Comparative transcriptomics of pathogenic and non-pathogenic Listeria species

Comparative RNA-seq analysis of two related pathogenic and non-pathogenic bacterial strains reveals a hidden layer of divergence in the non-coding genome as well as conserved, widespread regulatory structures called ‘Excludons', which mediate regulation through long non-coding antisense RNAs

Bacterial and cellular RNAs at work during Listeria infection.

International audienceListeria monocytogenes is an intracellular pathogen that can enter and invade host cells. In the course of its infection, RNA-mediated regulatory mechanisms provide a fast and versatile response for both the bacterium and the host. They regulate a variety of processes, such as environment sensing and virulence in pathogenic bacteria, as well as development, cellular differentiation, metabolism and immune responses in eukaryotic cells. The aim of this article is to summarize first the RNA-mediated regulatory mechanisms that play a role in the Listeria lifestyle and in its virulence, and then the host miRNA responses to Listeria infection. Finally, we discuss the potential cross-talk between bacterial RNAs and host RNA regulatory mechanisms as new mechanisms of bacterial virulence

A PNPase dependent CRISPR System in Listeria.

International audienceThe human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology"

RliB is a substrate for PNPase.

<p>A) <i>In vitro</i> PNPase activity assay. A 37 nt short P³²-labeled substrate RNA (RNA37*) was incubated alone (lane 1) or with 200 nM purified PNPase (lane 2). Three types of non-labeled competitor RNAs were then added: non-labeled RliB (lanes 3, 100 nM; lane 4, 200 nM; lane 5, 400 nM), non-labeled control RNA RsaA (lane 6, 100 nM; lane 7, 200 nM; lane 8, 400 nM) and non-labeled RNA37 substrate (lane 9, 100 nM; lane 10, 200 nM; lane 11, 400 nM). B) <i>In vitro</i> PNPase-mediated processing of RliB. On the left are indicated ladders T1 (RNase T1 hydrolysis) and L (alkaline hydrolysis). The full length P³²-5′ labeled RliB (RliB*) was incubated with increasing concentrations of purified PNPase (lane 1, no PNPase; lane 2, 25 nM; lane 3, 100 nM; lane 4, 400 nM). Competition experiments were performed in the presence of 400 nM non-labelled RliB (lane 5). C) <i>In vivo</i> PNPase-mediated processing of RliB. Northern blot performed on the total RNAs extracted from the <i>L. monocytogenes</i> EGD-e wild type bacteria (WT), strain deleted for <i>pnpA</i> (Δ<i>pnpA</i>), complemented strain (Δ<i>pnpA-pnpA</i>) and strain deleted for RliB (Δ<i>rliB</i>). Expression of RliB was detected by the P³² labeled <i>in vitro</i> transcribed RNA probe complementary to the full length RliB. The membrane was reprobed with P³² labeled <i>in vitro</i> transcribed RNA probe complementary to the tmRNA.</p

CRISPR DNA interference activity assay.

<p>A) Design of the plasmids. Two types of plasmids were used: i) protospacer plasmid (P) carrying the protospacer (highlighted in red) matching the spacer 3 in <i>L. monocytogenes</i> EGD-e strain RliB-CRISPR and spacer 5 in <i>L. monocytogenes</i> Finland strain RliB-CRISPR; ii) control plasmid (C) carrying the DNA fragment corresponding to the shuffled protospacer (highlighted in green). Black box marks the position of the protospacer adjacent motif (PAM). Position of the specific oligonucleotides used for the Q-PCR screening is indicated (P-fw, P-rev, C-fw, C-rev) B) Ratio (R). The number of colonies carrying plasmid P and the number of colonies carrying the plasmid C in the different <i>Listeria</i> strains and genetic backgrounds is represented as the ratio (R = n (colony P)/n (colony C). Shown are the mean and the standard deviation of five experiments performed for each bacterial strain. Two statistical tests were used: i) a t-test measuring if the ratio in the given strain is significantly different from 1 (*** - p = 0,0008; ns- not significant); ii) a t-test comparing the ratios of the WT strain and the Δ<i>rliB</i> and Δ<i>pnpA</i> strains (**- p = 0,0025; *- p = 0,0334).</p

Spacer composition of CRISPR arrays in <i>L. monocytogenes</i>.

<p>CRISPR systems present in complete and draft <i>L. monocytogenes</i> genomes are represented. For each <i>L. monocytogenes</i> strain spacers belonging to RliB-CRISPR, CRISPR-I and CRISPR-II are shown. Boxed numbers represent individual spacers conserved within an CRISPR array among the represented strains (for the RliB-CRISPR: 1-32, for the CRISPR-I: 1-70, for the CRISPR-II: 1- 92). Unique spacers are represented as not numbered boxes. Spacers matching bacteriophage sequences available in the Genbank are coloured according to the type of bacteriophage they target. The color code and corresponding bacteriophages are represented in the bottom part of the figure. Spacers matching prophages are highlighted as gray boxes. Self-targeting spacers, identified in complete <i>Listeria</i> genomes are highlighted with red box and the strain carrying the spacer is highlighted (•).</p

CRISPR-Cas systems in <i>L.monocytogenes</i> genomes.

<p>A) The three types of CRISPR-Cas systems found in <i>L.monocytogenes</i>. The sequences of the repeats are given. Points (•) indicate sites of repeat variability among <i>Listeria</i> strains. B) CRISPR-Cas systems in complete and draft <i>L. monocytogenes</i> genomes of the lineages I, II and III. For two strains indicated by an *, the sequencing results are too preliminary with a high number of contigs and RliB was not detected. However, these strains were included in the spacer analysis.</p

RliB-CRISPRs in EGD and Finland <i>L. monocytogenes</i> strains.

<p>A) Schematic representation of the loci harbouring RliB-CRISPR in <i>L.monocytogenes</i> EGD-e, EGD and Finland strains. In all three strains, RliB-CRISPR is located between genes <i>lmo0509</i> and <i>lmo0510</i>. Spacers that are identical between the strains are connected with black lines (S1EGD = S4Fin, S2EGD = S5Fin, S5EGD = S6Fin, S6EGD = S7Fin, S7EGD = S8Fin, S8EGD = S9Fin, S9EGD = S10Fin and S10EGD = S11Fin) and the sequence alignments between the spacers and bacteriophage genomes are shown below. B) Processing of the RliB-CRIPSRs. On the left is shown a northern blot performed on the total RNAs extracted from the wild type <i>L. monocytogenes</i> EGD strain (WT) and its isogenic mutants deleted for <i>pnpA</i> (Δ<i>pnpA</i>-EGD) or RliB-CRISPR (Δ<i>RliB</i>-EGD). On the right is shown a northern blot performed on the total RNAs extracted from the wild type <i>L. moncytogenes</i> Finland strain (WT) and its isogenic mutants deleted for <i>pnpA</i> (Δ<i>pnpA</i>-Fin) or RliB-CRISPR (Δ<i>RliB</i>-Fin). Expression of RliB-CRISPR was detected with the P³² labeled <i>in vitro</i> transcribed RNA probe complementary to the spacers S5 to S10 in EGD, and S6 to S11 in the Finland strain. The membranes were reprobed with P³² labeled <i>in vitro</i> transcribed RNA probe complementary to the tmRNA.</p

RliB interacts with PNPase.

<p>A) Protein affinity purification using 3′ biotinylated RliB. Two biotinylated bait RNAs were used: full length RliB (RliB-B) and a control RNA (<i>S. aureus</i> RNAIII). Coomassie stained SDS-PAGE gel of the two elution fractions is shown. B) Protein affinity purification using 5′ MS2 tagged RliB. Two MS2 tagged bait RNAs were used: full length RliB (RliB-MS2) and a control RNA (<i>S.aureus</i> RsaA). Coomassie stained SDS-PAGE gel of the fractions is shown. C) Interaction between purified PNPase and <i>in vitro</i> transcribed RliB. Gel retardation assay performed with P³²-5′ labelled RliB (RliB) and increasing concentrations of the purified PNPase protein (lane 1, no PNPase added; lane 2, 100 nM PNPase; lane 3, 250 nM PNPase; lane 4, 400 nM PNPase). Competition experiments were performed in the presence of non-labelled RliB (lane 5, 400 nM RliB) and non-labelled RsaA (lane 6, 400 nM RsaA). In these experiments, the concentration of PNPase was 400 nM.</p