Seminar Presentation

 Understanding the coordination between MreB and c-di-AMP in Listeria monocytogenes b-lactam resistance and pathogenesis 

The intracellular bacterial pathogen Listeria monocytogenes (Lm) requires c-di-AMP (cdA) for growth and infection, but must regulate its level to avoid toxic accumulation. cdA hydrolysis by phosphodiesterases (PDEs) is a major mechanism to maintain homeostasis. A mutant lacking cdA phosphodiesterases (ΔPDE), and thus having cdA accumulation, is highly attenuated for virulence and is sensitive to cell wall-targeting antibiotics like β-lactams. The mechanisms underlying this toxicity upon cdA accumulation are unknown. Thus, to profile these molecular defects in the ΔPDE mutant, we performed a genetic screen for suppressor mutants (mutants with high cdA levels but resistant to β-lactams) using the chemical mutagen ethyl methanesulfonate. Whole-genome sequencing revealed that among 10 suppressor mutants, 8 carried missense mutations in the gene encoding the MreB protein, an essential component of the Rod complex, involved in lateral peptidoglycan synthesis in Lm. Microscopy revealed that these suppressor mutants exhibited shorter, wider cells, consistent with reduced MreB function. Furthermore, β-lactam resistance in ΔPDE could be increased by chemical inhibition of MreB, mimicking the effect of suppressor mutations. Microscopy further revealed that ΔPDE cells exhibit a severe division defect under sub-lethal β-lactam stress, a defect that was also rescued by MreB mutations. Finally, the L2 plaque assay showed that all MreB suppressor mutants were also rescued for mammalian cell infectivity despite high cdA levels. Together, these observations suggest that cdA accumulation in Lm disrupts the balance between MreB-Rod complex activity and cell division, leading to defective cell wall homeostasis and attenuated virulence. Understanding how cdA regulates MreB activity will reveal fundamental principles of second-messenger coordination in bacteria and uncover new targets for antimicrobial intervention.