The horizontal transfer of Pseudomonas aeruginosa PA14 ICE PAPI-1 is controlled by a transcriptional triad between TprA, NdpA2 and MvaT.

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. Genome sequences reveal that most P. aeruginosa strains contain a significant number of accessory genes gathered in genomic islands. Those genes are essential for P. aeruginosa to invade new ecological niches with high levels of antibiotic usage, like hospitals, or to survive during host infection by providing pathogenicity determinants. P. aeruginosa pathogenicity island 1 (PAPI-1), one of the largest genomic islands, encodes several putative virulence factors, including toxins, biofilm genes and antibiotic-resistance traits. The integrative and conjugative element (ICE) PAPI-1 is horizontally transferable by conjugation via a specialized GI-T4SS, but the mechanism regulating this transfer is currently unknown. Here, we show that this GI-T4SS conjugative machinery is directly induced by TprA, a regulator encoded within PAPI-1. Our data indicate that the nucleotide associated protein NdpA2 acts in synergy with TprA, removing a repressive mechanism exerted by MvaT. In addition, using a transcriptomic approach, we unravelled the regulon controlled by Ndpa2/TprA and showed that they act as major regulators on the genes belonging to PAPI-1. Moreover, TprA and NdpA2 trigger an atypical biofilm structure and enhance ICE PAPI-1 transfer.

Alternatives to Fight Vancomycin-Resistant Staphylococci and Enterococci.

Gram positive pathogens are a significant cause of healthcare-associated infections, with Staphylococci and Enterococci being the most prevalent ones. Vancomycin, a last resort glycopeptide, is used to fight these bacteria but the emergence of resistance against this drug leaves some patients with few therapeutic options. To counter this issue, new generations of antibiotics have been developed but resistance has already been reported. In this article, we review the strategies in place or in development to counter vancomycin-resistant pathogens. First, an overview of traditional antimicrobials already on the market or in the preclinical or clinical pipeline used individually or in combination is summarized. The second part focuses on the non-traditional antimicrobials, such as antimicrobial peptides, bacteriophages and nanoparticles. The conclusion is that there is hitherto no substitute equivalent to vancomycin. However, promising strategies based on drugs with multiple mechanisms of action and treatments based on bacteriophages possibly combined with conventional antibiotics are hoped to provide treatment options for vancomycin-resistant Gram-positive pathogens.

Analysis of glycerol and dihydroxyacetone metabolism in Enterococcus faecium.

Glycerol (Gly) can be dissimilated by two pathways in bacteria. Either this sugar alcohol is first oxidized to dihydroxyacetone (DHA) and then phosphorylated or it is first phosphorylated to glycerol-3-phosphate (GlyP) followed by oxidation. Oxidation of GlyP can be achieved by NAD-dependent dehydrogenases or by a GlyP oxidase. In both cases, dihydroxyacetone phosphate is the product. Genomic analysis showed that Enterococcus faecium harbors numerous genes annotated to encode activities for the two pathways. However, our physiological analyses of growth on glycerol showed that dissimilation is limited to aerobic conditions and that despite the presence of genes encoding presumed GlyP dehydrogenases, the GlyP oxidase is essential in this process. Although E. faecium contains an operon encoding the phosphotransfer protein DhaM and DHA kinase, which are required for DHA phosphorylation, it is unable to grow on DHA. This operon is highly expressed in stationary phase but its physiological role remains unknown. Finally, data obtained from sequencing of a transposon mutant bank of E. faecium grown on BHI revealed that the GlyP dehydrogenases and a major intrinsic family protein have important but hitherto unknown physiological functions.

Targeted Genome Reduction of Pseudomonas aeruginosa Strain PAO1 Led to the Development of Hypovirulent and Hypersusceptible rDNA Hosts.

Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections, which is largely used as a model organism to study antibiotic resistance and pathogenesis. As other species of the genus, its wide metabolic versatility appears to be attractive to study biotechnological applications. However, its natural resistance to antibiotics and its capacity to produce a wide range of virulence factors argue against its biotechnological potential. By reducing the genome of the reference strain PAO1, we explored the development of four hypovirulent and hypersusceptible recombinant DNA hosts (rDNA hosts). Despite deleting up to 0.8% of the core genome, any of the developed strains presented alterations of fitness when cultured under standard laboratory conditions. Other features such as antibiotic susceptibility, cytotoxicity, in vivo pathogenesis, and expression of heterologous peptides were also explored to highlight the potential applications of these models. This work stands as the first stage of the development of a safe-platform strain of Pseudomonas aeruginosa that will be further optimized for biotechnological applications.

The DEAD-box RNA helicase CshA is required for fatty acid homeostasis in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen that can grow in a wide array of conditions: on abiotic surfaces, on the skin, in the nose, in planktonic or biofilm forms and can cause many type of infections. Consequently, S. aureus must be able to adapt rapidly to these changing growth conditions, an ability largely driven at the posttranscriptional level. RNA helicases of the DEAD-box family play an important part in this process. In particular, CshA, which is part of the degradosome, is required for the rapid turnover of certain mRNAs and its deletion results in cold-sensitivity. To understand the molecular basis of this phenotype, we conducted a large genetic screen isolating 82 independent suppressors of cold growth. Full genome sequencing revealed the fatty acid synthesis pathway affected in many suppressor strains. Consistent with that result, sublethal doses of triclosan, a FASII inhibitor, can partially restore growth of a cshA mutant in the cold. Overexpression of the genes involved in branched-chain fatty acid synthesis was also able to suppress the cold-sensitivity. Using gas chromatography analysis of fatty acids, we observed an imbalance of straight and branched-chain fatty acids in the cshA mutant, compared to the wild-type. This imbalance is compensated in the suppressor strains. Thus, we reveal for the first time that the cold sensitive growth phenotype of a DEAD-box mutant can be explained, at least partially, by an improper membrane composition. The defect correlates with an accumulation of the pyruvate dehydrogenase complex mRNA, which is inefficiently degraded in absence of CshA. We propose that the resulting accumulation of acetyl-CoA fuels straight-chained fatty acid production at the expense of the branched ones. Strikingly, addition of acetate into the medium mimics the cshA deletion phenotype, resulting in cold sensitivity suppressed by the mutations found in our genetic screen or by sublethal doses of triclosan.

Opposite effect of vancomycin and D-Cycloserine combination in both vancomycin resistant Staphylococcus aureus and enterococci.

The increasing spread of antibiotic resistant bacteria is a major human health concern. The challenging development of new effective antibiotics has led to focus on seeking synergistic antibiotic combinations. Vancomycin (VAN) is a glycopeptide antibiotic used to treat Staphylococcus aureus and enterococci infections. It is targeting D-Alanyl-D-Alanine dimers during peptidoglycan biosynthesis. D-cycloserine (DCS) is a D-Alanine analogue that targets peptidoglycan biosynthesis by inhibiting D-Alanine:D-Alanine ligase (Ddl). The VAN-DCS combination was found to be synergistic in VAN resistant S. aureus strains lacking van genes cluster. We hypothesize that this combination leads to opposite effects in S. aureus and enterococci strains harboring van genes cluster where VAN resistance is conferred by the synthesis of modified peptidoglycan precursors ending in D-Alanyl-D-Lactate. The calculated Fractional Inhibitory Concentration of VAN-DCS combination in a van- vancomycin-intermediate, VanA type, and VanB type strains were 0.5, 5 and 3, respectively. As a result, VAN-DCS combination leads to synergism in van-lacking strains, and to antagonism in strains harboring van genes cluster. The VAN-DCS antagonism is due to a mechanism that we named van-mediated Ddl inhibition bypass. Our results show that antibiotic combinations can lead to opposite effects depending on the genetic backgrounds.

Azetidine-Containing Alkaloids Produced by a Quorum-Sensing Regulated Nonribosomal Peptide Synthetase Pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine-containing alkaloids from P. aeruginosa PAO1, termed azetidomonamides, which are derived from a conserved, quorum-sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new S-adenosylmethionine-dependent enzyme to produce azetidine 2-carboxylic acid as an unusual building block of NRPS. The mutants of P. aeruginosa unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in Galleria mellonella model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.

Expression profiling in a mammalian host reveals the strong induction of genes encoding LysM domain-containing proteins in Enterococcus faecium.

Enterococcus faecium is an important health care-associated pathogen that is difficult to treat due to the high level of antibiotic resistance of clinical isolates. The identification of new potential therapeutic targets or vaccination strategies is therefore urgently needed. In this regard, we carried out a transcriptomic analysis of the E. faecium vancomycin-resistant strain AUS0004, comparing the gene expression of bacteria grown under laboratory conditions and bacteria isolated from an infection site. This analysis highlighted more than 360 genes potentially induced under infection conditions. Owing to their expression profiles, four LysM domain-containing proteins were characterized in more detail. The EFAU004_01059, 1150 and 494 proteins are highly homologous, whereas EFAU004_01209 has a unique domain-architecture and sequence. The analysis of corresponding mutants showed that all LysM proteins played relevant roles in the infection process of E. faecium in mice. The EFAU004_01209 mutant also displayed profound morphological modifications, suggesting it has a role in cell wall synthesis or cell division. Furthermore, the adhesion to kidney cells and growth of the mutant was affected in human urine. All these phenotypes and the surface exposure of EFAU004_01209 identify this protein as an interesting new drug target in E. faecium.

Characterization of Sviceucin from Streptomyces Provides Insight into Enzyme Exchangeability and Disulfide Bond Formation in Lasso Peptides.

Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

The C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus aureus.

Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes.

The CshA DEAD-box RNA helicase is important for quorum sensing control in Staphylococcus aureus.

DEAD-box RNA helicases are present in almost all living organisms and participate in various processes of RNA metabolism. Bacterial proteins of this large family were shown to be required for translation initiation, ribosome biogenesis and RNA decay. The latter is primordial for rapid adaptation to changing environmental conditions. In particular, the RhlB RNA helicase from E. coli was shown to assist the bacterial degradosome machinery. Recently, the CshA DEAD-box proteins from Bacillus subtilis and Staphylococcus aureus were shown to interact with proteins that are believed to form the degradosome. S. aureus can cause life-threatening disease, with particular concern focusing on biofilm formation on catheters and prosthetic devices, since in this form the bacteria are almost impossible to eradicate both by the immune system and antibiotic treatment. This persistent state relies on the expression of surface encoded proteins that allow attachment to various surfaces, and contrasts with the dispersal mode of growth that relies on the secretion of proteins such as hemolysins and proteases. The switch between these two states is mainly mediated by the Staphylococcal cell density sensing system encoded by agr. We show that inactivation of the cshA DEAD-box gene results in dysregulation of biofilm formation and hemolysis through modulation of agr mRNA stability. Importantly, inactivation of the agrA gene in the cshA mutant background reverses the defect, indicating that cshA is genetically upstream of agr and that a delicate balance of agr mRNA abundance mediated through stability control by CshA is critical for proper expression of virulence factors.

Unique biofilm signature, drug susceptibility and decreased virulence in Drosophila through the Pseudomonas aeruginosa two-component system PprAB.

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.

Inside the complex regulation of Pseudomonas aeruginosa chaperone usher systems.

Pseudomonas aeruginosa assembles several cell surface-associated organelles, including those of the chaperone usher (CU) pathway. Five different CU loci have been identified and characterized in various strains of P.aeruginosa. However, their potential functional redundancy, particularly in biofilm formation, is supported by the control of their expression by a complex and specific regulatory network. Here, we review recent findings relating to this network. The control exerted by this network involves transcriptional repressors and activators, a phase-variable mechanism, a second intracellular messenger (c-di-GMP) and chemosensory and two-component systems.

The PprA-PprB two-component system activates CupE, the first non-archetypal Pseudomonas aeruginosa chaperone-usher pathway system assembling fimbriae.

The opportunistic pathogen Pseudomonas aeruginosa has redundant molecular systems that contribute to its pathogenicity. Those assembling fimbrial structures promote complex organized community lifestyle. We characterized a new 5.8 kb genetic locus, cupE, that includes the conserved usher- and chaperone-encoding genes. This locus, widely conserved in different bacterial species, contains four additional genes encoding non-archetypal fimbrial subunits. We first evidenced that the cupE gene cluster was specifically expressed in biofilm conditions and was responsible for fibre assembly containing at least CupE1 protein, at the bacterial cell surface. These fimbriae not only played a significant role in the early stages (microcolony and macrocolony formation) but also in shaping 3D mushrooms during P. aeruginosa biofilm development. Using wide-genome transposon mutagenesis, we identified the PprAB two-component system (TCS) as a regulator of cupE expression, and further demonstrated the involvement of the PprAB TCS in direct CupE fimbrial assembly activation. Thus, this TCS represents a new regulatory element controlling the transition between planktonic and community lifestyles in P. aeruginosa.

Biological 'glue' and 'Velcro': molecular tools for adhesion and biofilm formation in the hairy and gluey bug Pseudomonas aeruginosa.

Pseudomonas aeruginosa contains an extraordinarily large number of loci encoding systems facilitating a communal lifestyle and binding to supports of various natures. These P. aeruginosa systems are reviewed here and may be categorized as classical or non-classical systems. They highlight the panoply of strategies that this hairy and gluey bacterium has developed for dealing with the diverse environments with which it is faced during various types of infection, involving complex regulatory networks that have not yet been fully elucidated but several aspects of which are discussed here.

Expression of Pseudomonas aeruginosa CupD fimbrial genes is antagonistically controlled by RcsB and the EAL-containing PvrR response regulators.

Pseudomonas aeruginosa is a gram-negative pathogenic bacterium with a high adaptive potential that allows proliferation in a broad range of hosts or niches. It is also the causative agent of both acute and chronic biofilm-related infections in humans. Three cup gene clusters (cupA-C), involved in the assembly of cell surface fimbriae, have been shown to be involved in biofilm formation by the P. aeruginosa strains PAO1 or PAK. In PA14 isolates, a fourth cluster, named cupD, was identified within a pathogenicity island, PAPI-I, and may contribute to the higher virulence of this strain. Expression of the cupA genes is controlled by the HNS-like protein MvaT, whereas the cupB and cupC genes are under the control of the RocS1A1R two-component system. In this study, we show that cupD gene expression is positively controlled by the response regulator RcsB. As a consequence, CupD fimbriae are assembled on the cell surface, which results in a number of phenotypes such as a small colony morphotype, increased biofilm formation and decreased motility. These behaviors are compatible with the sessile bacterial lifestyle. The balance between planktonic and sessile lifestyles is known to be linked to the intracellular levels of c-di-GMP with high levels favoring biofilm formation. We showed that the EAL domain-containing PvrR response regulator counteracts the activity of RcsB on cupD gene expression. The action of PvrR is likely to involve c-di-GMP degradation through phosphodiesterase activity, confirming the key role of this second messenger in the balance between bacterial lifestyles. The regulatory network between RcsB and PvrR remains to be elucidated, but it stands as a potential model system to study how the equilibrium between the two lifestyles could be influenced by therapeutic agents that favor the planktonic lifestyle. This would render the pathogen accessible for the immune system or conventional antibiotic treatment.