Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

ABSTRACT CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1 , an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence

Culture-Independent Characterization of the Digestive-Tract Microbiota of the Medicinal Leech Reveals a Tripartite Symbiosis

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the α-, γ-, and δ-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations

Calcium and Iron Regulate Swarming and Type III Secretion in Vibrio parahaemolyticus▿ †

Here, we probe the response to calcium during growth on a surface and show that calcium influences the transcriptome and stimulates motility and virulence of Vibrio parahaemolyticus. Swarming (but not swimming) gene expression and motility were enhanced by calcium. Calcium also elevated transcription of one of the organism's two type III secretion systems (T3SS1 but not T3SS2) and heightened cytotoxicity toward host cells in coculture. Calcium stimulation of T3SS gene expression has not been reported before, although low calcium is an inducing signal for the T3SS of many organisms. EGTA was also found to increase T3SS1 gene expression and virulence; however, this was demonstrated to be the consequence of iron rather than calcium chelation. Ectopic expression of exsA, encoding the T3SS1 AraC-type regulator, was used to define the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. To begin to understand the regulatory mechanisms modulating the calcium response, a calcium-repressed, LysR-type transcription factor named CalR was identified and shown to repress swarming and T3SS1 gene expression. Swarming and T3SS1 gene expression were also demonstrated to be linked by LafK, a σ54-dependent regulator of swarming, and additionally connected by a negative-feedback loop on the swarming regulon propagated by ExsA. Thus, calcium and iron, two ions pertinent for a marine organism and pathogen, play a signaling role with global consequences on the regulation of gene sets that are relevant for surface colonization and infection

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence

<i>Pseudomonas aeruginosa</i> exoproducts determine antibiotic efficacy against <i>Staphylococcus aureus</i>

<div><p>Chronic coinfections of <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i> frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on <i>S</i>. <i>aureus</i> antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of <i>P</i>. <i>aeruginosa</i> burn wound and cystic fibrosis (CF) lung isolates to demonstrate that <i>P</i>. <i>aeruginosa</i> alters <i>S</i>. <i>aureus</i> susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against <i>S</i>. <i>aureus</i>. We find that <i>P</i>. <i>aeruginosa</i> LasA endopeptidase potentiates lysis of <i>S</i>. <i>aureus</i> by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline <i>N</i>-oxide (HQNO) induces multidrug tolerance in <i>S</i>. <i>aureus</i> through respiratory inhibition and reduction of cellular ATP. We find that the production of each of these factors varies between clinical isolates and corresponds to the capacity of each isolate to alter <i>S</i>. <i>aureus</i> antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a <i>S</i>. <i>aureus</i> mouse burn infection is potentiated by the presence of a LasA-producing <i>P</i>. <i>aeruginosa</i> population. These findings demonstrate that antibiotic susceptibility is complex and dependent not only upon the genotype of the pathogen being targeted, but also on interactions with other microorganisms in the infection environment. Consideration of these interactions will improve the treatment of polymicrobial infections.</p></div

<i>P</i>. <i>aeruginosa</i>-mediated alteration of <i>S</i>. <i>aureus</i> antibiotic susceptibility.

<p><i>P</i>. <i>aeruginosa</i> exoproducts PYO, HQNO, and HCN inhibit <i>S</i>. <i>aureus</i> electron transport, leading to collapse of PMF and inhibition of the F₁F₀ ATPase leading to a decrease in <i>S</i>. <i>aureus</i> antibiotic susceptibility. Conversely, <i>P</i>. <i>aeruginosa</i> RLs intercalate into the plasma membrane-forming pores that permit aminoglycoside entry into the cell in a PMF-independent manner, while <i>P</i>. <i>aeruginosa</i> endopeptidase LasA cleaves pentaglycine crosslinks between peptidoglycan molecules of the cell wall, increasing vancomycin-mediated lysis of <i>S</i>. <i>aureus</i>. HCN, hydrogen cyanide; HQNO, 2-heptyl-4-hydroxyquinoline <i>N</i>-oxide; NAG, <i>N</i>-acetylglucosamine; NAM, <i>N</i>-acetylmuramic acid; PMF, proton-motive force; PYO, pyocyanin; RL, rhamnolipids.</p

<i>P</i>. <i>aeruginosa</i> supernatant potentiates killing by vancomycin via the LasA endopeptidase.

<p><i>S</i>. <i>aureus</i> HG003 was grown to mid-exponential phase and exposed to sterile supernatants for 30 min prior to addition of vancomycin 50 μg/ml. Where indicated, PAO1 supernatant was heat inactivated at 95°C for 10 min. (A) At indicated times, an aliquot was removed, washed, and plated to enumerate survivors or (B) 100 μl cells were added to a 96-well plate and lysis was measured at OD₆₀₀ every hour for 16 h. (C) LasA present in the supernatant of <i>P</i>. <i>aeruginosa</i> PAO1, PA14, CF isolates (blue) or burn isolates (green) was quantified by western blot and the ability of each supernatant to lyse heat-killed <i>S</i>. <i>aureus</i> HG003 cells after 2 h. All experiments were performed in biological triplicate. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.s003" target="_blank">S1 Data</a>. Error bars represent mean ± sd. CF, cystic fibrosis.</p

LC-MS/MS quantification of HQNO production in <i>P</i>. <i>aeruginosa</i> strains.

<p>LC-MS/MS quantification of HQNO production in <i>P</i>. <i>aeruginosa</i> strains.</p

<i>P</i>. <i>aeruginosa</i> secondary metabolites inhibit <i>S</i>. <i>aureus</i> aerobic respiration resulting in a drop in intracellular ATP and protection from ciprofloxacin killing.

<p>(A) <i>S</i>. <i>aureus</i> strain HG003 harboring plasmid P<i>pflB</i>∷<i>gfp</i> was grown to mid-exponential phase and treated with supernatant from <i>P</i>. <i>aeruginosa</i> PAO1, PA14, CF isolates (blue) or burn isolates (green), for 30 min. OD₆₀₀ and <i>gfp</i> expression levels were determined after 16 h using a Biotek Synergy H1 microplate reader. (B) Intracellular ATP was measured after 1.5 h incubation with supernatant. ***<i>p</i> < 0.0005 (one-way ANOVA with Tukey’s multiple comparison post-test). (C) <i>S</i>. <i>aureus</i> strain HG003 was grown to mid-exponential phase in MHB media and pre-treated with sterile supernatants from <i>P</i>. <i>aeruginosa</i> strains PA14 wild-type or its isogenic mutants or (D) physiologically-relevant concentrations of HQNO, PYO, or NaCN for 30 min prior to antibiotic challenge [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.ref026" target="_blank">26</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.ref027" target="_blank">27</a>]. At indicated times, an aliquot was washed and plated to enumerate survivors. All experiments were performed in biological triplicate. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.s003" target="_blank">S1 Data</a>. Error bars represent mean ± sd. CF, cystic fibrosis; CFU, colony-forming units; GFP, green fluorescent protein; HQNO, 4-hydroxyquinoline <i>N</i>-oxide; MHB, Mueller-Hinton broth; NaCN, sodium cyanide; OD, optical density; PYO, pyocyanin.</p

<i>P</i>. <i>aeruginosa</i> supernatant alters <i>S</i>. <i>aureus</i> antibiotic susceptibility.

<p><i>S</i>. <i>aureus</i> strain HG003 was grown to mid-exponential phase and exposed to sterile supernatants from <i>S</i>. <i>aureus</i> HG003 (red), <i>P</i>. <i>aeruginosa</i> laboratory strains PAO1 and PA14 (grey), <i>P</i>. <i>aeruginosa</i> CF clinical isolates (blue) or <i>P</i>. <i>aeruginosa</i> burn isolates (green) for 30 min prior to addition of (A) 50 μg/ml vancomycin, (B) 58 μg/ml tobramycin or (C) 2.34 μg/ml ciprofloxacin concentrations similar to the Cmax in humans. An aliquot was removed after 24 h, washed, and plated to enumerate survivors. The dotted red line represents the number of survivors in the control culture. All experiments were performed in biological triplicate and the number of survivors following antibiotic challenge in the presence of <i>P</i>. <i>aeruginosa</i> supernatant was compared to the HG003 supernatant-treated control. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.s003" target="_blank">S1 Data</a>. *p<0.05 (one-way ANOVA with Tukey’s multiple comparisons post-test analysis of surviving CFU). Error bars represent mean + sd. CF, cystic fibrosis; CFU, colony-forming units.</p