Listeria monocytogenes-Infected Phagocytes Can Initiate Central Nervous System Infection in Mice

Abstract

Listeria monocytogenes-infected phagocytes are present in the bloodstream of experimentally infected mice, but whether they play a role in central nervous system (CNS) invasion is unclear. To test whether bacteria within infected leukocytes could establish CNS infection, experimentally infected mice were treated with gentamicin delivered by surgically implanted osmotic pumps. Bacterial inhibitory titers in serum and plasma ranged from 1:16 to 1:256, and essentially all viable bacteria in the bloodstream of treated mice were leukocyte associated. Nevertheless, CNS infection developed in gentamicin-treated animals infected intraperitoneally or by gastric lavage, suggesting that intracellular bacteria could be responsible for neuroinvasion. This was supported by data showing that 43.5% of bacteria found with blood leukocytes were intracellular and some colocalized with F-actin, indicating productive intracellular parasitism. Experiments using an L. monocytogenes strain containing a chromosomal actA-gfpuv-plcB transcriptional fusion showed that blood leukocytes were associated with intracellular and extracellularly bound green fluorescent protein-expressing (GFP ؉ ) bacteria. Treatment with gentamicin decreased the numbers of extracellularly bound GFP ؉ bacteria significantly but did not affect the numbers of intracellular GFP ؉ bacteria, suggesting that the latter were the result of intercellular spread of GFP ؉ bacteria to leukocytes. These data demonstrate that infected leukocytes and the intracellular L. monocytogenes harbored within them play key roles in neuroinvasion. Moreover, they suggest that phagocytes recruited to infected organs such as the liver or spleen are themselves parasitized by intercellular spread of L. monocytogenes and then reenter the bloodstream and contribute to the systemic dissemination of bacteria.Listeria monocytogenes is a facultative intracellular bacterium that infects the central nervous system (CNS) of humans and domesticated animals (21, 22). Most human infections result from ingestion of contaminated food and typically manifest as febrile gastroenteritis (25). Immunosuppressed hosts, however, are much more likely to develop invasive listeriosis marked by bacteremia, CNS infection, and death (24,27). Although the precise mechanism(s) used by L. monocytogenes for entering the CNS are not clear, current theory indicates that neuroinvasive bacteria in general can enter the CNS by several different routes (37). These include invasion of microvascular endothelial cells, invasion of epithelial cells of the choroid plexus, and passage of bacteria through intercellular junctions. In addition, bacteria that are capable of intracellular survival can enter the CNS via phagocyte-facilitated infection, the major steps of which are adhesion of infected phagocytes to endothelium followed by cell-to cell spread of bacteria to endothelial cells and/or migration of infected phagocytes into the CNS (10).Experimental L. monocytogenes infection of mice shows that bacteria enter the CNS following prol...

The Ly-6Chigh Monocyte Subpopulation Transports Listeria monocytogenes into the Brain during Systemic Infection of Mice

“…Data supporting the latter mechanism come from histological studies of experimentally infected mice that identified infected phagocytes in the choroid plexus (11). In addition, studies from our laboratory showed that killing extracellular L. monocytogenes in blood with gentamicin did not prevent bacterial infection of the brain (12). This finding suggests that migration of parasitized leukocytes from the bloodstream into the CNS is instrumental in neuroinvasion.…”

“…This finding establishes that the Ly-6C high blood monocyte is the cell that harbors most of the cell-associated L. monocytogenes in vivo. Previous studies using gentamicin-treated mice infected with green fluorescence protein (GFP)-expressing L. monocytogenes demonstrated intracellular parasitism of blood leukocytes in vivo (12). To establish whether Ly-6C high monocytes were parasitized in vivo, we modified this system by also eliminating existing blood monocytes with clodronate-loaded liposomes and then labeling newly produced monocytes with liposomes containing DiD as previously described (20).…”

“…injection of 1-2 LD 50 of wild-type L. monocytogenes or 10 6 -10 7 CFU of other bacteria and then were euthanized at the indicated time with ketamine/xylazine (Vedco, St. Joseph, MO). Blood was collected into PBS containing 10 mM EDTA, and blood leukocytes were isolated as previously described (10,12). In experiments that required harvesting of bacteria, leukocytes, or RNA from the brain, the animals were perfused with 30 ml of PBS via the left ventricle to remove blood from the brain.…”

“…Eighteen hours later they underwent surgical implantation of Alzet osmotic pumps (Durect, Cupertino, CA) filled with gentamicin (Sigma-Aldrich; 100 mg/ml in PBS) as previously described (12) and were injected i.v. with 0.2 ml of clodronate liposome into the lateral tail vein to eliminate monocytes in vivo (20).…”

“…Gene deletion mutants of L. monocytogenes strain 10403s were obtained from D. Portnoy (University of California, Berkeley, CA) and included the listeriolysin O-deficient (⌬hly) DP-L2161 and actA-deficient (⌬actA) DP-L1942 (22,23). L. monocytogenes strain NF-L512 containing a chromosomal actA-gfpuv-plcB transcriptional fusion was obtained from N. Freitag (Seattle Biomedical Research Institute, Seattle, WA) (12). For experiments, 0.5 ml of stock culture was diluted in 4 ml of broth and then cultured for 4.5 h at 37°C.…”