Identification of a Novel Small RNA Modulating <em>Francisella tularensis</em> Pathogenicity

<div><p><em>Francisella tularensis</em> is a highly virulent bacterium responsible for the zoonotic disease tularemia. It is a facultative intracellular pathogen that replicates in the cytoplasm of host cells, particularly in macrophages. Here we show that <em>F. tularensis</em> live vaccine strain (LVS) expresses a novel small RNA (sRNA), which modulates the virulence capacities of the bacterium. When this sRNA, designated FtrC (for <em><u>F</u>rancisella <u>t</u>ularensis</em><u>R</u>NA <u>C</u>), is expressed at high levels, <em>F. tularensis</em> replicates in macrophages less efficiently than the wild-type parent strain. Similarly, high expression of FtrC reduces the number of viable bacteria recovered from the spleen and liver of infected mice. Our data demonstrate that expression of gene <em>FTL_1293</em> is regulated by FtrC. Furthermore, we show by <em>in vitro</em> gel shift assays that FtrC interacts specifically with <em>FTL_1293</em> mRNA and that this happens independently of the RNA chaperone Hfq. Remarkably, FtrC interacts only with full-length <em>FTL_1293</em> mRNA. These results, combined with a bioinformatic analysis, indicate that FtrC interacts with the central region of the mRNA and hence does not act by sterically hindering access of the ribosome to the mRNA. We further show that gene <em>FTL_1293</em> is not required for <em>F. tularensis</em> virulence <em>in vitro</em> or <em>in vivo</em>, which indicates that another unidentified FtrC target modulates the virulence capacity of the bacterium.</p> </div

Sequence, structure and expression of <i>ftrC</i>.

<p>(A) Sequence of <i>ftrC</i> in different <i>F. tulrensis</i> subspecies. LVS: <i>F. tularensis</i> subsp. <i>holarctica</i> strain LVS; SCHU: <i>F. tularensis</i> subsp. <i>tularensis</i> strain SCHU S4; media: <i>F. tularensis</i> subsp. <i>mediasiatica</i> strain FSC147; U112: <i>F. tularensis</i> subsp. <i>novicida</i> strain U112; philo: <i>F. philomiragia</i> strain ATCC 25071. The numbers after U112 indicate the four different copies of <i>ftrC</i>. Nucleotides that are conserved in all strains are marked with an * and those that differ from the LVS sequence are shown in red. (B) Predicted secondary structure of FtrC (using mFold). (C) Northern blot showing FtrC expression in wild-type LVS bacteria (lane 1) and in a strain overexpressing FtrC, LVS/p<i>ftrC</i>+ (lane 3). Lack of signal in lane 2 (LVSΔ<i>ftrC</i>) confirms absence of FtrC in the deletion strain. Size markers are indicated in nt at right. (D) Northern blot showing the stability of FtrC in LVS and LVSΔ<i>hfq</i> strains. Total RNA was isolated at different times (0–30 min, indicated at top) after addition of rifampicin and same amount of each sample was loaded onto gel. The estimated half-lifes (in min) are indicated. 5S RNA served as loading control.</p

FtrC specifically binds <i>FTL_1293</i> mRNA.

<p>(A) Putative duplex formation between FtrC and <i>FTL_1293</i> mRNA predicted by TargetRNA. The positions of basepairing nucleotides relative to transcript start (for FtrC) or AUG codon (for <i>FTL_1293</i>) are indicated at left and right to the sequences. (B) Gel shift assay of ³²P end-labeled <i>FTL_1293</i> mRNA incubated with increasing amounts of FtrC for 60 min before loading on a native 5% acrylamide gel. (C) ³²P end-labeled <i>FTL_1293</i> mRNA was incubated for the indicated time with FtrC before loading and RNA-RNA complex formation assessed by gel shift assay. When indicated, 10 µM Hfq was included during RNA-RNA incubation. Quantification of complex formation over time is presented at right. (D) ³²P end-labeled <i>FTL_1293</i> mRNA incubated with FtrC in the presence of increasing amount of Hfq or tRNA and interaction analyzed by native gel electrophoresis. Lane 1: no FtrC was added to reaction. (E) ³²P end-labeled full-length (<i>FTL_1293</i>) or truncated <i>FTL_1293</i> mRNA (<i>FTL_1293*</i>) incubated with or without FtrC and analyzed by native acrylamide gel electrophoresis. (F) Schematic representation of putative interaction between FtrC and <i>FTL_1293</i> mRNA (top) and the extent of the truncated mRNA (<i>FTL_1293*</i>) (bottom).</p

Expression of FtrC reduces the number of bacteria in spleen and liver of infected mice.

<p>The numbers of bacteria in the spleen and liver of mice infected with strain LVS/p<i>ftrC</i>+ or LVS/p6 were determined after 3 days of infection with approximately 500 bacteria. Student's t-test shows a significant difference between the numbers of bacteria in spleen and liver of mice infected with the two strains (p<0.0005).</p

FTL_1293 does not contribute to <i>F. tularensis</i> virulence.

<p>(A) Intracellular multiplication of LVS (black diamonds) and LVSΔ1293 (grey squares) in murine macrophages-like J774 cells. After 60 min the cells were washed and gentamycin added to kill extracellular bacteria (time 0). The number of intracellular bacteria was determined after lysis of macrophages cells and plating of lysate on agar plates. Results are from one experiment with triplicate samples. (B) Average viable numbers of LVS (black bars) and LVSΔ1293 (grey bars) in the spleen and liver of five mice after 3 and 4 days of infection by the i.p. route with approximately 100 bacteria.</p

Expression of FtrC reduces intracellular multiplication of <i>F. tularensis</i>.

<p>Murine macrophage-like cells J774, human THP1 cells, and murine bone marrow-derived macrophages (BMM) were incubated with LVS/p6 (diamonds) or LVS/p<i>ftrC</i>+ (squares) bacteria. After 60 min the cells were washed and gentamycin added to kill extracellular bacteria (time 0). The number of intracellular bacteria was determined after lysis of macrophages cells and plating of lysate on agar plates. Results are from one representative experiment (triplicate samples). Student's t-test showed difference in bacterial numbers (* designates p<0.05, and ** designates p<0.005).</p

Effect of overexpression of FtrC on the transcript level of four genes^a.

a<p>Four genes found to have consistently changed mRNA levels in DNA microarray study.</p>b<p>Fold difference in RNA level in LVSΔ<i>ftrC</i> relative to LVS/p<i>ftrC</i>+ strain.</p

The type I RM system locus containing <i>ftrC</i>.

<p>In <i>F. tularensis</i> subsp. <i>novicida</i> U112, the restriction subunit R (HsdR) is encoded by <i>FTN_1155</i> (blue), the specificity subunit S (HsdS) is encoded by <i>FTN_1154</i> (green) and the modification subunit M (HsdM) is encoded by <i>FTN_1152</i> (orange). The corresponding pseudogenes (indicated with gene names below arrows) in <i>F. tularensis</i> subsp. <i>holarctica</i> LVS and subsp. <i>tularensis</i> Schu S4 are represented using the same colors. The <i>ftrC</i> gene (red) is encoded on the opposite strand.</p

A New Family of Secreted Toxins in Pathogenic Neisseria Species

<div><p>The genus <i>Neisseria</i> includes both commensal and pathogenic species which are genetically closely related. However, only meningococcus and gonococcus are important human pathogens. Very few toxins are known to be secreted by pathogenic <i>Neisseria</i> species. Recently, toxins secreted via type V secretion system and belonging to the widespread family of contact-dependent inhibition (CDI) toxins have been described in numerous species including meningococcus. In this study, we analyzed loci containing the <i>maf</i> genes in <i>N. meningitidis</i> and <i>N. gonorrhoeae</i> and proposed a novel uniform nomenclature for <u>m</u>af <u>g</u>enomic <u>i</u>slands (MGIs). We demonstrated that <i>mafB</i> genes encode secreted polymorphic toxins and that genes immediately downstream of <i>mafB</i> encode a specific immunity protein (MafI). We focused on a MafB toxin found in meningococcal strain NEM8013 and characterized its EndoU ribonuclease activity. <i>maf</i> genes represent 2% of the genome of pathogenic <i>Neisseria</i>, and are virtually absent from non-pathogenic species, thus arguing for an important biological role. Indeed, we showed that overexpression of one of the four MafB toxins of strain NEM8013 provides an advantage in competition assays, suggesting a role of <i>maf</i> loci in niche adaptation.</p></div

Additional file 2: Table S1. of A widespread family of polymorphic toxins encoded by temperate phages

General characteristics of bacteriophages, results of the prediction of lifestyle, and of the detection of MuF. Table S2. General characteristics of bacterial genomes, prophages and information on whether they encode a MuF protein. Table S3. General characteristics of the HMM protein profiles. Table S4. General characteristics of the 614 MuF proteins from bacteriophages. Table S5. General characteristics of the 901 MuF proteins identified in bacterial genomes. Table S6. Information on the bacterial prophages where muf genes could be identified. Table S7. General characteristics of toxin domains. Table S8. List of the MuF proteins identified in the integrated reference catalog of the human gut microbiome [41]. (XLSX 1074 kb