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1.
Nucleic Acids Res ; 49(19): 10956-10974, 2021 11 08.
Article in English | MEDLINE | ID: mdl-34643711

ABSTRACT

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.


Subject(s)
Bacterial Proteins/genetics , Gene Transfer, Horizontal , Genomic Islands , Pseudomonas aeruginosa/genetics , Trans-Activators/genetics , Virulence Factors/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Chromosomes, Bacterial , Conjugation, Genetic , DNA Transposable Elements , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Regulon , Trans-Activators/metabolism , Transcription, Genetic , Virulence Factors/metabolism
2.
PLoS Genet ; 12(5): e1006032, 2016 05.
Article in English | MEDLINE | ID: mdl-27176226

ABSTRACT

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.


Subject(s)
Bacterial Proteins/genetics , Histidine Kinase/genetics , Pseudomonas aeruginosa/genetics , Transcription Factors/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Gene Expression Regulation, Bacterial , Histidine Kinase/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Isoforms , RNA, Small Untranslated/genetics , Signal Transduction/genetics , Transcription Factors/metabolism , Virulence
3.
Article in English | MEDLINE | ID: mdl-29530852

ABSTRACT

When overproduced, the multidrug efflux system MexEF-OprN increases the resistance of Pseudomonas aeruginosa to fluoroquinolones, chloramphenicol, and trimethoprim. In this work, we demonstrate that gain-of-function mutations in the regulatory gene mexT result in oligomerization of the LysR regulator MexT, constitutive upregulation of the efflux pump, and increased resistance in clinical isolates.


Subject(s)
Pseudomonas aeruginosa/drug effects , Amino Acid Substitution/genetics , Amino Acid Substitution/physiology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Gene Expression Regulation, Bacterial/genetics , Microbial Sensitivity Tests , Mutation/genetics , Pseudomonas aeruginosa/genetics
4.
PLoS Pathog ; 8(11): e1003052, 2012.
Article in English | MEDLINE | ID: mdl-23209420

ABSTRACT

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.


Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Secretion Systems/physiology , Biofilms/growth & development , Pseudomonas aeruginosa/physiology , Adhesins, Bacterial/genetics , Animals , Cell Line , Drosophila melanogaster , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism
5.
Mol Microbiol ; 79(5): 1353-66, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21205015

ABSTRACT

Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two-component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non-characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross-regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB-oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB-OprM pump. The concomitant RocS2-dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB-oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung.


Subject(s)
Bacterial Proteins/metabolism , Fimbriae, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/genetics , Fimbriae, Bacterial/genetics , Humans , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics
6.
Microbiology (Reading) ; 158(Pt 8): 1964-1971, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22628483

ABSTRACT

Bacterial gene regulation is controlled by complex regulatory cascades which integrate input environmental signals and adapt specific and adequate output cellular responses. These complex networks are far from being elucidated, in particular in Pseudomonas aeruginosa. In the present study, we developed bacterial two-hybrid genome fragment libraries of the P. aeruginosa PAO1 strain to identify potential partners involved in the HptB/HsbR/HsbA pathway. This powerful tool, validated by the interaction previously described between HsbR and HsbA proteins, allowed us to demonstrate that the HsbR response regulator dimerizes through its PP2C/ATPase C-terminal effector domain, an observation further confirmed by pull-down experiments. This will also allow us to identify further new partners in this cascade.


Subject(s)
Genome, Bacterial , Pseudomonas aeruginosa/genetics , Two-Hybrid System Techniques , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Genomic Library , Protein Binding , Protein Structure, Tertiary , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/metabolism
7.
Plasmid ; 67(3): 245-51, 2012 May.
Article in English | MEDLINE | ID: mdl-22212534

ABSTRACT

A method for replacing endogenous promoter by a constitutive promoter in Pseudomonas aeruginosa is described. Plasmid pKNG101, a broadly used shuttle suicide vector in P. aeruginosa, was improved to allow chromosomal introduction of a Plac promoter in front of any kind of gene especially those with unknown function. Using this strategy alleviates the need for cloning difficulties encountered in this bacteria and antibiotic marker selection.


Subject(s)
Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Genetic Loci , Pseudomonas aeruginosa/genetics , Cloning, Molecular , DNA, Bacterial/isolation & purification , Genes, Bacterial , Plasmids , Promoter Regions, Genetic , Pseudomonas aeruginosa/growth & development , Sequence Analysis, DNA
8.
Mol Microbiol ; 76(6): 1427-43, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20398205

ABSTRACT

Bacterial pathogenesis often depends on regulatory networks, two-component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine-tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti-anti-sigma factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS-dependent but HptB-independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine-tuned regulation of gene expression.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/pathogenicity , RNA, Bacterial/biosynthesis , RNA, Small Interfering/biosynthesis , Signal Transduction , Biofilms/growth & development , Biological Transport , Genes, Reporter , Models, Biological , RNA, Bacterial/genetics , RNA, Small Interfering/genetics , beta-Galactosidase/metabolism
9.
Environ Microbiol ; 13(3): 666-83, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21091863

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Biofilms/growth & development , Fimbriae, Bacterial/metabolism , Molecular Chaperones/metabolism , Pseudomonas aeruginosa/pathogenicity , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Fimbriae Proteins/physiology , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/ultrastructure , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/physiology
10.
Environ Microbiol ; 12(6): 1775-86, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20553556

ABSTRACT

Bacterial two-component regulatory systems (TCSs) sense environmental stimuli to adapt the lifestyle of microbial populations. For many TCSs the stimulus is a ligand of unknown chemical nature. Pseudomonas aeruginosa utilizes the closely related RetS and LadS sensor kinases to switch between acute and chronic infections. These sensor proteins antagonistically mediate biofilm formation through communication with a central TCS, GacA/GacS. Recently, it was shown that RetS modulates the GacS sensor activity by forming RetS/GacS heterodimers. LadS and RetS are hybrid sensors with a signalling domain consisting of a 7-transmembrane (7TMR) region and a periplasmic sensor domain (diverse intracellular signalling module extracellular 2, DISMED2). The 2.65 A resolution crystal structure of RetS DISMED2, called RetSp, reveals three distinct oligomeric states capable of domain swapping. The RetSp structure also displays two putative ligand binding sites. One is equivalent to the analogous site in the structurally-related carbohydrate binding module (CBM) but the second site is located at a dimer interface. These observations highlight the modular architecture and assembly of the RetSp fold and give clues on how homodimerization of RetS could be modulated upon ligand binding to control formation of a RetS/GacS heterodimer. Modelling the DISMED2 of LadS reveals conservation of only one ligand binding site, suggesting a distinct mechanism underlying the activity of this sensor kinase.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Protein Conformation , Pseudomonas aeruginosa/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Ligands , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Multimerization , Pseudomonas aeruginosa/genetics , Sequence Alignment , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism
11.
Sci Rep ; 10(1): 3077, 2020 02 20.
Article in English | MEDLINE | ID: mdl-32080219

ABSTRACT

c-di-GMP is a major player in the switch between biofilm and motile lifestyles. Several bacteria exhibit a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be demonstrated in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a polar peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells but scattered in the cytoplasm of cells lacking FimV. Overexpression of dgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a ΔfimV background. Therefore, our findings suggest that DgcP activity is regulated by FimV. The polar localization of DgcP might contribute to a local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.


Subject(s)
Bacterial Proteins/metabolism , Cyclic GMP/analogs & derivatives , Escherichia coli Proteins/metabolism , Phosphorus-Oxygen Lyases/metabolism , Biofilms , Cyclic GMP/metabolism , Escherichia coli Proteins/chemistry , Fimbriae, Bacterial/metabolism , Models, Biological , Mutation/genetics , Phenotype , Phosphorus-Oxygen Lyases/chemistry , Protein Binding , Protein Domains , Pseudomonas aeruginosa/cytology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/physiology
12.
Cell Stem Cell ; 26(5): 657-674.e8, 2020 05 07.
Article in English | MEDLINE | ID: mdl-32169166

ABSTRACT

Hematopoietic stem cells (HSCs) maintain life-long production of immune cells and can directly respond to infection, but sustained effects on the immune response remain unclear. We show that acute immune stimulation with lipopolysaccharide (LPS) induced only transient changes in HSC abundance, composition, progeny, and gene expression, but persistent alterations in accessibility of specific myeloid lineage enhancers occurred, which increased responsiveness of associated immune genes to secondary stimulation. Functionally, this was associated with increased myelopoiesis of pre-exposed HSCs and improved innate immunity against the gram-negative bacterium P. aeruginosa. The accessible myeloid enhancers were enriched for C/EBPß targets, and C/EBPß deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPß-dependent chromatin accessibility, resulting in HSC-trained immunity, during secondary infection. This establishes a mechanism for how infection history can be epigenetically inscribed in HSCs as an integral memory function of innate immunity.


Subject(s)
CCAAT-Enhancer-Binding Protein-beta , Epigenesis, Genetic , Hematopoietic Stem Cells/immunology , Immunity, Innate , CCAAT-Enhancer-Binding Protein-beta/genetics , Epigenomics , Humans , Myelopoiesis
13.
J Bacteriol ; 191(6): 1961-73, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19151143

ABSTRACT

Bacterial attachment to the substratum involves several cell surface organelles, including various types of pili. The Pseudomonas aeruginosa Tad machine assembles type IVb pili, which are required for adhesion to abiotic surfaces and to eukaryotic cells. Type IVb pili consist of a major subunit, the Flp pilin, processed by the FppA prepilin peptidase. In this study, we investigated the regulatory mechanism of the tad locus. We showed that the flp gene is expressed late in the stationary growth phase in aerobic conditions. We also showed that the tad locus was composed of five independent transcriptional units. We used transcriptional fusions to show that tad gene expression was positively controlled by the PprB response regulator. We subsequently showed that PprB bound to the promoter regions, directly controlling the expression of these genes. We then evaluated the contribution of two genes, tadF and rcpC, to type IVb pilus assembly. The deletion of these two genes had no effect on Flp production, pilus assembly, or Flp-mediated adhesion to abiotic surfaces in our conditions. However, our results suggest that the putative RcpC protein modifies the Flp pilin, thereby promoting Flp-dependent adhesion to eukaryotic cells.


Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Genes, Regulator , Operon , Pseudomonas aeruginosa/genetics , Bacterial Adhesion , Bacterial Proteins/metabolism , Cell Line , Epithelial Cells/microbiology , Fimbriae Proteins/genetics , Fimbriae Proteins/metabolism , Humans , Protein Binding , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/physiology
14.
Sci Rep ; 9(1): 6496, 2019 04 24.
Article in English | MEDLINE | ID: mdl-31019225

ABSTRACT

Biofilm formation is a complex process resulting from the action of imbricated pathways in response to environmental cues. In this study, we showed that biofilm biogenesis in the opportunistic pathogen Pseudomonas aeruginosa depends on the availability of RpoS, the sigma factor regulating the general stress response in bacteria. Moreover, it was demonstrated that RpoS is post-translationally regulated by the HsbR-HsbA partner switching system as has been demonstrated for its CrsR-CrsA homolog in Shewanella oneidensis. Finally, it was established that HsbA, the anti-sigma factor antagonist, has a pivotal role depending on its phosphorylation state since it binds HsbR, the response regulator, when phosphorylated and FlgM, the anti-sigma factor of FliA, when non-phosphorylated. The phosphorylation state of HsbA thus drives the switch between the sessile and planktonic way of life of P. aeruginosa by driving the release or the sequestration of one or the other of these two sigma factors.


Subject(s)
Bacterial Proteins/genetics , Biofilms , Gene Expression Regulation, Bacterial , Pseudomonas aeruginosa/genetics , Sigma Factor/genetics , Bacterial Proteins/metabolism , Models, Genetic , Phosphorylation , Protein Binding , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/physiology , Sigma Factor/metabolism
15.
Appl Environ Microbiol ; 74(11): 3419-25, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18408063

ABSTRACT

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.


Subject(s)
Bacillus subtilis/genetics , DNA Transposable Elements , Mutagenesis, Insertional/methods , Anti-Bacterial Agents/pharmacology , Bacteriocins/pharmacology , Drug Resistance, Bacterial/genetics , Fosfomycin/pharmacology , Gene Order , Peptides/pharmacology , Plasmids , Promoter Regions, Genetic , Recombination, Genetic , Spectinomycin/pharmacology , Xylose/metabolism
17.
Biomol NMR Assign ; 11(1): 25-28, 2017 04.
Article in English | MEDLINE | ID: mdl-27714507

ABSTRACT

Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen. It can infect vulnerable patients such as those with cystic fibrosis or hospitalized in intensive care units where it is responsible for both acute and chronic infection. The switch between these infections is controlled by a complex regulatory system involving the central GacS/GacA two-component system that activates the production of two small non-coding RNAs. GacS is a histidine kinase harboring one periplasmic detection domain, two inner-membrane helices and three H1/D1/H2 cytoplasmic domains. By detecting a yet unknown signal, the GacS histidine-kinase periplasmic detection domain (GacSp) is predicted to play a key role in activating the GacS/GacA pathway. Here, we present the chemical shift assignment of 96 % of backbone atoms (HN, N, C, Cα, Cß and Hα), 88 % aliphatic hydrogen atoms and 90 % of aliphatic carbon atoms of this domain. The NMR-chemical shift data, on the basis of Talos server secondary structure predictions, reveal that GacSp consists of 3 ß-strands, 3 α-helices and a major loop devoid of secondary structures.


Subject(s)
Histidine Kinase/chemistry , Histidine Kinase/metabolism , Nuclear Magnetic Resonance, Biomolecular , Periplasm/metabolism , Pseudomonas aeruginosa/cytology , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence
18.
Nat Microbiol ; 2: 17027, 2017 Mar 06.
Article in English | MEDLINE | ID: mdl-28263305

ABSTRACT

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.


Subject(s)
Cyclic GMP/analogs & derivatives , Immune Evasion , Lipopolysaccharides/metabolism , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Animals , Cyclic GMP/metabolism , Disease Models, Animal , Methyltransferases/metabolism , Protein Binding , Pseudomonas Infections/microbiology , Pseudomonas Infections/pathology , Zebrafish
19.
Sci Rep ; 7(1): 11262, 2017 09 12.
Article in English | MEDLINE | ID: mdl-28900144

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium responsible for both acute and chronic infections and has developed resistance mechanisms due to its ability to promote biofilm formation and evade host adaptive immune responses. Here, we investigate the functional role of the periplasmic detector domain (GacSPD) from the membrane-bound GacS histidine kinase, which is one of the key players for biofilm formation and coordination of bacterial lifestyles. A gacS mutant devoid of the periplasmic detector domain is severely defective in biofilm formation. Functional assays indicate that this effect is accompanied by concomitant changes in the expression of the two RsmY/Z small RNAs that control activation of GacA-regulated genes. The solution NMR structure of GacSPD reveals a distinct PDC/PAS α/ß fold characterized by a three-stranded ß-sheet flanked by α-helices and an atypical major loop. Point mutations in a putative ligand binding pocket lined by positively-charged residues originating primarily from the major loop impaired biofilm formation. These results demonstrate the functional role of GacSPD, evidence critical residues involved in GacS/GacA signal transduction system that regulates biofilm formation, and document the evolutionary diversity of the PDC/PAS domain fold in bacteria.


Subject(s)
Biofilms/growth & development , Histidine Kinase/chemistry , Histidine Kinase/metabolism , Periplasmic Proteins/chemistry , Periplasmic Proteins/metabolism , Pseudomonas aeruginosa/enzymology , Catalytic Domain , Histidine Kinase/genetics , Magnetic Resonance Spectroscopy , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Periplasmic Proteins/genetics , Point Mutation , Protein Conformation , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development
20.
Front Microbiol ; 12: 813062, 2021.
Article in English | MEDLINE | ID: mdl-34966380
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