Phosphatidylcholine-specific phospholipase C from Listeria monocytogenes is an important virulence factor in murine cerebral listeriosis

Abstract

Meningoencephalitis is a serious and often fatal complication of Listeria monocytogenes infection. The aim of the present study was to analyze the role of internalin A (InlA) and B, which are involved in the invasion of L. monocytogenes into cultivated host tissue cells, and that of phosphatidylcholine-specific phospholipase C (PlcB), which mainly promotes the direct cell-to-cell spread of L. monocytogenes, in murine cerebral listeriosis by use of an InlA/B (DeltainlAB2)- and a PlcB (DeltaplcB2)-deficient isogenic deletion mutant strain and the wild-type (WT) L. monocytogenes EGD. Listeria strains were directly applied to the brain, a technique which has been employed previously to study the pathogenesis of cerebral listeriosis (D. Schlüter, S. B. Oprisiu, S. Chahoud, D. Weiner, O. D. Wiestler, H. Hof, and M. Deckert-Schlüter, Eur. J. Immunol. 25:2384-2391, 1995). We demonstrated that PlcB, but not InlA or InlB, is an important virulence factor in cerebral listeriosis. Nonimmunized mice infected intracerebrally with the DeltaplcB2 strain survived significantly longer and had a reduced intracerebral bacterial load compared to mice infected with the DeltainlAB2 strain or WT bacteria. In addition, immunization with the WT prior to intracerebral infection significantly increased the survival rate of mice challenged intracerebrally with the DeltaplcB2 strain compared to that of mice infected with the WT or DeltainlAB2 strain. Histopathology revealed that the major difference between the various experimental groups was a significantly delayed intracerebral spread of the DeltaplcB2 mutant strain, indicating that cell-to-cell spread is an important pathogenic feature of cerebral listeriosis. Interestingly, irrespective of the Listeria mutant used, the apoptosis of hippocampal and cerebellar neurons and an internal hydrocephalus developed in surviving mice, indicating that these complications are not dependent on the virulence factors InlA/B and PlcB. In conclusion, this study points to PlcB as a virulence factor important for the intracerebral pathogenesis of murine L. monocytogenes meningoencephalitis.

FIG. 1

Survival rates of nonimmunized (upper panel) and immunized (lower panel) mice infected i.c. with L. monocytogenes WT, ΔinlAB2, and ΔplcB2. Nonimmunized mice infected i.c. with ΔplcB2 survived significantly longer than mice infected with WT or ΔinlAB2 (P < 0.05). Immunized mice infected i.c. with ΔplcB2 had a significantly increased survival rate compared to that of mice infected with WT or ΔinlAB2 L. monocytogenes (P < 0.05). Data represent survival rates of 10 mice per experimental group. The results of one of two experiments which gave comparable results are shown.

FIG. 2

Parasitic load of nonimmunized (upper panel) and immunized (lower panel) mice i.c. infected with L. monocytogenes WT, ΔinlAB2, and ΔplcB2. At each time point after infection, the i.c. bacterial load of mice infected with ΔplcB2 was significantly lower than that of mice infected with WT or ΔinlAB2 (P < 0.05). †, mice were already deceased. Five mice per group were analyzed, and the median ± the highest and lowest values of each group are shown.

FIG. 3

CNS pathology of nonimmunized mice i.c. infected with L. monocytogenes WT (a and b), ΔinlAB2 (c and d), and ΔplcB2 (e and f) at day 3 p.i. (a) Severe empyema of the ventricle (area above the arrows). The ependymal wall is largely destroyed and barely discernible, and the periventricular brain stem is infiltrated by numerous neutrophils and macrophages. (b) L. monocytogenes WT cluster in the largely destroyed plexus. The arrow points to a group of remarkably elongated WT bacteria. (c) ΔinlAB2 has also largely destroyed the ependyma of the ventricular wall (area left of the arrowheads) and has invaded the adjacent brain parenchyma. Brain stem neurons are surrounded by inflammatory leukocytes (arrow). (d) ΔinlAB2 cluster in the partially necrotic choroid plexus. Note that the elongated shape of the ΔinlAB2 strain (arrow) is identical to that of the WT in panel b. (e) ΔplcB2 has also infected the fourth ventricle, and the bacteria are accompanied by intraventricular neutrophils and macrophages. In contrast to WT (a) and ΔinlAB2 (c), the ependymal lining is largely intact (arrows). Very few bacteria are detectable in the periventricular tissue (arrowhead). (f) Some ΔplcB2 are detectable either as single or small groups of coccoid bacteria (arrowheads) in the choroid plexus. The choroid plexus is infiltrated by neutrophils and macrophages, but in contrast to the images shown in panels b and d, its structure is largely preserved. Specimens in panels a to f were stained with cresyl violet. Magnification is as follows: for panels a and e, ×260; b and d, ×1,250; c, ×520; f, ×1,470.

FIG. 4

CNS pathology of immunized mice i.c. infected with L. monocytogenes WT (a and b), ΔinlAB2 (c and d), and ΔplcB2 (e and f) at days 3 (a, c, and e) and 5 (b, d, and f) p.i. (a) Inflammation of the lateral ventricle (V) in a mouse infected with L. monocytogenes WT. Additional infiltrates are present in the adjacent brain parenchyma. (b) Small, circumscribed infiltrates are present in the brain stem in the vicinity of neurons. (c) In ΔinlAB2-infected mice, inflammation is also largely confined to the lateral ventricle, and small parts of the choroid plexus are preserved (asterisk). A significant part of the ependyma is still intact (arrows). (d) From days 3 to 5 p.i., disease progressed in ΔinlAB2-infected mice. Purulent ventriculitis and focal brain stem encephalitis (arrows) developed. (e) Only discrete infiltrates were detectable in the largely normal fourth ventricle in a ΔplcB2-infected mouse at day 3 p.i. The choroid plexus was largely preserved (arrowhead), and ventricular empyema was absent. The ependyma was only focally destroyed, and small, single infiltrates were present in the periventricular tissue (arrow). (f) At day 5 p.i., the inflammation was largely resolved, and only small residual infiltrates were present (arrow) in the wall of the fourth ventricle. Specimens in panels a to f were stained with cresyl violet. Magnification, ×260.

FIG. 5

CNS complications in L. monocytogenes meningoencephalitis at day 10 p.i. (a and b) A severe obstructive hydrocephalus developed at day 10 p.i. Note the massive enlargement of the third ventricle (a) compared to the normal size of the third ventricle of an immunized mouse i.c. infected with ΔplcB2 at day 1 p.i. (b). (c) Postinflammatory scarring of the ventricular wall of the fourth ventricle. Subependymal formation of a membranous gliotic tissue (large arrow). In this area, the ependyma is largely intact (arrowheads). In addition, some Purkinje cells in the adjacent cerebellum have small, pyknotic nuclei with condensed chromatin (small arrows), which is characteristic of apoptotic cells. (d) Apoptotic, TUNEL-positive neurons with small, pyknotic nuclei in the CA1 segment of the hippocampus (large arrow). Note the relatively sharp demarcation from the adjacent hippocampal segments (small arrows). Specimens in panels a to c were stained with cresyl violet, and panel d shows TUNEL staining. Magnification for panels a to d, ×260. Images shown in panels a, c, and d are from immunized mice i.c. infected with ΔplcB2 at day 10 p.i. Similar observations were made about the brains of surviving mice infected with L. monocytogenes WT and ΔinlAB2 at day 10 p.i.