Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors

Abstract

Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) plays a critical role in escape of this human pathogen from host cell vacuoles. Unlike classical bacterial PI-PLCs, the L. monocytogenes enzyme has very weak activity on glycosylphosphatidylinositol (GPI)-anchored proteins. Previous crystal structure analysis has revealed that a small ␤-strand (Vb) is present in Bacillus cereus PI-PLC and is absent in the enzyme from L. monocytogenes. This Vb ␤-strand in B. cereus PI-PLC forms contacts with the glycan linker of GPI anchors, which presumably increases its activity on GPI-anchored proteins. In this study, we show that, of all known bacterial PI-PLCs, those from listeriae are the only ones that lack the ␤-strand. Expression by L. monocytogenes of B. cereus PI-PLC, which has strong activity on GPI-anchored proteins, inhibited bacterial escape from a vacuole and cell-to-cell spread, resulting in greatly reduced virulence in mice. Deletion of the Vb ␤-strand from B. cereus PI-PLC abolished its ability to cleave GPI-anchored proteins, decreased its inhibitory effects, and increased its virulence in mice. These results strongly suggest that L. monocytogenes PI-PLC has evolved as an important determinant of L. monocytogenes pathogenesis by absence of the Vb ␤-strand, thus leading to greatly reduced activity on GPI-anchored proteins. glycosylphosphatidylinositol-anchored proteins

Membrane-damaging and cytotoxic sphingomyelinases and phospholipases

“…Mutants of L. monocytogenes that express B. cereus PI-specific PLase C, which has a strong activity on GPI protein anchors, inhibited bacterial escape from a vacuole and cell-to-cell spread, resulting in greatly reduced virulence in mice. These results support the hypothesis that L. moncocytogenes PI-specific PLase C has evolved for intracellular growth and virulence by reducing its activity on GPI-anchored proteins through loss of the Vb β-strand [113].…”

A Highly Dynamic ER-Derived Phosphatidylinositol-Synthesizing Organelle Supplies Phosphoinositides to Cellular Membranes

“…To take advantage of this substrate restriction, we used a bacterial PI-PLC for expression in mammalian cells to specifically manipulate PtdIns levels. The Listeria monocytogenes PLC has substantial activity against PtdIns in addition to its natural substrate, GPI (Wei et al, 2005). We cloned this enzyme from Listeria monocytogenes (strain 10403S), removed its N-terminal hydrophobic signal sequence to prevent its targeting to the secretory pathway, and fused it to mRFP.…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…9) consists primarily of three residues: His32 (general base), His82 (general acid), and Arg69, acting as a phosphate-activating residue (183). The higher capacity of the B. cereus PI-PLC to release GPI-anchored proteins from cellular membranes relative to the L. monocytogenes enzyme seems to be the result of the Vb strand insertion that extends the binding pocket and allows considerable additional interactions with glycan substrates (184).…”