Rapid Cytoskeletal Response of Epithelial Cells to Force Generation by Type IV Pili

ABSTRACT The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to an untreated monolayer,P. aeruginosa PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1. Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU. Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold. These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1. Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells. These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that P. aeruginosa exoproducts solely determine the steps subsequent to adhesion.

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Faculty Opinions recommendation of CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025856.327456 ◽

2005 ◽

Author(s):

John Atkinson

Keyword(s):

Epithelial Cells ◽

Type Iv Pili ◽

Type Iv ◽

Human Epithelial Cells

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Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells

mBio ◽

10.1128/mbio.02880-18 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 6

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.

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Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics

Journal of Bacteriology ◽

10.1128/jb.00859-16 ◽

2017 ◽

Vol 199 (8) ◽

Cited By ~ 12

Author(s):

Jan Ribbe ◽

Amy E. Baker ◽

Sebastian Euler ◽

George A. O'Toole ◽

Berenike Maier

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Molecular Motor ◽

Type Iv Pili ◽

Force Generation ◽

Surface Attachment ◽

Content Type ◽

Type Iv ◽

Critical Components ◽

Planktonic State

ABSTRACT For Pseudomonas aeruginosa, levels of cyclic di-GMP (c-di-GMP) govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ∼30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility, and exopolysaccharide (EPS) production (specifically, the diguanylate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA) showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides, particularly of Pel, is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that T4P-matrix interactions may be involved in biofilm formation by P. aeruginosa. Finally, our data support the previously proposed model of slingshot-like “twitching” motility of P. aeruginosa. IMPORTANCE Type IV pili (T4P) play various important roles in the transition of bacteria from the planktonic state to the biofilm state, including surface attachment and surface sensing. Here, we investigate adhesion, dynamics, and force generation of T4P after bacteria engage a surface. Our studies showed that two critical components of biofilm formation by Pseudomonas aeruginosa, T4P and exopolysaccharides, contribute to enhanced T4P-mediated force generation by attached bacteria. These data indicate a crucial role for the coordinated impact of multiple biofilm-promoting factors during the early stages of attachment to a surface. Our data are also consistent with a previous model explaining why pilus-mediated motility in P. aeruginosa results in characteristic “twitching” behavior.

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Modulation of Kingella kingae Adherence to Human Epithelial Cells by Type IV Pili, Capsule, and a Novel Trimeric Autotransporter

mBio ◽

10.1128/mbio.00372-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 29

Author(s):

Eric A. Porsch ◽

Thomas E. Kehl-Fie ◽

Joseph W. St. Geme

Keyword(s):

Epithelial Cells ◽

Young Children ◽

Type Iv Pili ◽

Upper Respiratory Tract ◽

Respiratory Epithelial Cells ◽

Kingella Kingae ◽

Content Type ◽

Type Iv ◽

Human Epithelial Cells ◽

Respiratory Epithelial

ABSTRACTKingella kingaeis an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. Colonization of the posterior pharynx is a key step in the pathogenesis ofK. kingaedisease. Previous work established that type IV pili are necessary forK. kingaeadherence to the respiratory epithelium. In this study, we set out to identify additional factors that influenceK. kingaeinteractions with human epithelial cells. We found that genetic disruption of the gene encoding a predicted trimeric autotransporter protein called Knh (KingellaNhhAhomolog) resulted in reduced adherence to human epithelial cells. In addition, we established thatK. kingaeelaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon calledctrABCDfor surface presentation. Furthermore, we discovered that the presence of a surface capsule interferes with Knh-mediated adherence to human epithelial cells by nonpiliated organisms and that maximal adherence in the presence of a capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. On the basis of the data presented here, we propose a novel adherence mechanism that allowsK. kingaeto adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance.IMPORTANCEKingella kingaeis a Gram-negative bacterium that is being recognized increasingly as a cause of joint and bone infections in young children. The pathogenesis of disease due toK. kingaebegins with bacterial colonization of the upper respiratory tract, and previous work established that surface hair-like fibers called type IV pili are necessary forK. kingaeadherence to respiratory epithelial cells. In this study, we set out to identify additional factors that influenceK. kingaeinteractions with respiratory epithelial cells. We discovered a novel surface protein called Knh that mediatesK. kingaeadherence and found that a surface-associated carbohydrate capsule interferes with the Knh-mediated adherence of organisms lacking pili. Further analysis revealed that pilus retraction is necessary for maximal Knh-mediated adherence in the presence of the capsule. Our results may lead to new strategies to prevent disease due toK. kingaeand potentially other pathogenic bacteria.

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Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells

Molecular Microbiology ◽

10.1046/j.1365-2958.1999.01459.x ◽

1999 ◽

Vol 32 (6) ◽

pp. 1316-1332 ◽

Cited By ~ 142

Author(s):

Alexey J. Merz ◽

Caroline A. Enns ◽

Magdalene So

Keyword(s):

Epithelial Cells ◽

Plaque Formation ◽

Type Iv Pili ◽

Type Iv

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Type IV Pili PromoteClostridium difficileAdherence and Persistence in a Mouse Model of Infection

Infection and Immunity ◽

10.1128/iai.00943-17 ◽

2018 ◽

Vol 86 (5) ◽

Cited By ~ 20

Author(s):

Robert W. McKee ◽

Naira Aleksanyan ◽

Elizabeth M. Garrett ◽

Rita Tamayo

Keyword(s):

Epithelial Cells ◽

Mouse Model ◽

Type Iv Pili ◽

Host Cells ◽

Content Type ◽

Type Iv ◽

Flagellum Biosynthesis ◽

Mouse Model Of Infection ◽

Null Mutants

ABSTRACTCyclic diguanylate (c-di-GMP) is a second messenger that regulates the transition from motile to sessile lifestyles in numerous bacteria and controls virulence factor production in a variety of pathogens. InClostridium difficile, c-di-GMP negatively regulates flagellum biosynthesis and swimming motility and promotes the production of type IV pili (TFP), biofilm formation, and surface motilityin vitro. Flagella have been identified as colonization factors inC. difficile, but the role of TFP in adherence to host cells and in colonization of the mammalian gut is unknown. Here we show that c-di-GMP promotes adherence to epithelial cellsin vitro, which can be partly attributed to the loss of flagella. Using TFP-null mutants, we demonstrate that adherence to epithelial cells is partially mediated by TFP and that this TFP-mediated adherence requires c-di-GMP regulation. In a mouse model of colonization, the TFP-null mutants initially colonized the intestine as well as the parental strain but were cleared more quickly. Moreover, compared to the parent strain,C. difficilestrains lacking TFP were particularly deficient in association with the cecal mucosa. Together these data indicate that TFP and their positive regulation by c-di-GMP promote attachment ofC. difficileto the intestinal epithelium and contribute to persistence ofC. difficilein the host intestine.

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