Abstract
Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.
Highlights
The peptidoglycan, built as a giant polymer of glycan chains crosslinked with short peptides, is essential for cell shape and survival in most bacteria
Some bacterial pathogens introduce structural modifications that interfere with immune recognition
Given the scarce material that can be obtained from non-proliferating intracellular bacteria [7,47], we drew upon ultra-sensitive UPLC chromatography coupled to untargeted MS/MS
Summary
Intracellular bacterial proliferation is attenuated by the pathogen within non-phagocytic cells located in the intestinal lamina propria [7] These physiological stages are understood as balanced attack and counter-attack strategies used by the host and pathogen. Typhimurium faces recognition of its peptidoglycan (PG) when persisting intracellularly despite the presence of cytosolic immune receptors that recognize molecular patterns of this cell wall component [18]. This lack of information is relevant since S. Typhimurium releases massive amounts of outer membrane vesicles (OMVs) while residing within the phagosome [19] These OMVs shed outside the phagosomal compartment may have as cargo peptidoglycan fragments present in the periplasm of intracellular bacteria. Whether these OMVs stimulate immune defenses from the intracellular environment, is unknown
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