Listeria monocytogenes σH Contributes to Expression of Competence Genes and Intracellular Growth

Abstract

The alternative sigma factor σ(H)has two functions in Gram-positive bacteria: it regulates sporulation and the development of genetic competence. Listeria monocytogenes is a nonsporulating species in which competence has not yet been detected. Nevertheless, the main competence regulators and a series of orthologous genes that form the competence machinery are present in its genome; some of the competence genes play a role in optimal phagosomal escape. In this study, strains overexpressing σ(H) and strains with a σ(H) deletion were used to elucidate the contribution of σ(H) to the expression of the competence machinery genes inL. monocytogenes Gene expression analysis showed that σ(H) is, indeed, involved in comG and come regulation. Unexpectedly, we observed a unique regulation scheme in which σ(H) and the transcription factor ComK were involved. Population-level analysis showed that even with the overexpression of both factors, only a fraction of the cells expressed the competence machinery genes. Although we could not detect competence, σ(H) was crucial for phagosomal escape, which implies that this alternative sigma factor has specifically evolved to regulate the L. monocytogenes intracellular life cycle.

Importance: Listeria monocytogenes can be an intracellular pathogen capable of causing serious infections in humans and animal species. Recently, the competence machinery genes were described as being necessary for optimal phagosomal escape, in which the transcription factor ComK plays an important role. On the other hand, our previous phylogenetic analysis suggested that the alternative sigma factor σ(H) might play a role in the regulation of competence genes. The present study shows that some of the competence genes belong to the σ(H) regulon and, importantly, that σ(H) is essential for intracellular growth, implying a unique physiological role of σ(H) among Firmicutes.

FIG 1

Consensus sequences recognized by ComK and σ^H in L. monocytogenes. (A) comG operon; (B) comE operon; (C) comF operon. Arrows, open reading frame; underlines, ComK box (16); boxes, σ^H promoter consensus sequence (in B. subtilis, AGGAWWT-12 to 14 residues-RGAAT, where W is A or T and R is A or G [32]; in L. monocytogenes, AGG … GAA [4]); dashed line, suggested σ^A (rpoD) promoter consensus sequence conserved among Listeria spp. (16). We could not find a conserved region similar to the σ^A-type consensus sequence in comE promoter regions.

FIG 2

Growth of the L. monocytogenes strains (in BHI at 180 rpm and 37°C) used in this study. Arrow, sampling point; wt, phage-cured EGDe strain; ΔcomK, a comK deletion mutant; ΔsigH, a sigH deletion mutant; ΔsigH+comK, a strain overexpressing comK in the sigH deletion background, +sigH, the wt strain overexpressing sigH; +sigH+comK, the wt strain overexpressing sigH and comK; ΔsigHΔcomK, a sigH and comK double deletion mutant. OD 600nm, optical density at 600 nm.

FIG 3

Semiquantitative RT-PCR of the comK, sigH, comGA, comEA, comFA, and rpoB genes. wt, phage-cured strain EGDe; ΔcomK, a comK deletion strain; ΔsigH, a sigH deletion strain; ΔsigH+comK, a strain overexpressing comK in the sigH deletion background; +sigH+comK, the wt strain overexpressing sigH and comK; ΔsigHΔcomK; a sigH and comK double deletion mutant. The sampling point is shown in Fig. 2. A representative result from 3 independent experiments is shown.

FIG 4

Cells expressing CFP under the control of the comGA promoter. wt, phage-cured strain EGDe; +sigH, the wt strain overexpressing sigH; +sigH+comK, the wt strain overexpressing sigH and comK; RN+sigH, S. aureus RN overexpressing L. monocytogenes sigH. Data represent the averages with SDs from 3 independent experiments. Significant differences are indicated. *, P = 0.002; **, P = 0.00001. ND, none detected. The sampling point is shown in Fig. 2.

FIG 5

Intracellular growth in RAW 264.7 macrophages. The numbers of CFU (left) were normalized using an IGC (right). (A) wt, phage-cured strain EGDe; ΔsigH, a sigH deletion strain; ΔcomK, a comK deletion strain; ΔsigHΔcomK, a sigH and comK double deletion mutant; (B) wt, phage-cured strain EGDe; ΔsigH, a sigH deletion strain; ΔsigH+comK, a strain overexpressing comK in the sigH deletion background; ΔcomK, a comK deletion strain; ΔcomK+sigH, a strain overexpressing sigH in the comK deletion background. The means from 3 independent experiments are shown.

FIG 6

Intracellular growth in HeLa cells. The numbers of CFU (left) were normalized using an IGC (right). wt, phage-cured strain EGDe; ΔsigH, a sigH deletion strain; ΔcomK, a comK deletion strain; ΔsigHΔcomK, a sigH and comK double deletion mutant. The means from 3 independent experiments are shown.

FIG 7

Semiquantitative RT-PCR of the comEC gene. wt, phage-cured strain EGDe; +sigH, the wt strain overexpressing sigH; ΔsigH, a sigH deletion strain; ΔsigH+comK, a strain overexpressing comK in the sigH deletion background. The sampling point is shown in Fig. 2.

FIG 8

Phagosomal escape at 3 hpi in RAW 264.7 macrophages. (A) Representative images from fluorescence microscopy observations. YFP, yellow fluorescent protein. (B) Average values of phagosomal escape percentages with SDs from 4 independent experiments. Significant differences are indicated. *, P = 0.01; **, P < 0.0001; ***, P < 0.00001. wt, phage-cured strain EGDe; ΔsigH, a sigH deletion strain; ΔcomK, a comK deletion strain; ΔsigHΔcomK, a sigH and comK double deletion mutant.

FIG 9

Representative TEM images of L. monocytogenes in RAW 264.7 macrophages. Macrophages were infected at an MOI of 10 and fixed at 3 hpi. (A) wt (phage-cured strain EGDe); (B) strain ΔcomK (a comK deletion mutant); (C) strain ΔsigH (a sigH deletion mutant). In panels B and C, the boxed areas in the panels on the left are enlarged in the two panels on the right.