scholarly journals Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa

2002 ◽  
Vol 184 (13) ◽  
pp. 3605-3613 ◽  
Author(s):  
Scott A. Beatson ◽  
Cynthia B. Whitchurch ◽  
Jennifer L. Sargent ◽  
Roger C. Levesque ◽  
John S. Mattick
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv ◽  
Allelic Deletion ◽  
Pyocyanin Production ◽  
Null Mutants

ABSTRACT Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

scholarly journals Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

2016 ◽  
Vol 12 ◽  
pp. 1428-1433 ◽  
Author(s):  
Bernardas Morkunas ◽  
Balint Gal ◽  
Warren R J D Galloway ◽  
James T Hodgkinson ◽  
Brett M Ibbeson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Cell Signalling ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Wide Range ◽  
Potential Applications ◽  
Therapeutic Context ◽  
Pyocyanin Production

Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

scholarly journals Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa

2007 ◽  
Vol 56 (6) ◽  
pp. 738-748 ◽  
Author(s):  
J. Andy Schaber ◽  
Adrienne Hammond ◽  
Nancy L. Carty ◽  
Simon C. Williams ◽  
Jane A. Colmer-Hamood ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Confocal Laser ◽  
Post Inoculation ◽  
Twitching Motility ◽  
Type Iv ◽  
Continuous Culture System

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.

scholarly journals Influence of Quorum Sensing and Iron on Twitching Motility and Biofilm Formation in Pseudomonas aeruginosa

2007 ◽  
Vol 190 (2) ◽  
pp. 662-671 ◽  
Author(s):  
Glenn M. Patriquin ◽  
Ehud Banin ◽  
Christie Gilmour ◽  
Rivka Tuchman ◽  
E. Peter Greenberg ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Minimal Medium ◽  
Homoserine Lactone ◽  
Planktonic Cell ◽  
Growth Yields ◽  
Twitching Motility ◽  
Wild Type ◽  
Biofilm Growth

ABSTRACT Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at FeCl3 concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of FeCl3 that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhlI, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhlI mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.

scholarly journals Pseudomonas aeruginosa Exhibits Directed Twitching Motility Up Phosphatidylethanolamine Gradients

2001 ◽  
Vol 183 (2) ◽  
pp. 763-767 ◽  
Author(s):  
Daniel B. Kearns ◽  
Jayne Robinson ◽  
Lawrence J. Shimkets
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Solid Surfaces ◽  
Twitching Motility ◽  
Wild Type ◽  
Directed Movement ◽  
Endogenous Factors ◽  
Type Iv ◽  
Cell Velocity ◽  
Dependent Form

ABSTRACT Pseudomonas aeruginosa translocates over solid surfaces by a type IV pilus-dependent form of multicellular motility known as twitching. We wondered whether cells utilize endogenous factors to organize twitching, and we purified from wild-type cells a lipid that caused directed movement. Wild-type P. aeruginosa, but not a pilJ pilus-deficient mutant, showed biased movement up gradients of phosphatidylethanolamine (PE) established in agar. Activity was related to the fatty acid composition of the lipid, as two synthetic PE species, dilauroyl and dioleoyl PE, were capable of directing P. aeruginosa motility while many other species were inactive. P. aeruginosa PE did not contain either laurate or oleate, implying that the native attractant species contains different fatty acids. Uniform concentrations of PE increased cell velocity, suggesting that chemokinesis may be at least partly responsible for directed movement. We speculate that PE-directed twitching motility may be involved in biofilm formation and pathogenesis.

scholarly journals Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Vincent Nieto ◽  
Abby R. Kroken ◽  
Melinda R. Grosser ◽  
Benjamin E. Smith ◽  
Matteo M. E. Metruccio ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Host Cell ◽  
Type Iv Pili ◽  
Fluorescent Imaging ◽  
Bacterial Motility ◽  
Twitching Motility ◽  
Wild Type ◽  
Content Type ◽  
Type Iv

ABSTRACT Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s−1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa. Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.

scholarly journals tonB3 Is Required for Normal Twitching Motility and Extracellular Assembly of Type IV Pili

2004 ◽  
Vol 186 (13) ◽  
pp. 4387-4389 ◽  
Author(s):  
Bixing Huang ◽  
Kelin Ru ◽  
Zheng Yuan ◽  
Cynthia B. Whitchurch ◽  
John S. Mattick
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iv Pili ◽  
Twitching Motility ◽  
Wild Type ◽  
Type Iv

ABSTRACT Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

scholarly journals Correlation of quorum sensing and virulence factors in Pseudomonas aeruginosa isolates in Egypt

2015 ◽  
Vol 9 (10) ◽  
pp. 1091-1099 ◽  
Author(s):  
Hamida M. Aboushleib ◽  
Hoda M. Omar ◽  
Rania Abozahra ◽  
Amel Elsheredy ◽  
Kholoud Baraka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Point Mutations ◽  
Inhibitory Effect ◽  
Clinical Isolates ◽  
Clove Oil ◽  
Twitching Motility ◽  
Pyocyanin Production

Introduction: Pseudomonas aeruginosa is one of the most virulent nosocomial pathogens worldwide. Quorum sensing (QS) regulates the production of pathogenic virulence factors and biofilm formation in P. aeruginosa. The four genes lasR, lasI, rhlR,and rhlI were found to regulate this QS system. In this study, we aimed to assess the correlation between these four genes and QS-dependent virulence factors and to detect the inhibitory effect of clove oil on QS. Methodology: Fifty P. aeruginosa clinical isolates were collected. Susceptibility to different antibiotics was tested. Virulence factors including biofilm formation, pyocyanin production, and twitching motility were phenotypically detected. QS genes were amplified by polymerase chain reaction (PCR), and one strain subsequently underwent sequencing. The inhibitory effect of clove oil on virulence factors was also tested. Results: A positive correlation was found between biofilm formation and the presence of lasR and rhlI genes. Twitching motility was positively correlated with the presence of lasR, lasI, and rhlI genes. On the other hand, no correlation was found between pyocyanin production and any of the studied genes. Only one isolate amplified all the tested QS gene primers, but it did not express any of the tested virulence factors phenotypically. Sequence analyses of this isolate showed that the four genes had point mutations. Conclusions: Results emphasize the importance of QS in P. aeruginosa virulence; however, QS-deficient clinical isolates occur and are still capable of causing clinical infections in humans. Also, clove oil has an obvious inhibitory effect on QS, which should be clinically exploited.

scholarly journals Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants

2003 ◽  
Vol 48 (6) ◽  
pp. 1511-1524 ◽  
Author(s):  
Mikkel Klausen ◽  
Arne Heydorn ◽  
Paula Ragas ◽  
Lotte Lambertsen ◽  
Anders Aaes-Jørgensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Type Iv Pili ◽  
Wild Type ◽  
Type Iv

scholarly journals Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

2005 ◽  
Vol 187 (3) ◽  
pp. 829-839 ◽  
Author(s):  
Poney Chiang ◽  
Marc Habash ◽  
Lori L. Burrows
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Distribution Patterns ◽  
Opportunistic Pathogen ◽  
Twitching Motility ◽  
Polar Localization ◽  
Type Iv ◽  
And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

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