Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.     

Phenotypic variation of Pseudomonas brassicacearum as a plant root-colonization strategy

Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.     

Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements.

Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase- and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE- and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.     

Phenotypic switching in fungi

Over the past three decades new fungal diseases have emerged that now constitute a major threat, especially for patients with chronic diseases and/or underlying immune deficiencies. Despite the epidemiologic data, the emergence of stable drug-resistant or hypervirulent fungal strains in human disease has not been demonstrated as seen in emerging viral and bacterial infections. Fungi are eukaryotic microbes that capitalize on a sophisticated built-in ability to generate phenotypic variability. This successful strategy allows them to undergo rapid adaptation in response to environmental challenges, such as individual body locations that may exhibit drastic differences in temperature and pH. Rapid microevolution can also confer drug resistance and protect them from the host’s immune response. This review explores phenotypic switching in pathogenic fungi, including Candida spp and Cryptococcus spp, and how phenotypic switching contributes to the pathogenesis of fungal diseases.     

Role of small RNAs in the interaction between Arabidopsis and Pseudomonas syringae

Endogenous small RNAs (miRNAs and siRNAs) regulate gene expression in diverse biological processes.Research with the Arabidopsis-Pseudomonas syringae system has shown that small RNAs contribute to plan...     

A pathogenic mechanism in Huntington's disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.     

Phenotypic switching in Cryptococcus neoformans

Cryptococcus neoformans strains exhibit considerable phenotype variability with regards to the capsular polysaccharide, sterol composition of the cell wall, and cell and colony morphology. Phenotypic changes can occur spontaneously during in vitro passage of strains or during chronic infection in vivo and may be associated with differences in virulence. Studies from our laboratory have demonstrated that phenotype variability can be the result of phenotypic switching. Phenotypic switching is defined as a reversible change of an observable colony phenotype that occurs at a frequency above the expected frequency for somatic mutations. This implies that phenotypic switching represents controlled and programmed changes in this pathogenic yeast rather than random mutations. We have shown that a phenotypic switch from a smooth colony phenotype to a mucoid colony phenotype occurs in vitro and in vivo during chronic infection of mice. More importantly we have now demonstrated that the switch is associated with an increase in virulence and a change in the host immune response. Implications of these findings for the pathogenesis of cryptococcosis are discussed.     

Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs

Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)RNAs. Because ncRNAs typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncRNPs with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncRNPs.     

Small regulatory RNAs in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is frequently associated with nosocomial infections, and can be life threatening in immunosuppressed, cancer and cystic fibrosis patients. Virulence in P. aeruginosa is combinatorial, and results from the activation of several genetic programs that regulate motility, attachment to the host epithelium as well as the synthesis of exotoxins. The pathogen has a high survival capacity in the host owing to its metabolic versatility, nutrient scavenging and resistance against both, antibiotics and immune defenses. Adaptive responses to various environmental stresses and stimuli are often regulated by small regulatory RNAs (sRNA). In this review, we summarize the current knowledge on the regulation and function of P. aeruginosa sRNAs that titrate regulatory proteins, base-pair with target mRNAs, and which are derived from CRISPR elements.     

Expression of Marker RNAs in Pseudomonas putida

A broad-host-range vector that expresses a unique artificial RNA in Pseudomonas putida has been developed. This vector was derived from the plasmid pBBR1MCS and incorporates regulatory regions from the Escherichia coli ribosomal operon, rrnB. These include the promoters P1 and P2, and the terminators T1 and T2. The gene for the artificial RNA was derived from Vibrio proteolyticus 5S rRNA. The artificial RNA product accumulates to a level that is 10–20% of the total 5S rRNA in P. putida. The RNA product is not incorporated into ribosomes and has a minimal effect on cell growth rate. In contrast, when wild-type V. proteolyticus 5S rRNA was expressed from the vector, it was incorporated into ribosomes. It is expected that this new vector system will allow artificial RNA expression systems to be readily developed for a large variety of species. Received: 16 June 1999 / Accepted: 11 August 1999     

Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs

Pseudomonas aeruginosa is a highly adaptable bacterium that thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen. This wide adaptability correlates with its broad genetic diversity. In this study, we used a deep-sequencing approach to explore the complement of small RNAs (sRNAs) in P. aeruginosa as the number of such regulatory molecules previously identified in this organism is relatively low, considering its genome size, phenotypic diversity and adaptability. We have performed a comparative analysis of PAO1 and PA14 strains which share the same host range but differ in virulence, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting. Interestingly, a number of these novel sRNAs are strain-specific or showed strain-specific expression, strongly suggesting that they could be involved in determining specific phenotypic traits.     

Phenotypic and genome-wide analysis of an antibiotic-resistant small colony variant (SCV) of Pseudomonas aeruginosa

Background

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.

Methodology/Principal Findings

One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10⁻⁵ on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.

Conclusions

By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.