The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread

Abstract

Listeria monocytogenes secretes two distinct phospholipases C, a phosphatidylinositol-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). In this study, single in-frame deletion mutants with mutations in each PLC and a double mutant lacking both PLCs were characterized with regard to virulence in mice, escape from a primary vacuole, and cell-to-cell spread in cell culture. The mutant lacking PI-PLC, previously shown to be twofold less virulent than the wild type in mice, had a minor defect in escape from a primary vacuole but was not notably affected in cell-to-cell spread. The mutant lacking PC-PLC was 20-fold less virulent in mice and was defective in cell-to-cell spread but had no measurable defect in escape from a primary vacuole. The mutant lacking both PLCs was 500-fold less virulent in mice and was severely diminished in its ability to escape from the primary vacuole and to spread cell to cell. Cellular levels of diacylglycerol and ceramide, products of PLC activity, accumulated beginning 3 to 4 h after infection of cells with wild-type bacteria. The bacterial PLCs were partially responsible for this activity, since cells infected with the mutant lacking both PLCs had a reduced increase in diacylglycerol and no increase in ceramide. Elevation of diacylglycerol in the absence of bacterial PLCs indicated that host cell phospholipase(s) was activated during infection. The results of this study were consistent with the two bacterial PLCs having overlapping functions throughout the course of intracellular infection. Furthermore, the PC-PLC, and possibly PI-PLC, appeared to be enzymatically active intracellularly.

Listeriolysin O Is Necessary and Sufficient to Induce Autophagy during Listeria monocytogenes Infection

“…The majority of wildtype L. monocytogenes escape the vacuole within the first 30 minutes following entry, however Py et al (2007) assayed escape at four hours post-infection [44]. Since L. monocytogenes begin to spread from cell to cell as early as three hours post-infection and phospholipase mutants have a known defect in escape from a secondary vacuole, it is possible that the defect in growth observed by Py et al (2007) was due to a defect in escape from vacuoles of secondarily infected cells [13,45,46] Autophagy plays a role in cellular homeostasis. For example, smooth ER, excess peroxisomes, damaged mitochondria and rough ER have been shown to be selectively targeted and removed from cells by the autophagic machinery [47][48][49][50].…”

“…The wild-type Listeria monocytogenes strain used was 10403S [54]. The Dhly (DP-L2161; [55]), DactA (DP-L3078; [56]) and DplcAB (DP-L1936; [46]) mutants contained in-frame deletions of the respective genes in the 10403s background.…”

Lipids in host–pathogen interactions: Pathogens exploit the complexity of the host cell lipidome

“…The combined action of phospholipase and a pore-forming protein in escape from the vacuole is also seen for the well-studied L. monocytogenes, an intracellular pathogen that replicates in the cytosol of host cells [63,64]. After invasion into cells, L. monocytogenes escapes from its vacuole by the combined action of the pore-forming protein listeriolysin O (LLO) and a phosphatidylinositol-specific PLC (PI-PLC), both secreted by the bacterium [63,64,66,67]. It has been suggested that LLO forms holes into the vacuole membrane, which allow L. monocytogenes phospholipase to access exposed membrane leaflet [63].…”

“…After such cell-to-cell spread the bacterium resides in a secondary vacuole surrounded by a double membrane. The escape from this double-membrane vacuole requires the coordinated action of LLO, PI-PLC and PC-PLC (a PC-preferring PLC also secreted by L. monocytogenes) [67,69,70]. Recent data indicate that the PI-PLC and PC-PLC act specifically in the initial degradation of the inner membrane of the double-membrane vacuole, after which the outer membrane is disrupted by LLO [71].…”

Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles

“…Listeria monocytogenes escape is enhanced by two bacterial C-type phospholipases, termed PlcA and PlcB (Smith et al ., 1995). Lm mutants deficient in a phosphatidylinositol-specific phospholipase C (PlcA), escape from vacuoles less frequently than wild-type Lm (Smith et al ., 1995).…”