Characterization ofListeria monocytogenesExpressing Anthrolysin O and Phosphatidylinositol-Specific Phospholipase C fromBacillus anthracis

Abstract

Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. Listeria monocytogenes and Bacillus anthracis are gram-positive bacteria in the low-GϩC lineage that cause rare but fatal infections of humans and animals. Although L. monocytogenes has been studied as a model facultative intracellular pathogen for decades, B. anthracis had, until recently, received less attention. Interestingly, the B. anthracis genome sequence has revealed the presence of a number of potential determinants of pathogenesis shared with those of L. monocytogenes, including a phosphatidylinositol-specific phospholipase C (PI-PLC), a phosphatidylcholine-preferring phospholipase C (PC-PLC), and listeriolysin O (LLO) (27). In L. monocytogenes, these determinants are integral to its intracellular growth cycle. LLO is essential for escape from a phagosome, the phospholipases contribute to that process, and all three are important for cell-to-cell spread (3, 10, 27, 34). Although B. anthracis is traditionally considered an extracellular pathogen, there is considerable evidence that spores germinate and grow in macrophages during the early phase of infection (8, 30). We expressed here the B. anthracis orthologs of L. monocytogenes LLO and PI-PLC in L. monocytogenes in order to characterize them in a well-studied model of intracellular pathogenesis. MATERIALS AND METHODSBacterial strains and growth conditions. The L. monocytogenes strains used in the present study are listed in Table 1. Unless otherwise specified, all bacterial strains were grown in brain heart infusion broth, and strains derived from L. monocytogenes ⌬dal ⌬dat (Lmdd) were supplemented with 100 g of D-alanine/ml to stationary phase at 30°C. Strains containing pKSV7 were selected and maintained with ampicillin (50 g/ml) in Escherichia coli and chloramphenicol (10 g/ml) in L. monocytogenes.Tissue-culture cells and growth media. The mouse macrophage-like cell line J774 and murine L2 fibroblasts were grown in Dulbecco modified Eagle medium plus 7.5% fetal bovine serum (FBS) and 1 mM L-glutamine. Bone marrowderived macrophages obtained from C57BL/6 mice were cultured in RPMI 1640-10% FBS-1 mM L-glutamine. All were incubated at 37°C with 5% CO 2 . For bacterial infec...

Listeriolysin O: a phagosome-specific lysin

“…However, although ivanolysin O facilitated growth of L. monocytogenes in the liver of infected mice, the mutant bacteria were substantially attenuated for virulence in the murine model of listeriosis. Expression of PFO or ALO, the CDCs from the extracellular pathogens C. perfringens and B. anthracis, respectively, in place of LLO also complemented for hemolytic activity and phagosomal escape, albeit less efficiently, but did not facilitate intracellular growth [13,64,65]. Subsequent to phagosomal escape, L. monocytogenes expressing PFO or ALO perforated the host plasma membrane and were released into the extracellular milieu [13,64,65].…”

“…LLO as a double edge sword: the importance of compartmentalization of LLO activity L. monocytogenes is exquisitely adapted for its intracellular life style and maintenance of the intracellular niche is critical for its pathogenesis and virulence [13,64,65,67]. To reach the cytosol, L. monocytogenes requires the pore-forming activity of LLO; bacteria that are unable to reach the cytosol are avirulent [8,9].…”

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

“…GILT is responsible for the activation of LLO in vivo, and mice lacking GILT (and which are deficient in generating MHC-II-restricted CD4-positive T-cell responses to relevant protein antigens) are resistant to L. monocytogenes infection (426). Interestingly, PFTs from several other bacteria also allow L. monocytogenes to escape from phagosomes; however, because these PFTs are not correctly regulated, they go on to attack the plasma membranes of host cells, thus impairing L. monocytogenes virulence in vivo (427,428).…”

Human α-Defensins Inhibit Hemolysis Mediated by Cholesterol-Dependent Cytolysins

“…LLO, a major virulence determinant of L. monocytogenes, enables ingested bacteria to escape a macrophage's phagosome and enter its cytoplasm (50). ALO can be substituted for LLO in this activity (68). PLY contributes to virulence early in pneumococcal pneumonia (52,53) and is a promising vaccine candidate.…”