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Rapid cytoskeletal response of epithelial cells to force generation by type IV pili.

ABSTRACT: Many bacterial pathogens interfere with cellular functions including phagocytosis and barrier integrity. The human pathogen Neissieria gonorrhoeae generates grappling hooks for adhesion, spreading, and induction of signal cascades that lead to formation cortical plaques containing f-actin and ezrin. It is unclear whether high mechanical forces generated by type IV pili (T4P) are a direct signal that leads to cytoskeletal rearrangements and at which time scale the cytoskeletal response occurs. Here we used laser tweezers to mimic type IV pilus mediated force generation by T4P-coated beads on the order of 100 pN. We found that actin-EGFP and ezrin-EGFP accumulated below pilus-coated beads when force was applied. Within 2 min, accumulation significantly exceeded controls without force or without pili, demonstrating that T4P-generated force rapidly induces accumulation of plaque proteins. This finding adds mechanical force to the many strategies by which bacteria modulate the host cell cytoskeleton.

SUBMITTER: Opitz D

PROVIDER: S-EPMC3038865 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Rapid cytoskeletal response of epithelial cells to force generation by type IV pili.

Opitz Dirk D Maier Berenike B

PloS one 20110214 2

Many bacterial pathogens interfere with cellular functions including phagocytosis and barrier integrity. The human pathogen Neissieria gonorrhoeae generates grappling hooks for adhesion, spreading, and induction of signal cascades that lead to formation cortical plaques containing f-actin and ezrin. It is unclear whether high mechanical forces generated by type IV pili (T4P) are a direct signal that leads to cytoskeletal rearrangements and at which time scale the cytoskeletal response occurs. He ...[more]

PMID: 21340023

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Project description:UNLABELLED:Kingella kingae is 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 of K. kingae disease. Previous work established that type IV pili are necessary for K. kingae adherence to the respiratory epithelium. In this study, we set out to identify additional factors that influence K. kingae interactions with human epithelial cells. We found that genetic disruption of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, we established that K. kingae elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called ctrABCD for 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 allows K. kingae to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. IMPORTANCE:Kingella kingae is 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 to K. kingae begins with bacterial colonization of the upper respiratory tract, and previous work established that surface hair-like fibers called type IV pili are necessary for K. kingae adherence to respiratory epithelial cells. In this study, we set out to identify additional factors that influence K. kingae interactions with respiratory epithelial cells. We discovered a novel surface protein called Knh that mediates K. kingae adherence 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 to K. kingae and potentially other pathogenic bacteria.

Project description:The rare actinomycete Actinoplanes missouriensis produces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili in A. missouriensis The pilus gene (pil) cluster, consisting of three apparently ?FliA-dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6?±?3 pili per zoospore (n?=?37 piliated zoospores) and a length of 0.62?±?0.35??m (n?=?206), via observation of fliC-deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encoding pilA deletion (?pilA) mutant. In addition, the deletion of pilT, which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and ?pilA zoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve the pil cluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth.IMPORTANCE Bacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomycete Actinoplanes missouriensis Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore.

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Rapid cytoskeletal response of epithelial cells to force generation by type IV pili.

Publications

Rapid cytoskeletal response of epithelial cells to force generation by type IV pili.

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