Identification of Listeria monocytogenes in vivo-induced genes by fluorescence-activated cell sorting

Abstract

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen capable of causing severe infections in immunocompromised or pregnant individuals, as well as numerous animal species. Genetic analysis of Listeria pathogenesis has identified several genes which are crucial for virulence. The transcription of most of these genes has been shown to be induced upon entry of Listeria into the host cell. To identify additional genes that are induced in vivo and may be required for L. monocytogenes pathogenesis, a fluorescence-activated cell-sorting technique was initiated. Random fragments of the L. monocytogenes chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were transformed into the L. monocytogenes actA mutant DP-L1942. Fluorescence-activated cell sorting (FACS) was used to isolate L. monocytogenes clones that exhibited increased GFP expression within macrophage-like J774 cells but had relatively low levels of GFP expression when the bacteria were extracellular. Using this strategy, several genes were identified, including actA, that exhibited such an expression profile. In-frame deletions of two of these genes, one encoding the putative L. monocytogenes uracil DNA glycosylase (ung) and one encoding a protein with homology to the Bacillus subtilis YhdP hemolysin-like protein, were constructed and introduced into the chromosome of wild-type L. monocytogenes 10403s. The L. monocytogenes 10403s ung deletion mutant was not attenuated for virulence in mice, while the yhdP mutant exhibited a three- to sevenfold reduction in virulence.

FIG. 1

Construction of plasmid pAMGFP. A promoterless gfp mut3 gene encoding the A. victoria GFP was removed from plasmid pGFPmut3 (9) and ligated into the BamHI-EcoRV sites of plasmid pAM401 (46). Sau3AI fragments of the Listeria 10403s chromosome were ligated into the BamHI site of pAMGFP to construct the library used for FACS for in vivo-induced promoters.

FIG. 2

Expression of pLLOGFP by L. monocytogenes increases the fluorescence of infected J774 cells. L. monocytogenes DP-L1942 containing plasmid pAMGFP or plasmid pLLOGFP was used to infect J774 cells at an MOI of 10. After 4 h, the infected J774 cells were analyzed by flow cytometry.

FIG. 3

FACS for in vivo-induced promoters. (A) Fluorescence of J774 cells infected with DP-L1942 (actA) containing the pAMGFP library. The solid line indicates that the sorting gate was set to collect the 1% of cells expressing the highest fluorescence. (B) Fluorescence of the population of bacteria released from the macrophages sorted in the step depicted in panel A. The 11% of bacteria expressing the lowest fluorescence was collected. (C) Fluorescence of J774 cells infected with bacteria from the step depicted in panel B. The line indicates that the sorting gate was set to collect the 0.6% of cells expressing the highest GFP.

FIG. 4

Flow cytometry analysis of individual clones expressing GFP. Overnight cultures of isolated clones were subcultured into fresh BHI medium (containing streptomycin and chloramphenicol) and grown to an OD₆₀₀ of approximately 0.1. These cultures were used to infect J774 cells at an MOI of approximately 200 and incubated for 4 h at 37°C. The inoculating cultures (BHI) and the infected J774 cells were analyzed by flow cytometry, and their fluorescence is shown (in black) relative to DP-L1942 containing the promoterless pAMGFP plasmid (in grey). The numbers represent the numbers of the individual clones. The fluorescence of bacteria containing the actA promoter in the GFPfusion plasmid that was isolated during the enrichment and the fluorescence of bacteria containing plasmid pLLOGFP are shown for reference. Clone 8 is a representative clone that expressed GFP both in BHI medium and within J774 cells.

FIG. 5

ClustalW 1.8 alignment (22) and box shade analysis of the B. subtilis YhdP (accession no. 007585), R. typhi TlyC (accession no. AAC62436), and L. monocytogenes YhdP (clone 104) protein sequences. The boxed gray shading indicates identical protein residues and the boxed light shading indicates similar protein residues.

FIG. 6

An L. monocytogenes yhdP mutant is attenuated in the mouse model of infection. L. monocytogenes 10403s and L. monocytogenes 10403s yhdP were used to infect groups of BALB/c mice intravenously with 2 × 10⁴ to 4 × 10⁴ L. monocytogenes organisms. Data represent the mean ± standard error of the mean (error bars) of CFU per organ from organs of nine mice pooled from three separate experiments. Student's t test was used in statistical analysis. The numbers of CFU per liver (P < 0.005) and per spleen (P < 0.025) in yhdP mutant-infected mice were reduced compared to those in L. monocytogenes 10403s-infected mice.