L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Grampositive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for Lrhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection.This work was funded by the Fundo Europeu de Desenvolvimento Regional (FEDER), through the Operational Competitiveness Programme (COMPETE), and by national funds through FCT (Fundacao para a Ciencia e a Tecnologia), under projects PTDC/SAU-IMU/111806/2009, PTDC/SAU-MIC/111581/2009FCOMP-01-0124-FEDER-015844, ERANet-Pathogenomics LISTRESS ERA-PTG/0003/2010, Infect-ERA/0001/2013 PROANTILIS, and by project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions", co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referencia Estrategico Nacional (QREN), through FEDER and FCT. FC, MLA, RP and GC were supported by FCT doctoral fellowships (SFRH/BD/61825/2009, SFRH/BD/28440/2006, SFRH/BD/89542/2012 and SFRH/BD/52207/2013). SS was supported by the Ciencia 2008 and FCT Investigator programs (COMPETE, POPH and FCT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript