14-3-3 proteins are required for the inhibition of Ras by exoenzyme S

14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-1 kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where ‘Xaa’ represents ‘any residue’, and ‘pSer’ is ‘phosphoserine’) . However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ibα and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

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Exoenzyme S binds its cofactor 14-3-3 through a non-phosphorylated motif

Biochemical Society Transactions ◽

10.1042/bst0300401 ◽

2002 ◽

Vol 30 (4) ◽

pp. 401-405 ◽

Cited By ~ 16

Author(s):

B. Hallberg

Keyword(s):

Specific Interaction ◽

Phage Display Library ◽

Bacterial Toxin ◽

Inositol Polyphosphate ◽

Checkpoint Control ◽

Exoenzyme S ◽

Infected Cells ◽

Adp Ribosyltransferase

14-3-3 proteins belong to a family of conserved molecules, which play a regulatory role and participate in signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine-phosphorylated ligands, such as the Raf-1 kinase and Bad, through recognition of the phosphorylated consensus motif, RSXpSXP (where pS is phosphoserine). Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and a small number of proteins, for example the 43 kDa inositol polyphosphate 5-phosphatase, glycoprotein Ib, p75NTR-associated cell-death executor (NADE) and the bacterial ADP-ribosyltransferase toxin exoenzyme S (ExoS). It has been suggested that specific residues of 14-3-3 proteins are required for activation of the bacterial toxin ExoS. An unphosphorylated peptide derived from a phage display library, known as the R18 peptide, and a synthetic peptide derived from ExoS inhibit the interaction between ExoS and 14-3-3. In this report we identify the amino acid sequence on ExoS which is responsible for its specific interaction with 14-3-3, both in vitro and in vivo. In addition, we believe that this interaction is critical for the ADP-ribosylation of an endogenous target, Ras, by ExoS both in vitro and in vivo. Loss of the 14-3-3-binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras and shows a reduced capacity to change the morphology of infected cells, together with reduced killing activity.

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In Vitro and In Vivo Activities of Levofloxacin against Biofilm-Producing Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1641 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1641-1645 ◽

Cited By ~ 80

Author(s):

Hiroko Ishida ◽

Yoshihisa Ishida ◽

Yuichi Kurosaka ◽

Tsuyoshi Otani ◽

Kenichi Sato ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Standard Method ◽

Phosphate Buffer ◽

Guinea Pigs ◽

Therapeutic Efficacy ◽

Killing Activity ◽

Biofilm Bacteria

ABSTRACT Interactions between biofilm cells of Pseudomonas aeruginosa and levofloxacin were studied. P. aeruginosa incubated for 6 days with Teflon sheets formed a biofilm on its surface. Against the biofilm bacteria, levofloxacin at an MIC determined by the standard method for the strain was highly bactericidal whereas gentamicin, ceftazidime, and ciprofloxacin showed no significant killing activity. Levofloxacin, ciprofloxacin, and gentamicin, but not ceftazidime, exhibited killing activity against nongrowing cells of the strain incubated in phosphate buffer. In addition, levofloxacin, ciprofloxacin, and ceftazidime, but not gentamicin, showed the ability to penetrate an agar containing alginate. These findings may explain the efficacy of levofloxacin and the ineffectiveness of gentamicin and ceftazidime against biofilm bacteria; however, the cause of the ineffectiveness of ciprofloxacin still remains to be determined. In experimental pneumonia in guinea pigs, in which the biofilm mode of growth of the strain was observed in the lung, only levofloxacin exhibited substantial therapeutic efficacy. These findings suggest the significant role of levofloxacin in therapy of biofilm bacterium-associated infectious diseases.

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Modification of Ras in Eukaryotic Cells by Pseudomonas aeruginosa Exoenzyme S

Infection and Immunity ◽

10.1128/iai.66.6.2607-2613.1998 ◽

1998 ◽

Vol 66 (6) ◽

pp. 2607-2613 ◽

Cited By ~ 58

Author(s):

Eileen M. McGuffie ◽

Dara W. Frank ◽

Timothy S. Vincent ◽

Joan C. Olson

Keyword(s):

Pseudomonas Aeruginosa ◽

Host Cells ◽

Eukaryotic Cells ◽

Exoenzyme S ◽

Ras Family ◽

Isogenic Mutant Strain ◽

Related Proteins ◽

Adp Ribosyltransferase

ABSTRACT Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa388 and an isogenic mutant strain, 388ΔexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388ΔexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388ΔexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.

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OsaR (PA0056) functions as a repressor of the gene fleQ encoding an important motility regulator in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00145-21 ◽

2021 ◽

Author(s):

Yibing Ma ◽

Yujie Liu ◽

Yutong Bi ◽

Xiao Han ◽

Yongxin Jin ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Site ◽

Biofilm Formation ◽

Transcriptional Regulator ◽

Regulatory Mechanisms ◽

Chronic Obstructive ◽

Transcriptional Level ◽

Obstructive Pulmonary Disease

FleQ plays a crucial role in motility and biofilm formation by regulating flagellar and exopolysaccharide biosynthesis in Pseudomonas aeruginosa . It has been reported that the expression of FleQ is transcriptionally downregulated by the virulence factor regulator Vfr. Herein we demonstrated that a LysR-type transcriptional regulator, OsaR, is also capable of binding to the promoter region of fleQ and repressing its transcription. Through gel shift and DNase I footprinting assays, the OsaR binding site was identified and characterized as a dual LysR-type transcriptional regulator box (AT-N 11 -AT-N 7 -A-N 11 -T). Mutation of the A-T palindromic base pairs in fleQ promoter not only reduced the binding affinity of OsaR in vitro , but also de-repressed fleQ transcription in vivo . The OsaR binding site was found to cover the Vfr binding site; knockout of osaR or vfr separately exhibited no effect on the transcriptional level of fleQ ; however, fleQ expression was repressed by overexpression of osaR or vfr . Furthermore, simultaneously deleting both osaR and vfr resulted in an upregulation of fleQ , but it could be complemented by the expression of either of the two repressors. In summary, our work revealed that OsaR and Vfr function as two transcriptional repressors of fleQ that bind to the same region of fleQ but work separately. IMPORTANCE Pseudomonas aeruginosa is a widespread human pathogen, which accounts for serious infections in the hospital, especially for lung infection in cystic fibrosis and chronic obstructive pulmonary disease patients. P. aeruginosa infection is closely associated with its motility and biofilm formation, which are both under the regulation of the important transcription factor FleQ. However, the upstream regulatory mechanisms of fleQ have not been fully elucidated. Therefore, our research identifying a novel regulator of fleQ as well as new regulatory mechanisms controlling its expression will be significant for better understanding the intricate gene regulatory mechanisms related to P. aeruginosa virulence and infection.

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A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo

Pathogens ◽

10.3390/pathogens10040401 ◽

2021 ◽

Vol 10 (4) ◽

pp. 401

Author(s):

Pauline Nogaret ◽

Fatima El El Garah ◽

Anne-Béatrice Blanc-Potard

Keyword(s):

Pseudomonas Aeruginosa ◽

Animal Model ◽

Quorum Sensing ◽

Drug Testing ◽

Drug Efficacy ◽

Embryo Viability ◽

Immersion Method ◽

Opportunistic Human Pathogen

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

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Antimicrobial and antibiofilm activities of meropenem loaded-mesoporous silica nanoparticles against carbapenem-resistant Pseudomonas aeruginosa

Journal of Biomaterials Applications ◽

10.1177/08853282211003848 ◽

2021 ◽

pp. 088532822110038

Author(s):

Mohammad Yousef Memar ◽

Mina Yekani ◽

Hadi Ghanbari ◽

Edris Nabizadeh ◽

Sepideh Zununi Vahed ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Mesoporous Silica ◽

Silica Nanoparticles ◽

Mesoporous Silica Nanoparticles ◽

Carbapenem Resistant ◽

Toxicity In Vitro ◽

Different Levels

The aims of the present study were the determination of antimicrobial and antibiofilm effects of meropenem-loaded mesoporous silica nanoparticles (MSNs) on carbapenem resistant Pseudomonas aeruginosa ( P. aeruginosa) and cytotoxicity properties in vitro. The meropenem-loaded MSNs had shown antibacterial and biofilm inhibitory activities on all isolates at different levels lower than MICs and BICs of meropenem. The viability of HC-04 cells treated with serial concentrations as MICs and BICs of meropenem-loaded MSNs was 92–100%. According to the obtained results, meropenem-loaded MSNs display the significant antibacterial and antibiofilm effects against carbapenem resistant and biofilm forming P. aeruginosa and low cell toxicity in vitro. Then, the prepared system can be an appropriate option for the delivery of carbapenem for further evaluation in vivo assays.

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Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00559-19 ◽

2019 ◽

Vol 202 (8) ◽

Cited By ~ 2

Author(s):

Courtney E. Price ◽

Dustin G. Brown ◽

Dominique H. Limoli ◽

Vanessa V. Phelan ◽

George A. O’Toole

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Physical Space ◽

Late Time ◽

Protective Effects ◽

Content Type ◽

Alginate Production ◽

The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

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Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6866 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6866-6875 ◽

Cited By ~ 20

Author(s):

D C Hagen ◽

L Bruhn ◽

C A Westby ◽

G F Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Dna Sequences ◽

Point Mutations ◽

Transcription Activation ◽

Specific Gene ◽

Binding Assays ◽

Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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