Staphylococcus aureus is an opportunistic pathogen responsible for a wide range of diseases in humans. During infections, this bacterium is exposed to various stresses that target its cell wall, such as oxidative or acid environments as well as various cell wall-acting antimicrobials. Staphylococcus aureus has effective regulatory systems for responding to environmental stresses, enabling the expression of factors necessary for its survival. Bacterial small RNAs (sRNAs) play a crucial role in this adaptation process. In this study, we show that RsaOI, an S. aureus sRNA, accumulates under acid stress conditions. This response is mediated via the two-component system VraSR, which is associated with the cell wall damage response. As a component of the VraSR regulon, RsaOI contributes to the survival of S. aureus under acid stress and affects its susceptibility to glycopeptide antibiotics. Our findings reveal that RsaOI targets the lacABCDFEG operon, which encodes components of tagatose pathway, a unique mechanism responsible for galactose metabolism in S. aureus. By antisense base pairing near the ribosome binding site of lacD, RsaOI inhibits the expression of this gene, encoding tagatose-6-phosphate aldolase. This regulation disrupts the tagatose pathway, impairing galactose utilization in S. aureus. These findings highlight the role of RsaOI in the mediation between cell wall stress responses and a specific metabolic pathway.

SprA1 and SprA2 are small hydrophobic peptides that belong to the type I toxin-antitoxin systems expressed by Staphylococcus aureus. Both peptides induce S. aureus death when overexpressed. Although they share 71% of amino acids sequence similarity, SprA2 exhibits stronger hemolytic activity than SprA1. In this study, we investigated the mode of action of these toxins on both prokaryotic-like and eukaryotic-like membranes. We first confirmed that SprA2, like SprA1, is an alpha-helical peptide located at the S. aureus membrane. By overexpressing each toxin, we demonstrated that SprA1 forms stable pores in the S. aureus membrane, evidenced by concomitant membrane depolarization, permeabilization and ATP release leading to growth arrest, whereas SprA2 forms transient pores, causing concomitant membrane depolarization, ATP release, and growth arrest. We showed that the unique cysteine residue present in SprA1 and SprA2 is required for toxicity through disulfide bond formation. Next, we found that both synthetic peptides induce slight leakage in anionic DOPC-DOPG lipid vesicles mimicking prokaryotic membranes, concomitant with lipid vesicles aggregation and/or fusion. Moreover, we observed that SprA1 permeabilizes S. aureus protoplasts, via its ability to form stable pores, whereas SprA2 permeabilizes and lyses them. However, no permeabilization of intact bacteria was detected after the addition of SprA1 and SprA2 in the extracellular medium. Finally, we confirmed that SprA2 has strong activity on zwitterionic DOPC lipid vesicles mimicking eukaryotic membranes, without inducing aggregation. This work highlights the strong selectivity of SprA2 for eukaryotic membranes, suggesting that this toxin may play a role in S. aureus virulence.

Staphylococcus aureus is a widespread livestock and human pathogen that colonizes diverse microenvironments within its host. Its adaptation to the environmental conditions encountered within humans relies on coordinated gene expression. This requires a sophisticated regulatory network, among which regulatory RNAs (usually called sRNAs) have emerged as key players over the last 30 years. In S. aureus, sRNAs regulate target genes at the post-transcriptional level through base–pair interactions. The functional characterization of a subset revealed that they participate in all biological processes, including virulence, metabolic adaptation, and antibiotic resistance. In this review, we report 30 years of S. aureus sRNA studies, from their discovery to the in-depth characterizations of some of them. We also discuss their actual in vivo contribution, which is still lagging behind, and their place within the complex regulatory network. These shall be key aspects to consider in order to clearly uncover their in vivo biological functions.

The increasing interest for Galleria mellonella larvae as an infection model is evidenced by the number of papers reporting its use, which increases exponentially since the early 2010s. This popularity was initially linked to limitation of conventional animal models due to financial, technical and ethical aspects. In comparison, alternative models (e.g. models using Caenorhabditis elegans, Drosophila melanogaster or G. mellonella) were cheap, simple to use and not limited by ethical regulation. Since then, similar results have been established with G. mellonella model comparatively to vertebrates, and it is more and more often used as a robust model per se, not only as an alternative to the murine model. This review attempts to summarize the current knowledge supporting the development of this model, both on immunological and microbiological aspects. For that, we focus on investigation of virulence and new therapies for the most important pathogenic bacteria. We also discuss points out directions for standardization, as well as recent advances and new perspectives for monitoring host-pathogen interactions.

SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the sarA regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a ΔsarA mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of sprG2, a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and in silico methodologies are effective to characterize transcriptional factor (TF) regulons and functions. IMPORTANCE Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of sprG2, a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

Bacterial type I toxin-antitoxin (TA) systems are widespread, and consist of a stable toxic peptide whose expression is monitored by a labile RNA antitoxin. We characterized Staphylococcus aureus SprA2/SprA2(AS) module, which shares nucleotide similarities with the SprA1/SprA1(AS) TA system. We demonstrated that SprA2/SprA2(AS) encodes a functional type I TA system, with the cis-encoded SprA2(AS) antitoxin acting in trans to prevent ribosomal loading onto SprA2 RNA. We proved that both TA systems are distinct, with no cross-regulation between the antitoxins in vitro or in vivo. SprA2 expresses PepA2, a toxic peptide which internally triggers bacterial death. Conversely, although PepA2 does not affect bacteria when it is present in the extracellular medium, it is highly toxic to other host cells such as polymorphonuclear neutrophils and erythrocytes. Finally, we showed that SprA2(AS) expression is lowered during osmotic shock and stringent response, which indicates that the system responds to specific triggers. Therefore, the SprA2/SprA2(AS) module is not redundant with SprA1/SprA1(AS), and its PepA2 peptide exhibits an original dual mode of action against bacteria and host cells. This suggests an altruistic behavior for S.aureus in which clones producing PepA2 in vivo shall die as they induce cytotoxicity, thereby promoting the success of the community.

The opportunistic pathogen Staphylococcus aureus expresses transcription factors (TFs) and regulatory small RNAs (sRNAs) which are essential for bacterial adaptation and infectivity. Until recently, the study of S. aureus sRNA gene expression regulation was under investigated, but it is now an expanding field. Here we address the regulation of Srn_3610_SprC sRNA, an attenuator of S. aureus virulence. We demonstrate that SarA TF represses srn_3610_sprC transcription. DNase I footprinting and deletion analyses show that the SarA binding site on srn_3610_sprC belongs to an essential 22 bp DNA region. Comparative analysis also revealed another possible site, this time in the srn_9340 promoter. SarA specifically binds these two sRNA promoters with high affinity in vitro and also represses their transcription in vivo Chromatin immunoprecipitation (ChIP) assays confirmed SarA attachment to both promoters. ChIP and electrophoretic mobility shift assays targeting σA RNA polymerase subunit or using bacterial RNA polymerase holoenzyme suggested that SarA and the σA bind srn_3610_sprC and srn_9340 promoters in a mutually exclusive way. Beyond the mechanistic study of SarA repression of these two sRNAs, this work also suggests that some S. aureus sRNAs belong to the same regulon and act jointly in responding to environmental changes.

Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg-gingipain A (RgpA), Lys-gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time-dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain-containing proteins.

Oral epithelia represent the first physical and chemical barrier against bacterial invasion and colonization of the underlying tissues This protection results from the production of epithelial innate immune responses, including the secretion of cationic antimicrobial peptides with a large spectrum of activity against pathogenic microorganisms Among these antimicrobial cationic peptides, beta-defensin 2 (hBD-2) is expressed in the gingival epithelia upon stimulation by microorganisms or inflammatory mediators such as interleukin-1 beta or tumour necrosis factor-alpha The aim of the present study was to investigate the effect of AV119, a patented blend of two sugars from avocado, on the induction of hBD-2 in two epithelial cell lines and a primo-culture of gingival epithelial cells Culture supernatant from epithelial cells treated with AV 119 was also evaluated for its antimicrobial activity against the periodontopathogen Porphyromonas gingivalis Cell ELISA assays revealed that AV119 induces the production of hBD-2 by all the epithelial cells tested Minimal Inhibition Concentration assay also showed that the culture supernatant of epithelial cells treated with AV119 possesses antibacterial activity In conclusion, our data revealed that AV119 component, through hBD2 induction and antibacterial activity, could be considered for potential use in the control of oral mucosal infections and reduction of microbial tissue invasion during periodontitis

The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

DevelopmentofSNAP-tag-mediated live cell labelingas an alternative toGFP inPorphyromonasgingivalis Ophélie Nicolle, Astrid Rouillon, Helene Guyodo, Zohreh Tamanai-Shacoori, Fatiha Chandad, Vincent Meuric & Martine Bonnaure-Mallet Equipe de Microbiologie, UPRES-EA 1254, Université Européenne de Bretagne, Université de Rennes I, Campus Médecine, Pharmacie, UFR Odontologie, Rennes, France; Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec City, QC, Canada; Pôle Odontologie et Chirurgie Buccale, CHU Rennes, Rennes, France; and Pôle Microorganismes, CHU Rennes, Rennes, France

Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O6-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3*. These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.

Background and objectives:Porphyromonas gingivalis is frequently identified to type by evaluation of fimA polymorphisms and less often by pulsed-field gel electrophoresis (PFGE) because of the technical intricacies of PFGE. To compare these techniques, we genotyped P. gingivalis clinical isolates as to (i) their fimA type and (ii) their whole genome restriction profile (PFGE analysis).Material and methods:Thirty-two P. gingivalis strains were isolated from 16 unrelated periodontitis patients. Two strains were isolated from each patient. Strains were subjected to a fimA-typing polymerase chain reaction (PCR) assay. Strains that could not be typed by PCR were submitted to sequencing of the entire fimA gene. The PFGE profiles of clinical strains were compared using bioinformatic analysis.Results:Seven of the 32 isolates were not typeable by PCR and so their entire fimA gene was sequenced. The sequencing identified each strain as belonging to a single fimA type. In one case, sequencing of the fimA gene did not agree with the result obtained using fimA PCR typing. With the exception of one patient, each patient presented isolates bearing the same fimA type. However, in three patients, isolates with the same fimA type presented different PFGE pulsotypes.Conclusion:The P. gingivalis typing using fimA PCR has limitations in typeability and discriminatory power. A typing technique for P. gingivalis that is easy to perform but that presents adequate typeability and discriminatory power is needed if we want to better understand the epidemiology of periodontal disease.

Activity of the Met4 transcription factor is antagonized by the SCF(Met30) ubiquitin ligase by degradation-dependent and degradation-independent mechanisms, in minimal and rich nutrient conditions, respectively. In this study, we show that the heavy metal Cd2+ over-rides both mechanisms to enable rapid Met4-dependent induction of metabolic networks needed for production of the antioxidant and Cd2+-chelating agent glutathione. Cd2+ inhibits SCF(Met30) activity through rapid dissociation of the F-box protein Met30 from the holocomplex. In minimal medium, dissociation of SCF(Met30) complex is sufficient to impair the methionine-induced degradation of Met4. In rich medium, dissociation of the SCF(Met30) complex is accompanied by a deubiquitylation mechanism that rapidly removes inhibitory ubiquitin moieties from Met4. Post-translational control of SCF(Met30) assembly by a physiological stress to allow rapid induction of a protective gene expression program represents a novel mode of regulation in the ubiquitin system.

The ubiquitin system has been recently implicated in various aspects of transcriptional regulation, including proteasome-dependent degradation of transcriptional activators. In yeast, the activator Met4 is inhibited by the SCF(Met30) ubiquitin ligase, which recognizes and oligo-ubiquitylates Met4. Here, we demonstrate that in minimal media, Met4 is ubiquitylated and rapidly degraded in response to methionine excess, whereas in rich media, Met4 is oligo-ubiquitylated but remains stable. In the latter growth condition, oligo-ubiquitylated Met4 is not recruited to MET gene promoters, but is recruited to the SAM genes, which are required for production of S-adenosylmethionine, an unstable metabolite that is not present in rich medium. Thus, ubiquitylation not only regulates Met4 by distinct degradation-dependent and -independent mechanisms, but also controls differential recruitment of a single transcription factor to distinct promoters, thereby diversifying transcriptional activator specificity.

Saccharomyces cerevisiae SCFMet30 ubiquitin–protein ligase controls cell cycle function and sulfur amino acid metabolism. We report here that the SCFMet30 complex mediates the transcriptional repression of the MET gene network by triggering degradation of the transcriptional activator Met4p when intracellular S‐adenosylmethionine (AdoMet) increases. This AdoMet‐induced Met4p degradation is dependent upon the 26S proteasome function. Unlike Met4p, the other components of the specific transcriptional activation complexes that are assembled upstream of the MET genes do not appear to be regulated at the protein level. We provide evidence that the interaction between Met4p and the F‐box protein Met30p occurs irrespective of the level of intracellular AdoMet, suggesting that the timing of Met4p degradation is not controlled by its interaction with the SCFMet30 complex. We also demonstrate that Met30p is a short‐lived protein, which localizes within the nucleus. Furthermore, transcription of the MET30 gene is regulated by intracellular AdoMet levels and is dependent upon the Met4p transcription activation function. Thus Met4p appears to control its own degradation by regulating the amount of assembled SCFMet30 ubiquitin ligase.