Legionella pneumophila 6S RNA optimizes intracellular multiplication

Abstract

Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which 6 were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed in postexponential phase, and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of sigma(70)-containing RNA polymerase. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA, as well as many genes involved in acquisition of nutrients and genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.

Fig. 1.

Detection of predicted sRNAs by microarray. Hierarchical clustering of normalized signal intensities of the predicted sRNAs during (in order, from left): wild-type exponential (E) and postexponential (PE) phases of growth in rich AYE medium at 37 °C; rpoS mutant (rpoS); after hydrogen peroxide stress (H₂O₂, 0.1 mM, 10 min); after salt stress (NaCl, 100 mM, 10 min); during growth at 30 °C in rich AYE medium; and after heat shock (HS, 30 °C to 37 °C in rich AYE medium). Black bars denote a cluster of putative sRNAs with strong and highly reproducible signal intensity that were studied further. For each condition, the signals from six replicates were analyzed.

Fig. 2.

Genomic context and expression of five of the detected sRNAs. For each identified sRNA, a northern blot was performed to confirm expression and regulation observed by microarray. E, exponential phase; PE, postexponential phase. Experiments were performed at least three times, and quantification of the northern blots is shown in Fig. S2. 5S rRNA is used as a loading control.

Fig. 3.

The sixth detected sRNA is the L. pneumophila 6S RNA. Genomic context of 6S RNA gene ssrS is shown in A. Northern blot was used to monitor expression of 6S RNA during growth in rich AYE medium over 24 h (B). The growth phase represented by each time point can be visualized on the growth-curve graph shown above the northern blot. This experiment was performed three times, and quantification of the northern blots is shown in Fig. S2. (C) Structure prediction of L. pneumophila 6S RNA reveals a structure similar to E. coli 6S RNA. (D) L. pneumophila 6S RNA coimmunoprecipitates with the core RNA polymerase subunit RpoB. 6S RNA and the negative control 5S rRNA were visualized by northern blot and the result was confirmed by RT-PCR. Lysates were obtained from PE-phase bacteria; the ssrS deletion strain was used as a control for specificity. Immunoprecipitaion with protein A Sepharose beads alone (Beads) and α-FliC antibody were used as negative controls. This experiment was performed four times with similar results.

Fig. 4.

Identification of genes regulated by 6S RNA. A 70-mer microarray representing all annotated ORFs was used to identify genes differentially regulated in a ΔssrS strain compared with the wild-type strain during PE phase of growth (A). Three biological replicates were used. Genes negatively (B) or positively (C) affected by the presence of 6S RNA were classified by their known or putative function, according to the L. pneumophila genome annotation. Shown is the fraction of affected genes in the different categories. FUN, genes of unknown function; OMP, outer membrane protein.

Fig. 5.

The ssrS mutant strain is unable to compete with the wild type during intracellular multiplication. THP-1 cells (A) or A. castellanii cells (B) were infected with a 1:1 mixture of differentially marked ssrS⁺ and ΔssrS strains, as well as a mixture of icm⁺/dot⁺ and dotA mutant as control (see text for details). The mixtures were also grown in rich AYE medium at 37 °C (C) and exposed to cycles of dilution (1:100) into fresh AYE medium after growth to PE phase (D). Mixtures of bacterial strains are as follows: black, ssrS⁺-Cm^r/ssrS⁺-Kn^r; blue, ΔssrS-Cm^r/ΔssrS-Kn^r; red, ssrS⁺-Cm^r/ΔssrS-Kn^r; green, ssrS⁺-Kn^r/ΔssrS-Cm^r; purple, ssrS⁺-Cm^r/ΔdotA-Kn^r. Values represent the average ± SD of three experiments; *P < 0.0001.