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 σ 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.Legionella pneumophila genome | microarray | sRNA | sRNA prediction | competition assay

Analysis of gene expression from theWolbachiagenome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

“…Alternatively, as 6S RNA appears to play a critical role in translational adaptation under conditions of nutrient stress (for which a primary signal is nucleotide availability ) (Wassarman 2007), increased production in the gonad may reflect the imperative for Wolbachia to synchronize its replication rate with that of the host. In accordance with this hypothesis, 6S RNA is required for optimal intracellular replication of Legionella pneumophila (Faucher et al 2010). The elevated expression of RNase P in the soma was also surprising, considering its canonical function in the catalysis of precursor tRNA maturation (Kazantsev and Pace 2006).…”

Virulence Phenotypes of Legionella pneumophila Associated with Noncoding RNA lpr0035

“…A 6S RNA, homologous to RNAs known to regulate 70 RNA polymerase, was predicted by a computational approach and shown to positively regulate expression of Dot/Icm effector proteins and to be required for optimal intracellular multiplication in Acanthamoeba castellanii and in THP-1-derived macrophages (18). With the advent of deep sequencing technologies, hundreds of additional L. pneumophila ncRNAs have been identified (43,52), and functions for the vast majority of these are unknown.…”

“…Recently, noncoding RNAs (ncRNAs) have been implicated in those regulatory networks (18,19,39,42). Initially, Legionella ncRNAs were predicted from bioinformatics or identified as Legionella homologs of ncRNAs found in other bacteria (18,32).…”

“…Recently, noncoding RNAs (ncRNAs) have been implicated in those regulatory networks (18,19,39,42). Initially, Legionella ncRNAs were predicted from bioinformatics or identified as Legionella homologs of ncRNAs found in other bacteria (18,32). Homologs of Escherichia coli ncRNAs RsmY and RsmZ bind to and inhibit L. pneumophila CsrA, which negatively affects expression of Dot/Icm effector proteins in a pathway regulated by RpoS, LetA, and PmrA (28,39,42).…”

Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

“…This finding is further corroborated by two separate transcriptome studies that compared the gene expression profiles of L. pneumophila during exponential and postexponential growth phases in buffered yeast extract (BYE) broth, where in both instances, oxyR Lp was found to be upregulated ϳ3-fold during the postexponential growth phase (44,45). However, OxyR Lp seems to exhibit regulatory control over two noncoding small RNAs (sRNAs), LprA and LprB, during the exponential growth phase, indicating that there is a role for OxyR Lp during this growth phase as well (46). Thus, it was of interest to investigate whether the transcriptional changes observed for oxyR Lp translate to differing OxyR Lp levels between the growth phases in L. pneumophila.…”