Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

ABSTRACT Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosa in vitro. Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa. Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa. Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

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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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cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

Journal of Bacteriology ◽

10.1128/jb.00135-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 9

Author(s):

Alice Berry ◽

Kook Han ◽

Julian Trouillon ◽

Mylène Robert-Genthon ◽

Michel Ragno ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Transcriptional Activation ◽

Opportunistic Pathogen ◽

Secretion System ◽

Intracellular Camp ◽

Virulence Determinants ◽

Cell Infection ◽

Content Type

ABSTRACT The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

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Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽

10.1099/mic.0.28368-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 143-152 ◽

Cited By ~ 12

Author(s):

Ciara M. Shaver ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Disease Severity ◽

Type Iii Secretion ◽

Disease Process ◽

Effector Proteins ◽

Host Cells ◽

Secreted Proteins ◽

Secretion Systems ◽

Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

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Flagellar Motility Is a Key Determinant of the Magnitude of the Inflammasome Response to Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.00054-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2043-2052 ◽

Cited By ~ 40

Author(s):

Yash R. Patankar ◽

Rustin R. Lovewell ◽

Matthew E. Poynter ◽

Jeevan Jyot ◽

Barbara I. Kazmierczak ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune System ◽

Innate Immune System ◽

Innate Immune ◽

Bacterial Motility ◽

Inflammasome Activation ◽

Flagellar Motility ◽

Content Type

ABSTRACTWe previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronicPseudomonas aeruginosainfections, enables bacteria to evade association and ingestion ofP. aeruginosaby phagocytes bothin vitroandin vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation byP. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotileP. aeruginosa. NonmotileP. aeruginosaelicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cellsin vitro. Importantly, nonmotileP. aeruginosaalso elicits reduced IL-1β levelsin vivoin comparison to those elicited by wild-typeP. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

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Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components

Infection and Immunity ◽

10.1128/iai.00778-16 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 22

Author(s):

Erin E. Zwack ◽

Eric M. Feeley ◽

Amanda R. Burton ◽

Baofeng Hu ◽

Masahiro Yamamoto ◽

...

Keyword(s):

Binding Proteins ◽

Membrane Damage ◽

Effector Proteins ◽

Host Cells ◽

Target Cells ◽

Inflammasome Activation ◽

Secretion Systems ◽

Content Type ◽

Endosomal Membrane ◽

Galectin 3

ABSTRACT Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (Gbp Chr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.

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Condensins are essential for Pseudomonas aeruginosa corneal virulence through their control of phenotypic programs

10.1101/2020.12.08.416081 ◽

2020 ◽

Author(s):

Hang Zhao ◽

April L. Clevenger ◽

Phillip S. Coburn ◽

Michelle C. Callegan ◽

Valentin Rybenkov

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Intracellular Signaling ◽

Primary Metabolism ◽

Essential Factor ◽

Secretion Systems ◽

Biofilm Growth ◽

Structural Chromosome

AbstractPseudomonas aeruginosa is a significant opportunistic pathogen responsible for a variety of human infections. Its high pathogenicity resides in a diverse array of virulence factors and an ability to adapt to hostile environments. We report that these factors are tied to the activity of condensins, SMC and MksBEF, which primarily function in structural chromosome maintenance. This study revealed that both proteins are required for P. aeruginosa virulence during corneal infection. The reduction in virulence was traced to broad changes in gene expression. Transcriptional signatures of smc and mksB mutants were largely dissimilar and non-additive, with the double mutant displaying a distinct gene expression profile. Affected regulons included those responsible for lifestyle control, primary metabolism, surface adhesion and biofilm growth, iron and sulfur assimilation, and denitrification. Additionally, numerous virulence factors were affected, including type 3 and type 6 secretion systems, hemagglutinin, pyocin and macroglobulin production, and a host of virulence regulators. in vitro properties of condensin mutants mirrored their transcriptional profiles. MksB-deficient cells were impaired in pyocyanin, c-di-GMP production, and sessile growth whereas smc mutants mildly upregulated c-di-GMP, secreted fewer proteases and were growth deficient under nutrient-limiting conditions. Moreover, condensin mutants displayed an abnormal regulation upon transition to stationary phase. These data reveal that condensins are integrated into the control of multiple genetic programs related to epigenetic and virulent behavior, establishing condensins as an essential factor in P. aeruginosa ocular infections.Author SummaryBacterial pathogenicity is a complex phenomenon dependent on the ability of a bacterium to thrive in a hostile environment while combating the host using an array of virulence factors. This study reports that pathogenicity is also tied to structural chromosome maintenance through condensins, proteins that are responsible for the global organization of the chromosome. We show that the two Pseudomonas aeruginosa condensins, SMC and MksB, act as global regulators of gene expression. The inactivation of SMC and MksB induces opposite regulatory programs in the cell that resemble those observed during the acute and chronic phases of infection. A substantial portion of this regulation is mediated by the intracellular signaling network of P. aeruginosa. Accordingly, virulence regulation is altered in condensin mutants. The results were validated by genetic, phenotypic and virulence studies of condensin mutants. Overall, these data establish condensins as an essential factor during ocular P. aeruginosa infections revealing their involvement in the regulatory virulence network and the control of the bacterial lifestyle.

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Pseudomonas aeruginosa AmrZ Binds to Four Sites in thealgDPromoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation

Journal of Bacteriology ◽

10.1128/jb.00259-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2673-2681 ◽

Cited By ~ 11

Author(s):

Binjie Xu ◽

Randal J. Soukup ◽

Christopher J. Jones ◽

Richard Fishel ◽

Daniel J. Wozniak

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Transcriptional Activation ◽

Binding Sites ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Upstream Region ◽

Content Type ◽

Alginate Biosynthesis

ABSTRACTDuring late stages of cystic fibrosis pulmonary infections,Pseudomonas aeruginosaoften overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, ourin vitrofluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgDand thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgDtranscription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD.IMPORTANCEOverproduction of the exopolysaccharide alginate provides protection toPseudomonas aeruginosaagainst antimicrobial treatments and is associated with chronicP. aeruginosainfections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely complex promoter.

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No Correlation between Biofilm Formation, Virulence Factors, and Antibiotic Resistance in Pseudomonas aeruginosa: Results from a Laboratory-Based In Vitro Study

Antibiotics ◽

10.3390/antibiotics10091134 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1134

Author(s):

Márió Gajdács ◽

Zoltán Baráth ◽

Krisztina Kárpáti ◽

Dóra Szabó ◽

Donatella Usai ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Virulence Factors ◽

Biofilm Formation ◽

Bacterial Infections ◽

Pearson Correlation ◽

Pigment Production ◽

Virulence Determinants ◽

Secretion Systems

Pseudomonas aeruginosa (P. aeruginosa) possesses a plethora of virulence determinants, including the production of biofilm, pigments, exotoxins, proteases, flagella, and secretion systems. The aim of our present study was to establish the relationship between biofilm-forming capacity, the expression of some important virulence factors, and the multidrug-resistant (MDR) phenotype in P. aeruginosa. A total of three hundred and two (n = 302) isolates were included in this study. Antimicrobial susceptibility testing and phenotypic detection of resistance determinants were carried out; based on these results, isolates were grouped into distinct resistotypes and multiple antibiotic resistance (MAR) indices were calculated. The capacity of isolates to produce biofilm was assessed using a crystal violet microtiter-plate based method. Motility (swimming, swarming, and twitching) and pigment-production (pyoverdine and pyocyanin) were also measured. Pearson correlation coefficients (r) were calculated to determine for antimicrobial resistance, biofilm-formation, and expression of other virulence factors. Resistance rates were the highest for ceftazidime (56.95%; n = 172), levofloxacin (54.97%; n = 166), and ciprofloxacin (54.64%; n = 159), while lowest for colistin (1.66%; n = 5); 44.04% (n = 133) of isolates were classified as MDR. 19.87% (n = 60), 20.86% (n = 63) and 59.27% (n = 179) were classified as weak, moderate, and strong biofilm producers, respectively. With the exception of pyocyanin production (0.371 ± 0.193 vs. non-MDR: 0.319 ± 0.191; p = 0.018), MDR and non-MDR isolates did not show significant differences in expression of virulence factors. Additionally, no relevant correlations were seen between the rate of biofilm formation, pigment production, or motility. Data on interplay between the presence and mechanisms of drug resistance with those of biofilm formation and virulence is crucial to address chronic bacterial infections and to provide strategies for their management.

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Characterization of Pyrin Dephosphorylation and Inflammasome Activation in Macrophages as Triggered by the Yersinia Effectors YopE and YopT

Infection and Immunity ◽

10.1128/iai.00822-18 ◽

2019 ◽

Vol 87 (3) ◽

Cited By ~ 9

Author(s):

Natasha P. Medici ◽

Maheen Rashid ◽

James B. Bliska

Keyword(s):

Immune Responses ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

Effector Proteins ◽

Host Cells ◽

Cell Physiology ◽

Inflammasome Activation ◽

Wild Type ◽

Content Type

ABSTRACT Pathogenic Yersinia species deliver Yop effector proteins through a type III secretion system into host cells. Among these effectors, YopE and YopT are Rho-modifying toxins, which function to modulate host cell physiology and evade immune responses. YopE is a GTPase-activating protein (GAP) while YopT is a protease, and they inhibit RhoA by different modes of action. Modifications to RhoA are sensed by pyrin, which, once activated, assembles a caspase-1 inflammasome, which generates cytokines such as interleukin-1β (IL-1β) and cell death by pyroptosis. In Yersinia-infected macrophages, YopE or YopT triggers inflammasome assembly only in the absence of another effector, YopM, which counteracts pyrin by keeping it inactive. The glucosyltransferase TcdB from Clostridium difficile, a well-studied RhoA-inactivating toxin, triggers activation of murine pyrin by dephosphorylation of Ser205 and Ser241. To determine if YopE or YopT triggers pyrin dephosphorylation, we infected lipopolysaccharide (LPS)-primed murine macrophages with ΔyopM Yersinia pseudotuberculosis strains expressing wild-type (wt) or YopE mutant variants or YopT. By immunoblotting pyrin after infection, we observed that wt YopE triggered dephosphorylation of Ser205 and inflammasome activation. Pyrin dephosphorylation was reduced if a YopE variant had a defect in stability or RhoA specificity but not membrane localization. We also observed that wt YopT triggered pyrin dephosphorylation but more slowly than YopE, suggesting that YopE is dominant in this process. Our findings provide evidence that RhoA-modifying toxins trigger activation of pyrin by a conserved dephosphorylation mechanism. In addition, by characterization of YopE and YopT, we show that different features of effectors, such as RhoA specificity, affect the efficiency of pyrin dephosphorylation.

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Phosphatidylinositol 4,5-Bisphosphate-Dependent Oligomerization of the Pseudomonas aeruginosa Cytotoxin ExoU

Infection and Immunity ◽

10.1128/iai.00402-17 ◽

2017 ◽

Vol 86 (1) ◽

Cited By ~ 3

Author(s):

Angelica Zhang ◽

Jeffrey L. Veesenmeyer ◽

Alan R. Hauser

Keyword(s):

Pseudomonas Aeruginosa ◽

Effector Proteins ◽

Single Amino Acid ◽

Target Cells ◽

Phospholipase Activity ◽

Content Type ◽

Cell Functions ◽

Severe Infections ◽

Membrane Localization Domain

ABSTRACTThePseudomonas aeruginosatype III secretion system delivers effector proteins directly into target cells, allowing the bacterium to modulate host cell functions. ExoU is the most cytotoxic of the known effector proteins and has been associated with more severe infections in humans. ExoU is a patatin-like A2phospholipase requiring the cellular host factors phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and ubiquitin for its activationin vitro. We demonstrated that PI(4,5)P2also induces the oligomerization of ExoU and that this PI(4,5)P2-mediated oligomerization does not require ubiquitin. Single amino acid substitutions in the C-terminal membrane localization domain of ExoU reduced both its activity and its ability to form higher-order complexes in transfected cells andin vitro. Combining inactive truncated ExoU proteins partially restored phospholipase activity and cytotoxicity, indicating that ExoU oligomerization may have functional significance. Our results indicate that PI(4,5)P2induces the oligomerization of ExoU, which may be a mechanism by which this coactivator enhances the phospholipase activity of ExoU.

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