Charlotte Michaux

Scopus Publications

Improved RNA stability estimation through Bayesian modeling reveals most Salmonella transcripts have subminute half-lives
Laura Jenniches, Charlotte Michaux, Linda Popella, Sarah Reichardt, Jörg Vogel, Alexander J. Westermann, and Lars Barquist
Proceedings of the National Academy of Sciences
RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in posttranscriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 min, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with cross-linking and immunoprecipitation followed by RNA sequencing (CLIP-seq) revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3′ untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for ProQ in the oxidative stress response. Our findings provide insights into posttranscriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

Intoxication of antibiotic persisters by host RNS inactivates their efflux machinery during infection
Séverin Ronneau, Charlotte Michaux, Rachel T. Giorgio, and Sophie Helaine
Public Library of Science (PLoS)
The host environment is of critical importance for antibiotic efficacy. By impacting bacterial machineries, stresses encountered by pathogens during infection promote the formation of phenotypic variants that are transiently insensitive to the action of antibiotics. It is assumed that these recalcitrant bacteria—termed persisters—contribute to antibiotic treatment failure and relapsing infections. Recently, we demonstrated that host reactive nitrogen species (RNS) transiently protect persisters against the action of β-lactam antibiotics by delaying their regrowth within host cells. Here, we discovered that RNS intoxication of persisters also collaterally sensitizing them to fluoroquinolones during infection, explaining the higher efficiency of fluoroquinolones against intramacrophage Salmonella. By reducing bacterial respiration and the proton-motive force, RNS inactivate the AcrAB efflux machinery of persisters, facilitating the accumulation of fluoroquinolones intracellularly. Our work shows that target inactivity is not the sole reason for Salmonella persisters to withstand antibiotics during infection, with active efflux being a major contributor to survival. Thus, understanding how the host environment impacts persister physiology is critical to optimize antibiotics efficacy during infection.

Decline in nitrosative stress drives antibiotic persister regrowth during infection
Séverin Ronneau, Charlotte Michaux, and Sophie Helaine
Elsevier BV

Grad-seq analysis of Enterococcus faecalis and Enterococcus faecium provides a global view of RNA and protein complexes in these two opportunistic pathogens
Charlotte Michaux, Milan Gerovac, Elisabeth E Hansen, Lars Barquist, and Jörg Vogel
Oxford University Press (OUP)
AbstractEnterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA–protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA–protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA–protein complexes such as the 6S RNA–RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/).

The RNA-Binding Protein ProQ Promotes Antibiotic Persistence in Salmonella
Alisa Rizvanovic, Charlotte Michaux, Margherita Panza, Zeynep Iloglu, Sophie Helaine, E. Gerhart H. Wagner, and Erik Holmqvist
American Society for Microbiology
Bacteria can avoid eradication by antibiotics through a phenomenon known as persistence. Persister cells arise through phenotypic heterogeneity and constitute a small fraction of dormant cells within a population of actively growing bacteria, which is susceptible to antibiotic killing.

Antibiotic tolerance and persistence have distinct fitness trade-offs
Charlotte Michaux, Séverin Ronneau, Rachel T. Giorgio, and Sophie Helaine
Public Library of Science (PLoS)
Genetically susceptible bacteria can escape the action of bactericidal antibiotics through antibiotic tolerance or persistence. However, one major difference between the two phenomena is their distinct penetrance within an isogenic population. While with antibiotic persistence, susceptible and persister cells co-exist, antibiotic tolerance affects the entire bacterial population. Here, we show that antibiotic tolerance can be achieved in numerous non-specific ways in vitro and during infection. More importantly, we highlight that, due to their impact on the entire bacterial population, these tolerance-inducing conditions completely mask persistence and the action of its molecular determinants. Finally, we show that even though tolerant populations display a high survival rate under bactericidal drug treatment, this feature comes at the cost of having impaired proliferation during infection. In contrast, persistence is a risk-limiting strategy that allows bacteria to survive antibiotic treatment without reducing the ability of the population to colonize their host. Altogether, our data emphasise that the distinction between these phenomena is of utmost importance to improve the design of more efficient antibiotic therapies.

Cellular RNA Targets of Cold Shock Proteins CspC and CspE and Their Importance for Serum Resistance in Septicemic Escherichia coli
Yael Yair, Charlotte Michaux, Dvora Biran, Jörg Bernhard, Jörg Vogel, Lars Barquist, and Eliora Z. Ron
American Society for Microbiology
Virulent Escherichia coli strains that cause infections outside the intestinal tract—extraintestinal pathogenic E. coli (ExPEC)—constitute a major clinical problem worldwide. They are involved in several distinct conditions, including urinary tract infections, newborn meningitis, and sepsis.

Erratum: Correction to: Studying Antibiotic Persistence During Infection (Methods in molecular biology (Clifton, N.J.))
Charlotte Michaux, Séverin Ronneau, and Sophie Helaine
Springer US

Studying Antibiotic Persistence During Infection
Charlotte Michaux, Séverin Ronneau, and Sophie Helaine
Springer US

Single-Nucleotide RNA Maps for the Two Major Nosocomial Pathogens Enterococcus faecalis and Enterococcus faecium
Charlotte Michaux, Elisabeth E. Hansen, Laura Jenniches, Milan Gerovac, Lars Barquist, and Jörg Vogel
Frontiers Media SA
Enterococcus faecalisandfaeciumare two major representative clinical strains of the Enterococcus genus and are sadly notorious to be part of the top agents responsible for nosocomial infections. Despite their critical implication in worldwide public healthcare, essential and available resources such as deep transcriptome annotations remain poor, which also limits our understanding of post-transcriptional control small regulatory RNA (sRNA) functions in these bacteria. Here, using the dRNA-seq technique in combination with ANNOgesic analysis, we successfully mapped and annotated transcription start sites (TSS) of bothE. faecalis V583andE. faecium AUS0004at single nucleotide resolution. Analyzing bacteria in late exponential phase, we capture ~40% (E. faecalis) and 43% (E. faecium) of the annotated protein-coding genes, determine 5′ and 3′ UTR (untranslated region) length, and detect instances of leaderless mRNAs. The transcriptome maps revealed sRNA candidates in both bacteria, some found in previous studies and new ones. Expression of candidate sRNAs is being confirmed under biologically relevant environmental conditions. This comprehensive global TSS mapping atlas provides a valuable resource for RNA biology and gene expression analysis in the Enterococci. It can be accessed online atwww.helmholtz-hiri.de/en/datasets/enterococcusthrough an instance of the genomic viewer JBrowse.

Bacterial persisters and infection: Past, present, and progressing
Bridget Gollan, Grzegorz Grabe, Charlotte Michaux, and Sophie Helaine
Annual Reviews
Persisters are nongrowing, transiently antibiotic-tolerant bacteria within a clonal population of otherwise susceptible cells. Their formation is triggered by environmental cues and involves the main bacterial stress response pathways that allow persisters to survive many harsh conditions, including antibiotic exposure. During infection, bacterial pathogens are exposed to a vast array of stresses in the host and form nongrowing persisters that survive both antibiotics and host immune responses, thereby most likely contributing to the relapse of many infections. While antibiotic persisters have been extensively studied over the last decade, the bulk of the work has focused on how these bacteria survive exposure to drugs in vitro. The ability of persisters to survive their interaction with a host is important yet underinvestigated. In order to tackle the problem of persistence of infections that contribute to the worldwide antibiotic resistance crisis, efforts should be made by scientific communities to understand and merge these two fields of research: antibiotic persisters and host-pathogen interactions. Here we give an overview of the history of the field of antibiotic persistence, report evidence for the importance of persisters in infection, and highlight studies that bridge the two areas.

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE
Charlotte Michaux, Erik Holmqvist, Erin Vasicek, Malvika Sharan, Lars Barquist, Alexander J. Westermann, John S. Gunn, and Jörg Vogel
Proceedings of the National Academy of Sciences
Significance Interactions between RNA and protein molecules are critical for many cellular processes. Bacterial cells rely on RNA–protein interactions to regulate gene expression in response to an ever-changing environment. To understand such regulation, it is key to identify the processes controlled by RNA-binding proteins. In this study, we have taken a RNA ligand-centered approach to chart the physiological processes controlled by a class of RNA-binding proteins harboring the highly conserved cold-shock domain. This approach revealed cold-shock proteins CspC and CspE to be critical for the stress response and virulence in the enterobacterial pathogen Salmonella enterica serovar Typhimurium, emphasizing RNA-binding proteins as major players in bacterial infection.

New Insight into Cold Shock Proteins: RNA-binding Proteins Involved in Stress Response and Virulence
Charlotte Michaux and Jean-Christophe Giard
John Wiley & Sons, Inc.

The polyamine N-acetyltransferase-like enzyme PmvE plays a role in the virulence of Enterococcus faecalis
Cecilia Martini, Charlotte Michaux, Francesca Bugli, Alessandro Arcovito, Federica Iavarone, Margherita Cacaci, Francesco Paroni Sterbini, Axel Hartke, Nicolas Sauvageot, Maurizio Sanguinetti,et al.
American Society for Microbiology
ABSTRACT We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001 , renamed pmvE (for p olyamine m etabolism and v irulence of E. faecalis ). PmvE shares strong homologies with N 1 -spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535- pmvE (V19/p3535- pmvE ), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N -acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis , likely through its involvement in the polyamine metabolism.

Identification of peptidoglycan-Associated proteins as vaccine candidates for enterococcal infections
Felipe Romero-Saavedra, Diana Laverde, Dominique Wobser, Charlotte Michaux, Aurélie Budin-Verneuil, Benoit Bernay, Abdellah Benachour, Axel Hartke, and Johannes Huebner
Public Library of Science (PLoS)
Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.

Involvement of Enterococcus faecalis small RNAs in stress response and virulence
Charlotte Michaux, Axel Hartke, Cecilia Martini, Swantje Reiss, Dirk Albrecht, Aurélie Budin-Verneuil, Maurizio Sanguinetti, Susanne Engelmann, Torsten Hain, Nicolas Verneuil,et al.
American Society for Microbiology
ABSTRACT Candidate small RNAs (sRNAs) have recently been identified in Enterococcus faecalis , a Gram-positive opportunistic pathogen, and six of these candidate sRNAs with unknown functions were selected for a functional study. Deletion mutants and complemented strains were constructed, and their virulence was tested. We were unable to obtain the ef0869-0870 mutant, likely due to an essential role, and the ef0820-0821 sRNA seemed not to be involved in virulence. In contrast, the mutant lacking ef0408-0409 sRNA, homologous to the RNAII component of the toxin-antitoxin system, appeared more virulent and more able to colonize mouse organs. The three other mutants showed reduced virulence. In addition, we checked the responses of these mutant strains to several stresses encountered in the gastrointestinal tract or during the infection process. In parallel, the activities of the sRNA promoters were measured using transcriptional fusion constructions. To attempt to identify the regulons of these candidate sRNAs, proteomics profiles of the mutant strains were compared with that of the wild type. This showed that the selected sRNAs controlled the expression of proteins involved in diverse cellular processes and the stress response. The combined data highlight the roles of certain candidate sRNAs in the adaptation of E. faecalis to environmental changes and in the complex transition process from a commensal to a pathogen.

Physiological roles of small RNA molecules
Charlotte Michaux, Nicolas Verneuil, Axel Hartke, and Jean-Christophe Giard
Microbiology Society
Unlike proteins, RNA molecules have emerged lately as key players in regulation in bacteria. Most reviews hitherto focused on the experimental and/or in silico methods used to identify genes encoding small RNAs (sRNAs) or on the diverse mechanisms of these RNA regulators to modulate expression of their targets. However, less is known about their biological functions and their implications in various physiological responses. This review aims to compile what is known presently about the diverse roles of sRNA transcripts in the regulation of metabolic processes, in different growth conditions, in adaptation to stress and in microbial pathogenesis. Several recent studies revealed that sRNA molecules are implicated in carbon metabolism and transport, amino acid metabolism or metal sensing. Moreover, regulatory RNAs participate in cellular adaptation to environmental changes, e.g. through quorum sensing systems or development of biofilms, and analyses of several sRNAs under various physiological stresses and culture conditions have already been performed. In addition, recent experiments performed with Gram-positive and Gram-negative pathogens showed that regulatory RNAs play important roles in microbial virulence and during infection. The combined results show the diversity of regulation mechanisms and physiological processes in which sRNA molecules are key actors.

Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis
Charlotte Michaux, Luis Felipe Romero Saavedra, Fany Reffuveille, Benoît Bernay, Didier Goux, Axel Hartke, Nicolas Verneuil, and Jean-Christophe Giard
Microbiology Society
CspR has been characterized recently as a cold-shock RNA-binding protein in Enterococcus faecalis, a natural member of the gastro-intestinal tract capable of switching from a commensal relationship with the host to an important nosocomial pathogen. In addition to its involvement in the cold-shock response, CspR also plays a role in the long-term survival and virulence of E. faecalis. In the present study, we demonstrated that anti-CspR immune rabbit serum protected larvae of Galleria mellonella against a lethal challenge of the WT strain. These results suggested that CspR might have a surface location. This hypothesis was verified by Western blot that showed detection of CspR in the total as well as in the surface protein fraction. In addition, identification of surface polypeptides by proteolytic shaving of intact bacterial cells followed by liquid chromatography-MS-MS revealed that cold-shock proteins (EF1367, EF2939 and CspR) were present on the cell surface. Lastly, anti-CspR immune rabbit serum was used for immunolabelling and detected with colloidal gold-labelled goat anti-rabbit IgG in order to determine the immunolocalization of CspR on E. faecalis WT strain. Electron microscopy images confirmed that the cold-shock protein RNA-binding protein CspR was present in both cytoplasmic and surface parts of the cell. These data strongly suggest that CspR, in addition to being located intracellularly, is also present in the extracellular protein fraction of the cells and has important functions in the infection process of Galleria larvae.

CspR, a cold shock RNA-binding protein involved in the long-term survival and the virulence of enterococcus faecalis
Charlotte Michaux, Cecilia Martini, Koki Shioya, Sandra Ahmed Lecheheb, Aurélie Budin-Verneuil, Pascal Cosette, Maurizio Sanguinetti, Axel Hartke, Nicolas Verneuil, and Jean-Christophe Giard
American Society for Microbiology
ABSTRACT By coprecipitation, we identified RNA-binding proteins in the Gram-positive opportunistic pathogen Enterococcus faecalis known to be deficient of the RNA chaperone Hfq. In particular, we characterized one belonging to the cold shock protein (Csp) family (Ef2925) renamed CspR for cold shock protein RNA binding. Compared to the wild-type strain, the Δ cspR mutant was less virulent in an insect infection model ( Galleria mellonella ) and exhibited a decreased persistence in mouse kidneys and a low survival rate in peritoneal macrophages. As expected, we found that the Δ cspR mutant strain was more impaired in its growth than the parental strain under cold conditions and in its long-term survival under nutrient starvation. All these phenotypes were restored after complementation of the Δ cspR mutant. In addition, Western blot analysis showed that CspR was overexpressed under cold shock conditions and in the stationary phase. Since CspR may act as an RNA chaperone, putative targets were identified using a global proteomic approach completed with transcriptomic assays. This study revealed that 19 proteins were differentially expressed in the Δ cspR strain (9 upregulated, 10 downregulated) and that CspR mainly acted at the posttranscriptional level. These data highlight for the first time the role of the RNA-binding protein CspR as a regulator in E. faecalis and its requirement in stress response and virulence in this important human pathogen.

SlyA regulator is involved in bile salts stress response of Enterococcus faecalis
Charlotte Michaux, Cecilia Martini, Aurélie Hanin, Yanick Auffray, Axel Hartke, and Jean-Christophe Giard
Oxford University Press (OUP)
SlyA is a newly transcriptional regulator identified in Enterococcus faecalis that is involved in the virulence, persistence in mouse kidneys and liver, and survival inside peritoneal macrophages. In this study we searched for environmental conditions that affect expression of the corresponding gene. Of the several stress conditions tested, only bile salts (0.08%) significantly induced transcription of slyA. In addition, the growth of ΔslyA mutant strain was significantly impaired in the presence of bile salts. To increase knowledge of SlyA regulon, real-time quantitative PCR was performed and revealed that expression of EF_3005, which encodes a choloylglycine hydrolase, is negatively regulated by SlyA.

Genome-wide identification of small RNAs in the opportunistic pathogen enterococcus faecalis V583
Kouki Shioya, Charlotte Michaux, Carsten Kuenne, Torsten Hain, Nicolas Verneuil, Aurélie Budin-Verneuil, Thomas Hartsch, Axel Hartke, and Jean-Christophe Giard
Public Library of Science (PLoS)
Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.

SlyA is a transcriptional regulator involved in the virulence of Enterococcus faecalis
Charlotte Michaux, Maurizio Sanguinetti, Fany Reffuveille, Yanick Auffray, Brunella Posteraro, Michael S. Gilmore, Axel Hartke, and Jean-Christophe Giard
American Society for Microbiology
ABSTRACT Phylogenetic analysis of the crystal structure of the Enterococcus faecalis SlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalis biology, we dissected the genetic organization of the slyA -EF_3001 locus and constructed a slyA deletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the Δ slyA mutant is more virulent in an insect infection model ( Galleria mellonella ), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA -EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the Δ slyA mutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

Charlotte Michaux

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