A prfA transposon mutant of Listeria monocytogenes F2365, a serotype 4b strain, is able to survive in the gastrointestinal tract but does not cause systemic infection of the spleens and livers of intragastrically inoculated mice

Abstract

prfA is a member of the Crp/Fnr family of global regulatory genes in Listeria monocytogenes that has been shown previously to regulate several key virulence determinants both in vitro and in parenterally inoculated laboratory rodents. However, the role of prfA in the ability of L. monocytogenes to cause infection via the gastrointestinal (GI) tract has not been clearly established. In this study, we used a prfA transposon mutant of L. monocytogenes F2365, a serotype 4b strain, to assess the role of prfA in the pathogenesis of gastrointestinal listeriosis in mice. We found that the prfA mutant was able to survive in the GI tract (i.e., cecum) of mice, albeit in numbers somewhat less than those of the wild-type parent strain of L. monocytogenes. However, mice inoculated with the prfA mutant did not exhibit systemic infection of the spleen and liver, as was noted for mice inoculated with the wild-type parent strain. Survival of the prfA mutant in synthetic gastric fluid at pH 2.5 or 5 was somewhat reduced compared to that of the wild-type strain, as was its ability to invade and multiply within differentiated human intestinal epithelial cells (Caco-2 cells). Prior infection with the prfA mutant gave mice some protection against a subsequent challenge with virulent L. monocytogenes, although much less than that gained by prior gastrointestinal infection with the wild-type parent strain. These findings indicate that the global regulatory gene prfA is dispensable for colonization of the GI tract in mice but not for systemic infection.

FIG. 1.

The prfA mutant (□) is avirulent, compared to the wild-type parent strain (▪) of L. monocytogenes, for i.g.-inoculated mice. Female A/J mice were inoculated i.g. with approximately 10⁷ CFU of the indicated strains of L. monocytogenes as described in Materials and Methods. Three days later the mice were euthanatized, and the numbers of viable CFU in the blood and in homogenates of the spleen and liver were determined by plating on blood agar. The limit of detection was 1.0 log₁₀ CFU. Samples that did not yield colonies were assigned a value of 0.95 log₁₀ CFU for calculation of the mean (± SEM) CFU per gram of tissue or per milliliter of blood for six mice per group. *, P < 0.05; **, P < 0.01.

FIG. 2.

Comparison of the recovery of viable listeriae from the spleens, livers, blood, ceca, and gall bladders of mice at 3 days after i.g. inoculation with the indicated number of CFU of the wild-type parent (10⁴, 10⁵, and 10⁶ CFU) or prfA mutant (10⁶, 10⁸, and 10⁹ CFU) strain of L. monocytogenes. Results are the mean (± SEM) log₁₀ CFU per gram of tissue or per milliliter of blood for six mice per group. Data for the gall bladders are per entire organ homogenate.

FIG. 3.

Fecal shedding (A) and recovery of listeriae from the ceca (B) of mice inoculated i.g. with 10⁶ CFU of the wild-type (WT) parent or prfA mutant strain of L. monocytogenes. Fresh fecal pellets were collected daily into a sterile tube from mice and homogenized in 1.0 ml of PBS. The homogenates were plated on modified Oxoid agar and incubated at 37°C. Ceca were removed, homogenized in sterile PBS, and plated on Oxoid agar as described in the text. Results are the mean (± SEM) log₁₀ CFU per gram of fecal pellet or cecal tissue (wet weight). Results from mice inoculated with the two strains are similar except at day 2 (fecal CFU) and day 5 (cecal CFU), where they are significantly different (P < 0.05). Results of fecal shedding for the mice inoculated with the wild-type strains are not illustrated for day 3 because the mice did not defecate when handled, perhaps reflecting the severity of infection on that day.

FIG. 4.

Prior i.g. infection with the prfA mutant strain of L. monocytogenes provides limited protection against subsequent i.g. challenge with the wild-type parent strain of L. monocytogenes. Mice that had been inoculated previously with 10⁸ CFU of the prfA mutant strain of L. monocytogenes (▪) and naive control mice (□) were challenged i.g.18 days later with 5 × 10⁶ CFU of wild-type L. monocytogenes. Three days later the mice were euthanatized, and the mean (± SEM) log₁₀ CFU per gram of tissue or per milliliter of blood was determined (six mice per group).

FIG. 5.

Prior i.g. infection with the wild-type parent strain of L. monocytogenes provides strong protection against subsequent i.g. challenge with the same strain of L. monocytogenes. Mice that had been inoculated previously with 10⁵ CFU of the wild-type strain of L. monocytogenes (▪) and naive control mice (□) were challenged i.g. 14 days later with 5 × 10⁶ CFU of wild-type L. monocytogenes. Three days later the mice were euthanatized, and the mean (± SEM) log₁₀ CFU per gram of tissue (spleen, liver, or cecum), per milliliter of blood, or per entire organ (gall bladder) was determined (six mice per group).

FIG. 6.

Survival of the prfA mutant (open symbols) and wild-type parent strain (closed symbols) of L. monocytogenes in synthetic gastric fluid adjusted to pH 2.5 (⋄, ⧫), 5 (□, ▪), or 7 (○, •) as described in Materials and Methods. Approximately 2.5 ×10⁶ CFU of the respective strains of L. monocytogenes was suspended in 2.5 ml (final volume) of synthetic gastric fluid in a 24-well tissue culture plate. At 30, 60, and 120 min of incubation at 37°C, samples were removed (0.5 ml), diluted, and plated on blood agar. Results are the mean (± SEM) log₁₀ CFU per milliliter (three wells per test condition).

FIG. 7.

Invasion (left) and intracellular multiplication (right) of the prfA mutant and wild-type (WT) parent strains of L. monocytogenes in differentiated Caco-2 cells. Caco-2 cells were incubated for 1 h at 37°C with 1 ml of medium containing 1 × 10⁷ CFU of L. monocytogenes. Following incubation, the medium was removed, the monolayers were washed five times with HBSS, and the infected cells were incubated for an additional 2.5 h in HBSS with 20 μg/ml gentamicin (Sigma) to kill any extracellular listeriae. The cells were washed five times with warm HBSS and lysed with 1% Triton X-100 (Acros Organics) in PBS. Serial dilutions of the cell lysates were plated in duplicate on blood agar plates and incubated at 37°C for 48 h. To assess intracellular listerial growth, the infected monolayers were incubated for 24 h in DMEM with the supplements indicated above and 20 μg/ml gentamicin. Results are the mean (± SEM) log₁₀ CFU per milliliter (three wells per test condition) from one representative experiment of three that were performed.