Interplay of flagellar motility and mucin degradation stimulates the uassociation of Pseudomonas aeruginosa with human epithelial colorectal adenocarcinoma (Caco-2) cells

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

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c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility

eLife ◽

10.7554/elife.01402 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 68

Author(s):

Bridget R Kulasekara ◽

Cassandra Kamischke ◽

Hemantha D Kulasekara ◽

Matthias Christen ◽

Paul A Wiggins ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Division ◽

Histidine Kinase ◽

Molecular Basis ◽

Individual Cell ◽

Cell Heterogeneity ◽

Flagellar Motility ◽

Bacterial Populations ◽

Phosphorylation State ◽

Asymmetric Partitioning

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors.

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Extracellular Signals of a Human Epithelial Colorectal Adenocarcinoma (Caco-2) Cell Line Facilitate the Penetration of Pseudomonas aeruginosa PAO1 Strain through the Mucin Layer

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2017.00415 ◽

2017 ◽

Vol 7 ◽

Cited By ~ 4

Author(s):

Naoki Hayashi ◽

Atsushi Yokotani ◽

Masami Yamamoto ◽

Mariko Kososhi ◽

Mayu Morita ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Line ◽

Colorectal Adenocarcinoma ◽

Pseudomonas Aeruginosa Pao1 ◽

Extracellular Signals ◽

Mucin Layer ◽

Pao1 Strain

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Ethanol decreases Pseudomonas aeruginosa flagellar motility through a cyclic-di-GMP- and stator-dependent pathway

10.1101/430884 ◽

2018 ◽

Author(s):

Kimberley A. Lewis ◽

Amy E. Baker ◽

Annie I. Chen ◽

Colleen E. Harty ◽

Sherry L. Kuchma ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Microscopic Analysis ◽

Fold Increase ◽

Bioactive Metabolites ◽

Flagellar Motility ◽

Planktonic Cells ◽

Motile Cells ◽

Diguanylate Cyclases ◽

Surface Sensing ◽

In The Wild

AbstractPseudomonas aeruginosa frequently encounters microbes that produce bioactive metabolites including ethanol. At concentrations that do not affect growth, we found that ethanol reduces P. aeruginosa motility by 30% in a swim agar assay and this decrease is accompanied by a 2.5-fold increase in levels of cyclic diguanylate (c-di-GMP), a second messenger that represses motility, in planktonic cells. A screen of mutants lacking genes involved in c-di-GMP metabolism identified SadC and GcbA as diguanylate cyclases involved in swim zone reduction by ethanol and ethanol-induced c-di-GMP production. The reduction of swimming in response to ethanol also required the stator set, MotAB, two PilZ-domain proteins (FlgZ and PilZ), PilY1-a proposed surface-sensing protein, and PilMNOP, which comprises the pilus alignment complex and these proteins have been previously implicated in the control of motility on agar surfaces. Microscopic analysis of the fraction of quiescent cells in swim medium showed that ethanol decreased the portion of motile cells in the wild type, but had opposite effects in the ∆pilY1, ∆pilMNOP, ∆motAB, and ∆pilZ∆flgZ mutants. Together, these data indicate ethanol induces a regulated change in motility in planktonic cells at concentrations similar to those produced by other microbes. We propose that this ethanol-responsiveness may contribute to the co-localization of P. aeruginosa with ethanol-producing microbes.

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Ethanol Decreases Pseudomonas aeruginosa Flagellar Motility through the Regulation of Flagellar Stators

Journal of Bacteriology ◽

10.1128/jb.00285-19 ◽

2019 ◽

Vol 201 (18) ◽

Cited By ~ 6

Author(s):

Kimberley A. Lewis ◽

Amy E. Baker ◽

Annie I. Chen ◽

Colleen E. Harty ◽

Sherry L. Kuchma ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Single Cells ◽

Microscopic Analysis ◽

Soft Agar ◽

Biologically Active ◽

Time Interval ◽

Flagellar Motility ◽

Content Type ◽

Swimming Motility ◽

Potential Marker

ABSTRACT Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol. IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

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Role of Motility and Flagellin Glycosylation in the Pathogenesis of Pseudomonas aeruginosa Burn Wound Infections

Infection and Immunity ◽

10.1128/iai.73.7.4395-4398.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4395-4398 ◽

Cited By ~ 110

Author(s):

Shiwani K. Arora ◽

Alice N. Neely ◽

Barbara Blair ◽

Stephen Lory ◽

Reuben Ramphal

Keyword(s):

Pseudomonas Aeruginosa ◽

Mouse Model ◽

Wound Infections ◽

Flagellar Motility ◽

Burn Wound ◽

First Report ◽

Flagellin Glycosylation ◽

Mouse Model Of Infection

ABSTRACT In this study, we tested the contribution of flagellar motility, flagellin structure, and its glycosylation in Pseudomonas aeruginosa using genetically defined flagellar mutants. All mutants and their parent strains were tested in a burned-mouse model of infection. Motility and glycosylation of the flagellum appear to be important determinants of flagellar-mediated virulence in this model. This is the first report where genetically defined flagellar variants of P. aeruginosa were tested in the burned-mouse model of infection.

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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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Flagellar stators stimulate c-di-GMP production by Pseudomonas aeruginosa

10.1101/483339 ◽

2018 ◽

Cited By ~ 1

Author(s):

Amy E. Baker ◽

Shanice S. Webster ◽

Andreas Diepold ◽

Sherry L. Kuchma ◽

Eric Bordeleau ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Feedback Loop ◽

Bacterial Cells ◽

Diguanylate Cyclase ◽

Flagellar Motility ◽

Swarming Motility ◽

Surface Attachment ◽

Bidirectional Interactions ◽

Gmp Production

AbstractFlagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in Pseudomonas aeruginosa and other microbes is cyclic diguanylate (c-di-GMP). High levels of this second messenger repress motility and stimulate biofilm formation. C-di-GMP levels regulate motility in P. aeruginosa in part by influencing the localization of its two flagellar stator sets, MotAB and MotCD. Here we show that just as c-di-GMP can influence the stators, stators can impact c-di-GMP levels. We demonstrate that the swarming motility-driving stator MotC physically interacts with the transmembrane region of the diguanylate cyclase SadC. Furthermore, we demonstrate that this interaction is capable of stimulating SadC activity. We propose a model by which the MotCD stator set interacts with SadC to stimulate c-di-GMP production in conditions not permissive to motility. This regulation implies a positive feedback loop in which c-di-GMP signaling events cause MotCD stators to disengage from the motor; then disengaged stators stimulate c-di-GMP production to reinforce a biofilm mode of growth. Our studies help define the bidirectional interactions between c-di-GMP and the motility machinery.Importance.The ability of bacterial cells to control motility during early steps in biofilm formation is critical for the transition to a non-motile, biofilm lifestyle. Recent studies have clearly demonstrated the ability of c-di-GMP to control motility via a number of mechanisms, including through controlling transcription of motility-related genes and modulating motor function. Here we provide evidence that motor components can in turn impact c-di-GMP levels. We propose that communication between motor components and c-di-GMP synthesis machinery allows the cell to have a robust and sensitive switching mechanism to control motility during early events in biofilm formation.

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