Pierre Fechter

• PhD
• Researcher at French National Centre for Scientific Research

The rising prevalence of bacterial infections, antibiotic resistance, and emerging pathogens underscores the urgent need for innovative diagnostic approaches. Aptamers, short nucleic acid sequences with high specificity and affinity for their targets, are promising candidates for diagnostic applications due to their ability to detect a wide range o...

The proliferation and prevalence of antibiotic-resistant bacteria despite modern medicine is considered as one of the most alarming threats to global health. The fear that antibiotics that work today might not work tomorrow makes it imperative to search and develop novel drugs or therapeutic strategies to fight against micro-organisms resistant to...

Pseudomonas aeruginosa (PA) is a common cause of chronic infections, particularly feared by cystic fibrosis patients. PA colonizes the lung where it adapts to the local environment, and/or to treatments by drugs. This genotypic and phenotypic adaptation, in turns, influences its interaction with its environment, like bacteria from the microbiota. A...

Background Metals are trace elements, vital in some instances or toxic in others. Due to this toxicity, they have been used since ancient time as antimicrobials, and prescribed when plant-only remedies were not efficient enough. These remedies could still contain secrets that may lead to the discovery of new therapeutically interesting combinations...

History can be a vast source of information, as long as we can have access to manuscripts dealing with pharmacopeia. The medieval Arab period is considered the Golden Age of ancient medicine, bringing together the knowledge of many ancient civilizations while continuously improving them. We have based this review on symptoms reflecting bacterial in...

Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. The pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, one with a high affinity for iron, and the second with a lower affinity, respectively. Furthermore, the production of both siderophores involve a positive auto-regulato...

Much data shows that biological metals other than iron can interfere with iron acquisition by siderophores in bacteria. Siderophores are small iron chelators produced by the microorganism to obtain access to iron. Here, we show that Co2+ is imported into Pseudomonas aeruginosa cells in a complex with the siderophore pyochelin (PCH) by the ferri-PCH...

Nucleic acid aptamers are often referred to as chemical antibodies. Because they possess several advantages, like their smaller size, temperature stability, ease of chemical modification, lack of immunogenicity and toxicity, and lower cost of production, aptamers are promising tools for clinical applications. Aptamers against cell surface protein b...

Chlamydia trachomatis is a bacterial human pathogen responsible for the development of trachoma, an infection leading to blindness, and is also the cause of the main bacterial sexually transmitted infection worldwide. We designed a new inhibitor of this bacterium with, however, some prerequisites using (i) the iron dependency of the bacterium, (ii)...

ncRNAs are key players in the adaptation of bacteria to new environments, by modulating the composition of the membrane upon changes in the environment. Nevertheless, monitoring the changes in surface protein expression is still a challenge, since these proteins are present in low abundance, and are difficult to extract. Here is described a method...

Cyclometallated iridium(III) dipyridylamine complexes present an antibacterial activity on P. aeruginosa, a highly resistant pathogenic bacteria. This activity is increased when the complex is conjugated to biotin, a bacterial nutrient,...

Staphylococcus aureus is a major opportunistic and versatile pathogen. Because the bacteria rapidly evolve multi-resistances towards antibiotics, there is an urgent need to find novel targets and alternative strategies to cure bacterial infections. Here, we provide a brief overview on the knowledge acquired on S. aureus ribosomes, which is one of t...

In Staphylococcus aureus, peptidoglycan metabolism plays a role in the host inflammatory response and pathogenesis. Transcription of the peptidoglycan hydrolases is activated by the essential two-component system WalKR at low cell density. During stationary growth phase, WalKR is not active and transcription of the peptidoglycan hydrolase genes is...

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family o...

Cells adapt to environmental changes by efficiently adjusting gene expression programs. Staphylococcus aureus, an opportunistic pathogenic bacterium, switches between defensive and offensive modes in response to quorum sensing signal. We identified and studied the structural characteristics and dynamic properties of the core regulatory circuit gove...

We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a s...

A plethora of RNAs with regulatory functions has been discovered in many non-pathogenic and pathogenic bacteria. In Staphylococcus aureus, recent findings show that a large variety of RNAs control target gene expression by diverse mechanisms and many of them are expressed in response to specific internal or external signals. These RNAs comprise tra...

Author Summary Gene expression is regulated at multiple levels, including the decision of whether or not to translate a mRNA. This phenomenon, known as translational regulation, allows rapid changes in cellular concentrations of proteins and is well suited to the adjustment of cellular growth in response to stress and environmental changes. Many ba...

The presence of regulatory sequences in the 3' untranslated region (3'-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3'-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the majo...

RNA molecules with regulatory functions in pathogenic bacteria have benefited from a renewed interest these two last decades. In Staphylococcus aureus, recent genome-wide approaches have led to the discovery that almost 10-20% of genes code for RNAs with critical regulatory roles in adaptive processes. These RNAs include trans-acting RNAs, which mo...

Mapping of the 5′ end and secondary structure probing of rnc mRNA. (A) Determination of the 5′ end of rnc mRNA by primer extension. Total RNA was extracted from different stages of growth of the wild type strain (RN6390). Primer extension was done with 10 µg of RNA. Lanes 1, 4: 240 min of growth; lanes 2, 3: 150 and 180 min of growth, respectively....

Examples of secondary structure motifs as found in several intergenic regions. The genomic organization is depicted and red arrows represent ncRNA genes. Examples of secondary structure motifs found in several ncRNAs as predicted by contrafold [20] and RNAFold [21]. (TIF)

Reads corresponding to small non coding RNAs (sRNAs) and their antisense RNAs (asRNA). Co-immunoprecipitation (coIP) was done flag tagged E135A (EL78), WT (EL79) and D63A (EL80) RNase III. In yellow: control experiment was carried out with the untagged WT protein (RN6390).Total RNAs were extracted from cultures grown at exponential (4 h, Exp) and l...

Reads corresponding to mRNAs. Co-immunoprecipitation (coIP) was done with flag tagged E135A (EL78), WT (EL79) and D63A (EL80) RNase III. In yellow: control experiment was carried out with untagged RNase III (RN6390). Total RNAs were prepared from exponential (4 h, Exp) and late exponential (6 h, Late Exp) phase of growth. CDS is for coding sequence...

Reads corresponding to antisense RNAs complementary to mRNAs. Co-immunoprecipitation (coIP) was done with flag tagged E135A (EL78), WT (EL79) and D63A (EL80) RNase III. In yellow: control experiment was carried out with untagged RNase III (RN6390). Total RNAs were prepared from cultures grown at the exponential (4 h, Exp) and late exponential (6 h,...

Transcriptional start sites (TSS) of several RNAs that were co-immunoprecipitated with RNase III. (a) Numbering according to N315 genome. (b) Start site as defined by primer extension (PE) analysis using the indicated primer (+1 site detected in wt and Δrnc strains was identical unless otherwise indicated). (c, d) Start site and size of RNAs as def...

Mutant RNase III E135A binds to the co-immunoprecipitated RNAs in vitro. (A) Binding of the mutant E135A RNase III to various RNAs assessed by gel retardation assays. The assays were performed with in vitro transcribed unlabeled RNA fragments (50–100 nM), which were incubated with increasing concentrations of E135A enzyme. The complexes were resolv...

Secondary structures of mRNA fragments co-immunoprecipitated with RNase III. The RNA fragment of srrA-srrB mRNA co-immunoprecipitated with the mutant enzymes corresponded to the translational coupling site. The stop codon of srrA is depicted in green, the start codon, and the Shine and Dalgarno sequence (SD) of srrB are given in red. UTR stands for...

Oligonucleotides used in this study. (a) With bold letters the mutated nucleotides are indicated; (b) With italics the enzyme restriction sites are indicated; (c) With small letters the sequence hybridizing to pQE30 vector is indicated; (d) With small bold letters the Flag-tag is indicated; (e) Underlined is the T7 promoter sequence. Cand is for ca...

Supplementary Material and Methods. (DOCX)

Examples of the distribution of cDNA reads represented with the Integrated Genome Browser. Genomic annotation is given at the top of each profile panel. The ncRNA genes are shown by black arrows. (+) and (−) indicate leading and lagging strand, respectively. CoIP RNA was from RN6390 parental strain, and from the mutant Δrnc strain transformed with...

List of reads corresponding to rRNA and tRNA operons and to their antisense RNAs. Co-immunoprecipitation (coIP) was done with flag tagged E135A (strain EL78), WT (strain EL79) and D63A (strain EL80) RNase III. In yellow: control experiment was carried out with the untagged WT protein (RN6390). Total RNAs were prepared from cultures grown at the exp...

Strains and plasmids used in this study. (DOCX)

Analysis of the secondary structure of cspA mRNA using enzymatic probing. (A) Enzymatic hydrolysis was performed using in vitro transcribed cspA mRNAs having a long (cspAL) or short (cspAS) 5′UTR. Increasing concentrations of enzymes were added: RNase V1 (0.0001, 0.001 and 0.002 U), RNase T1 (0.1, 0.2 and 0.4 U) and RNase T2 (0.0125, 0.025 and 0.12...

Effect of RNase III on the expression of several ncRNAs and antisense RNAs from Staphylococcus aureus. (A) The expression of housekeeping non-coding RNAs (4.5S and 6S RNA) was monitored in various strains: RN6390, the isogenic Δrnc mutant strain (Δrnc), the Δrnc mutant strain transformed with plasmid expressing the mutant E135A RNase III (Δrnc-E135...

Read numbers and Mapping statistics. NC_002745 = S. aureus N315 genome; NC_003140 = S. aureus N315 plasmid. Immunoprecipitation experiments were carried out in RN6390 strain (wild-type and referent strain) as a control, and in the mutant Δrnc strain transformed with a plasmid expressing either the WT flag-tagged RNase III (IP_EL79), the mutant D63A...

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family o...

Staphylococcus aureus is one of the major human pathogens, which causes numerous community-associated and hospital-acquired infections. The regulation of the expression of numerous virulence factors is coordinated by complex interplays between two component systems, transcriptional regulatory proteins, and regulatory RNAs. Recent studies have ident...

Chemical probing is often used to gain knowledge on the secondary and tertiary structures of RNA molecules either free or engaged in complexes with ligands. The method monitors the reactivity of each nucleotide towards chemicals of various specificities reflecting the hydrogen bonding environment of each nucleotide within the RNA molecule. In addit...

Staphylococcus aureus is a common commensal bacterial species that is usually found in the nose and on the skin of 30 % of healthy adults. However, the bacterium is a remarkably versatile pathogen that is one of the main causes of community as well as hospital-acquired infections (Cheung et al., 2004; Novick, 2003). S. aureus is responsible for sys...

Non-coding (nc)RNAs are important players in most biological processes. Although small RNAs such as microRNAs and small interfering RNAs have emerged as exceptionally important regulators of gene expression, great numbers of larger ncRNAs have also been identified. Many of these are abundant and differentially expressed but their functions have in...

Secondary structure of Staphylococcus aureus RNAIII. The 3′ domain is squared. Arrows indicate the deletions of the hairpin 13 (RNAIII-Δ13), or of the three hairpins 7 to 9 (RNAIII-Δ7–9). The hairpin 7 used in the RNase III hydrolysis is shown. The secondary structure of RNAIII was experimentally defined [38]. The drawing is adapted from Figure 1A...

Comparison of the secondary structures of the RNAIII targets. (A) The secondary structures of spa mRNA [11], SA1000 and rot mRNA [12], and coa mRNA. The nucleotides base-paired to RNAIII are in red. The conserved G-rich sequence in the mRNAs, which interact with the C-rich motif of hairpin loops 7, 13, 14 of RNAIII are encircled in pink. (B) Sequen...

Northern blot analysis on coa mRNA prepared from exponential phase cultures (OD600nm 0.5) from various S. aureus strains. The blot was hybridized with an RNA probe antisense to coa mRNA: RN6390 (WT, rnaIII+); S. aureus strain lacking rnaIII gene, WA400 (ΔrnaIII); S. aureus strain lacking rnc gene, Δrnc strain (LUG774); WA400 transformed with a plas...

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression...

Bacteria use various means of RNA-mediated gene regulation. Regulatory RNAs include mRNA leaders that affect expression in cis or in trans, non-coding RNAs that trap regulatory proteins or interact with one or multiple target mRNAs, and RNAs that protect the bacteria against foreign and invasive DNA. The aim of this review is to outline the basic p...

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs ar...

Toeprinting was developed to study the formation of ribosomal initiation complexes in bacteria. This approach, based on the inhibition of reverse transcriptase elongation, was used to monitor the effect of ribosomal components and translational factors on the formation of the active ribosomal initiation complex. Moreover, this method offers an easy...

A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5' leader regions of mRNAs have direct effects on translation initiation of the downstrea...

Staphylococcus aureus ribonuclease III (Sa-RNase III) belongs to the enzyme family known to process double-stranded RNAs consisting of two turns of the RNA helix. Although the enzyme is thought to play a role in ribosomal RNA processing and gene regulation, the deletion of the rnc gene in S. aureus does not affect cell growth in rich medium. S. aur...

RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3' domain of RNAIII coordinately represses at the post-transcriptional level, the expression...

Bacteria exploit functional diversity of RNAs in a wide range of regulatory mechanisms to control gene expression. In last few years, small RNA molecules have been discovered at a staggering rate in bacteria, mainly in Escherichia coli. While functions of many of these RNA molecules are still not known, several of them behave as key effectors of ad...

Most cellular and eukaryotic viral mRNAs have a cap structure at their 5' end that is critical for efficient translation. Cap structures also aid in mRNA transport from nucleus to cytoplasm and, in addition, protect the mRNAs from degradation by 5' exonucleases. Cap function is mediated by cap-binding proteins that play a key role in translational...

The RNA-dependent RNA polymerase complex of influenza A virus consists of three subunits, PB1, PB2 and PA. To investigate the function of the PA subunit, we mutated evolutionarily conserved amino acids to alanines in the C-terminal third of PA of influenza A/WSN/33 that shows the highest degree of conservation between PA proteins of influenza A, B...

mRNAs are capped at their 5'-end by a unique cap structure containing N7-methyl guanine. Recognition of the cap structure is of paramount importance in some of the most central processes of gene expression as well as in some viral processes, such as priming of influenza virus transcription. The recent resolution of the structure of three evolutiona...

The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA...

Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl-tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base-pair C1–G72 in the acceptor stem together with the anticodon triplet. The anticodon re...

tRNA-like domains are found at the 3' end of genomic RNAs of several genera of plant viral RNAs. Three groups of tRNA mimics have been characterized on the basis of their aminoacylation identity (valine, histidine and tyrosine) for aminoacyl-tRNA synthetases. Folding of these domains deviates from the canonical tRNA cloverleaf. The closest sequence...

Residues specifying aminoacylation by yeast tyrosyl-tRNA synthetase (TyrRS) of the tRNA-like structure present at the 3'-end of brome mosaic virus (BMV) RNA were determined by the in vitro approach using phage T7 transcripts. They correspond to nucleotides equivalent to base-pair C1-G72 and discriminator base A73 in the amino acid-acceptor branch o...

The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on the presence of modified residues in tRNA(Tyr), although such residues stabilize its structure. Thus, the major tyrosine identity determinants were searched by the in vitro approach using unmodified transcripts produced by T7 RNA polymerase. On the basis of the ty...

A limitation for a universal use of T7 RNA polymerase for in vitro tRNA transcription lies in the nature of the often unfavorable 5'-terminal sequence of the gene to be transcribed. To overcome this drawback, a hammerhead ribozyme sequence was introduced between a strong T7 RNA polymerase promoter and the tDNA sequence. Transcription of this constr...