Listeria monocytogenes is a facultative intracellular bacterium that has predilection for causing central nervous systemic infections in humans and d

SUMMARY: Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteriaListeria monocytogenes,Mycobacterium tuberculosis, andBrucellaandSalmonellaspp. and obligate intracellular microbes of the Rickettsiaceae family andTropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing thatL. monocytogenescan invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens.

Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b⁺ mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C^high monocyte subset and that Ly-6C^high and Ly-6C^neg-low monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or ΔactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C^high subset. In contrast, this was not evident after infection with Δhly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b⁺ leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C^high monocytes. Kinetic analysis showed a significant influx of Ly-6C^high monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6C^high monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.

In vitro data suggest that blood-borneListeria monocytogenesorganisms enter the central nervous system (CNS) by direct invasion of endothelial cells or by cell-to-cell spread from infected phagocytes to endothelial cells. However, a role for infected phagocytes in neuroinvasion and dissemination ofL. monocytogenesin vivo has not been confirmed experimentally. Experiments described here tested whetherL. monocytogenes-infected peripheral blood leukocytes (PBL) circulated in bacteremic mice and could establish organ infection in vivo. A mean of 30.5% of bacteria cultured from whole blood were PBL associated, and microscopy showed that 22.2% of monocytes and 1.6% of neutrophils were infected. PBL-associated bacteria spread to endothelial cells in vitro, indicating their potential for virulence in vivo. To test this possibility, mice were injected intravenously with infected PBL and CFU of bacteria in liver, spleen, and brain were quantified and compared with values for mice injected with broth-grown bacteria and in vitro-infected macrophage cell lines. An inoculum of infected macrophage cell lines led to greater numbers of bacteria in the liver than the numbers produced by a similar inoculum of broth-grown bacteria. In contrast, brain infection was best established by infected PBL. Results of intraperitoneal injection of infected peritoneal cells compared with results of injection with infected J774A.1 cells suggested that unrestricted intracellular bacterial replication within J774A.1 cells contributed to excessive liver infection in those mice. These data show dissemination of intracellularL. monocytogenesand indicate that phagocyte-facilitated invasion has a role in CNS infection in vivo. Heterogeneity with regard to bactericidal activity as well as to other phagocyte characteristics is a critical feature of this mechanism.

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 anL. monocytogenesstrain containing a chromosomalactA-gfpuv-plcBtranscriptional 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 intracellularL. monocytogenesharbored 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 ofL. monocytogenesand then reenter the bloodstream and contribute to the systemic dissemination of bacteria.