns with other studies showing that host cell glycans, which have structures similar to mucus, are targets of Salmonella chitinases. Furthermore, we unraveled the regulation of ChiA expression by a transcriptional regulator (STM0017) that we named ChiR. Finally, we showed that ChiA is actively secreted by a type 10 secretion system (T10SS) encoded within the chiA operon. Former studies have shown that the secretion of cargo proteins by T10SS in Gram-negative bacteria is facilitated by specified peptidoglycan hydrolases. They showed that these peptidoglycan hydrolases accumulate within the periplasm by traversing the inner membrane with the help of holins. Subsequently, the hydrolases cleave the peptidoglycan layer which enables the secretion of proteins through the bacterial cell wall. However, the final release mechanism through the bacterial outer membrane is currently unknown and needs further investigations in the future. In a second study, we focused on cytolysin A (ClyA), a barely described toxin known to be encoded only in strictly human-adapted typhoidal Salmonella serovars such as Typhi and Paratyphi A. ClyA is a pore-forming toxin and its homologue in Escherichia coli is well-described regarding its pore formation characteristics and regulation of expression. Using S. enterica serovar Paratyphi A, we demonstrated that ClyA represents a mainly intracellularly induced toxin that is exclusively expressed and secreted from bacteria residing within the SCV. Since its expression within the SCV correlates with the activity of a T10SS, which is responsible for the secretion of typhoid toxin, we hypothesized that the same secretion system also secretes ClyA. Using a TtsA peptidoglycan hydrolase mutant, we showed that ClyA secretion is completely abolished in this T10SS deficient mutant, thereby verifying our hypothesis. Once secreted, ClyA exhibit specific cytolytic activities towards macrophage-like THP-1 and U937 cells, but lacked activity towards epithelial cells such as HeLa, CaCo-2 and HT29-MTX cells. We postulate that during infection, ClyA is mainly produced within infected epithelial cells and aids Salmonella infection by lysing attracted macrophages after its release from epithelial producer cells. However, this assumption and the mechanistic details regarding the susceptibilities of different cell types need to be investigated in further studies. In summary, this work elucidates the expression, regulation, and secretion of ChiA and ClyA, two important yet barely described virulence factors of S. enterica. Additionally, it demonstrates the relevance of two similar but slightly different T10SS and their impacts on the pathogenicity of S. enterica.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 15. Juli 2025
• 1. Bericht­erstatter:in: Josenhans, Christine