Differential regulation of Listeria monocytogenes internalin and internalin-like genes by sigmaB and PrfA as revealed by subgenomic microarray analyses

Abstract

The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated internalins. To determine the contributions of the alternative sigma factor sigma(B) and the virulence gene regulator PrfA to internalin gene expression, a subgenomic microarray was designed to contain two probes for each of 24 internalin-like genes identified in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic Delta sigB strain; (ii) 10403S and an isogenic Delta prfA strain; (iii) a (G155S) 10403S derivative that expresses the constitutively active PrfA (PrfA*) and the Delta prfA strain; and (iv) 10403S and an isogenic Delta sigB Delta prfA strain. Sigma(B)- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse-transcriptase polymerase chain reaction. For the 24 internalin-like genes examined, (i) both sigma(B) and PrfA contributed to transcription of inlA and inlB, (ii) only sigma(B) contributed to transcription of inlC2, inlD, lmo0331, and lmo0610; (iii) only PrfA contributed to transcription of inlC and lmo2445; and (iv) neither sigma(B) nor PrfA contributed to transcription of the remaining 16 internalin-like genes under the conditions tested.

FIG. 1.

Correlation between quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) (TaqMan) results and microarray results based on (A) the average of both probes, (B) probe 1 only, and (C) probe 2 only. Fold changes in expression in the 10403S parent strain relative to the 10403S ΔsigB strain (○), the PrfA* strain relative to the 10403SΔprfA strain (♦), and the 10403S parent strain relative to the 10403SΔsigBΔprfA strain (▴) were log transformed and plotted against each other to evaluate correlations. Quantitative RT-PCR data for inlA, inlB, inlC, inlC2, inlD, inlE, inlG, and prfA transcript levels for the 10403S–ΔsigB comparison as well as inlA, inlB, inlC2, inlD, inlG, and plcA transcript levels for the 10403S–ΔsigBΔprfA comparison have been reported elsewhere (McGann et al., 2007b).

FIG. 2.

Transcript levels determined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) for selected genes identified as differentially expressed between the 10403S parent strain and the ΔsigB strain based on one or both probes in the microarray. lmo1290 was included as a negative control; the microarray did not reveal any differences in transcript levels between 10403S and the ΔsigB strain for this gene. Transcript levels were quantified by qRT-PCR and normalized (see Methods), and are represented on the y-axis as log₁₀ values. Dark and light bars represent the 10403S parent strain and the ΔsigB strain, respectively. Data represent the mean of results from qRT-PCR experiments using three independent RNA isolations; error bars represent one standard deviation. An asterisk indicates that transcript levels for a given gene are significantly different between 10403S and the ΔsigB strain.

FIG. 3.

Representation of (A) the structures of the four internalins encoded by σ^B-dependent genes, including the promoter region DNA sequences for these genes, and (B) the structures of the two internalins encoded by PrfA-dependent genes, including the promoter region sequences for these genes. The numbers within the gray shaded areas indicate the number of leucine-rich repeat units within each coding region (Raffelsbauer et al., ; Hamon et al., 2006). The LPXTG motif for proteins covalently anchored to the cell wall is also indicated. The total number of amino acids in each protein is listed next to the protein name. The DNA sequences corresponding with (A) the σ^B promoter sequences and (B) the PrfA binding domains (PrfA-box) are underlined and in bold and their distance from each start codon for each reading frame is shown. The PrfA box upstream of plcA, which has a perfect palindromic sequence, and upstream of prfA, which differs from the perfect palindrome by three mismatches, are shown for comparison. Mismatches are underlined and in italics. No σ^B promoter sequence is given for inlD in panel A as the inlC2D operon appears to be transcribed from the σ^B promoter upstream of inlC2, which is shown here.

FIG. 4.

Transcript levels determined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) for all genes identified as differentially expressed between the prfA* (G155S) strain and the ΔprfA strain based on one or both probes in the microarray. Dark and light bars represent the prfA* (G155S) and ΔprfA strains, respectively. lmo2470 was included as a negative control; the microarray did not reveal any differences in transcript levels between the prfA* (G155S) and the ΔprfA strain for this gene. Transcript levels were quantified by qRT-PCR and normalized (see Methods), and are represented on the y-axis as log₁₀ values. Data represent the mean of results from qRT-PCR experiments using three independent RNA isolations; error bars represent one standard deviation. An asterisk indicates that transcript levels for a given gene are significantly different between the prfA* and the ΔprfA strains.

FIG. 5.

Transcript levels (based on quantitative reverse-transcriptase polymerase chain reaction [qRT-PCR] data) under different conditions and in different strains (prfA wildtype, prfA*, and ΔprfA) for (A) inlA and inlB and (B) prfA, plcA, and opuCA. Transcript levels are shown for (i) 10403S prfA wildtype in the vacuole and cytoplasm of Caco-2 cells (prfA, plcA, and opuCA only; previously reported by Kazmierczak et al., 2006); (ii) 10403S prfA wildtype (cells grown to OD = 0.4 and then exposed to brain heart infusion (BHI) with 0.2% charcoal for 2 hours) (previously reported by McGann et al., 2007b); (iii) 10403S prfA wildtype (OD = 0.4 cells grown in BHI for another 2 hours); (iv) ΔprfA (OD = 0.4 cells grown in BHI for another 2 hours); and (v) 10403S prfA* (OD = 0.4 cells grown in BHI for another 2 hours). Transcript levels were quantified using qRT-PCR, normalized (see Methods), and represented on the y-axis as log₁₀ values. Data represent the mean of results from qRT-PCR experiments using three independent RNA isolations; error bars represent one standard deviation. Boxes labeled with different letters indicate transcript levels that differed significantly (p < 0.05), while boxes labeled with identical letters indicate transcript levels that did not differ significantly (as determined by Tukey's multiple comparison procedure).

FIG. 6.

Transcript levels determined by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) for selected genes identified as differentially expressed between 10403S and the isogenic ΔsigBΔprfA strain (based on one or both probes in the microarray; Table 1).lmo1290 and lmo2445 were included as negative controls; the microarray did not reveal any differences in transcript levels between 10403S and the ΔsigBΔprfA strain for these genes. Transcript levels were quantified by qRT-PCR and normalized (see Methods), and represented on the y-axis as log₁₀ values. Dark and light bars represent 10403S and the ΔsigBΔprfA strain, respectively. Data represent the mean of results from qRT-PCR experiments using three independent RNA isolations; error bars represent one standard deviation. An asterisk indicates that transcript levels for a given gene are significantly different between 10403S and the ΔsigBΔprfA strain.