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Vibrio Cholerae Use Pili and Flagella Synergistically to Effect Motility

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Vibrio Cholerae Use Pili and Flagella Synergistically to Effect Motility ARTICLE Received 20 Feb 2014 | Accepted 5 Aug 2014 | Published 19 Sep 2014 DOI: 10.1038/ncomms5913 Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment Andrew S. Utada1,*, Rachel R. Bennett2,*, Jiunn C.N. Fong3, Maxsim L. Gibiansky1, Fitnat H. Yildiz3, Ramin Golestanian2 & Gerard C.L. Wong1 We show that Vibrio cholerae, the causative agent of cholera, use their flagella and mannose- sensitive hemagglutinin (MSHA) type IV pili synergistically to switch between two com- plementary motility states that together facilitate surface selection and attachment. Flagellar rotation counter-rotates the cell body, causing MSHA pili to have periodic mechanical contact with the surface for surface-skimming cells. Using tracking algorithms at 5 ms resolution we observe two motility behaviours: ‘roaming’, characterized by meandering trajectories, and ‘orbiting’, characterized by repetitive high-curvature orbits. We develop a hydrodynamic model showing that these phenotypes result from a nonlinear relationship between trajectory shape and frictional forces between pili and the surface: strong pili–surface interactions generate orbiting motion, increasing the local bacterial loiter time. Time-lapse imaging reveals how only orbiting mode cells can attach irreversibly and form microcolonies. These observations suggest that MSHA pili are crucial for surface selection, irreversible attachment, and ultimately microcolony formation. 1 Department of Bioengineering, Department of Chemistry and Biochemistry, California NanoSystems Institute, University of California, Los Angeles, California 90095-1600, USA. 2 Rudolf Peierls Centre for Theoretical Physics, University of Oxford, 1 Keble Road, Oxford OX1 3NP, UK. 3 Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, California 95064, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to F.H.Y. (email: [email protected]) or to R.G. (email: [email protected]) or to G.C.L.W. (email: [email protected]). NATURE COMMUNICATIONS | 5:4913 | DOI: 10.1038/ncomms5913 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5913 ibrio cholerae is a vibroid-shaped gram-negative bacter- attachment correlate with the positions of eventual microcolonies, ium found in coastal and brackish waters, and is the which indicates that purely TFP-driven motility plays a minor Vcausative agent of the diarrhoeal disease cholera1–3. role in determining positions of microcolonies, unlike the case for Seasonal cholera epidemics occur as environmental conditions P. aeruginosa. These observations suggest that MSHA pili–surface become favourable for growth of the native reservoirs of binding is crucial to arresting cell motion during near-surface V. cholerae2,4,5. V. cholerae can attach to the surfaces of phyto- swimming and the transition to irreversible attachment and plankton, zooplankton and crustaceans4,6 as surface-attached microcolony formation. biofilm communities3,4,6–8, which are composed of cells adhered to a surface through extracellular polymeric substances that Results 9,10 consist primarily of polysaccharides and adhesive proteins . Near-surface motility of WT cells. Cells must first swim to a This biofilm lifestyle affords greater protection from environ- surface before they can attach and the hydrodynamics of these 9,11,12 mental variability, predation and antimicrobials . interactions have been studied extensively17–23. Bacteria The model organism Pseudomonas aeruginosa reversibly attach swimming near surfaces experience hydrodynamic forces that 9 to surfaces in a vertical orientation and move along random both attract them towards the surface and cause them to swim in trajectories with type IV pili (TFP)-driven ‘walking motility’ in circular trajectories15,18,20,24,25. Both polytrichous Escherichia coli 13 the early stages of biofilm formation . These cells can progress to and monotrichous vibrios have been observed to swim in an irreversibly attached state where the cell axis is oriented clockwise (CW) circular patterns near surfaces15,18,20,24,26. parallel to the surface. Such horizontal cells can move by TFP- Hydrodynamic models show that a torque on the cell body is driven ‘crawling’ or ‘twitching’ motility, which has much more induced by viscous drag forces felt by the flagellum as it sweeps directional persistence. Recent work has shown that P. aeruginosa past a surface; this surface-induced torque deflects the swimming PAO1 cells interact with a network of Psl polysaccharides direction of cells into curved CW paths20. secreted onto the surface that allows them to self-organise in a V. cholerae are among the fastest bacterial swimmers. They are manner reminiscent of ‘rich-get-richer’ economies, ultimately equipped with a Na þ motor that enables them to swim at speeds 14 resulting in the formation of microcolonies . V. cholerae also use up to 100 mmsÀ 1 (ref. 27). We record bright-field movies using a TFP to engage non-nutritive, abiotic surfaces. Despite having high-speed camera and use cell-tracking algorithms28 (Methods) three different types of TFP, mannose-sensitive hemagglutinin to track and analyze the motion of V. cholerae on glass at (MSHA) pili, virulence-associated toxin co-regulated pili and different times after inoculation. The trajectories of the centroids chitin-regulated pili (ChiRP), V. cholerae do not appear to have a of all WT cells tracked in this period are shown in Fig. 1a. 9,15 twitching surface motility mode and it is unclear how they Tracking all cells in the field of view reveals many cells that form microcolonies. Although V. cholerae lack a twitching mode, appear to travel in tight orbital paths. A large number of mobile it is known that MSHA pili and flagella play important roles in bacteria visit the surface, but relatively few attach irreversibly biofilm development; MSHA pili (DmshA) and flagellar (DflaA) during this initial period. A binary image of the stationary cells mutants require much more time than wild type (WT) to develop taken during the same period and location is shown Fig. 1b. 16 into biofilms in static environments . The details, however, are at To extract the orbital motion evident in the WT ensemble (see present also unknown. Fig. 1a), we limit our search to mobile cells, defined as cells that Here we show that V. cholerae use their polar flagellum and travel a minimum distance of at least one cell length, that are MSHA pili synergistically to scan a surface mechanically before visible for at least 1 s, and have a mean-squared displacement irreversible attachment and microcolony formation. Flagellum (MSD) slope41 (Methods). The MSD as a function of time rotation causes the Vibrio cell body to counter-rotate along its interval, t, is a statistical measure of the average shape of the major axis, which in principle allows MSHA appendages to have trajectories. The MSD(t) of purely diffusive particles generally has periodic mechanical contact with the surface for surface- a slope of 1, while that of purely ballistic motion has a slope of 2. skimming cells. We apply cell-tracking algorithms to high-speed We filter this database of mobile-cell trajectories using a threshold movies of V. cholerae taken at 5 ms resolution to reconstruct the value of the radius of gyration (Rgyr), which is a statistical motility history of every cell that comes within 1 mm of the 29 measure of the spatialP extentÀÁ of an ensemble of points . The Rgyr surface in a 160 mm  120 mm field of view, and observe two 2 is defined as R2 ¼ 1 N ~R À~R , where N is the number of distinct near-surface motility modes: ‘roaming’ motility, which is gyr N i¼1 i cm ~ characterized by long persistence length trajectories, and ‘orbit- points in the track, Ri is the position vector of the ith point on the ing’ motility, which is characterized by near-circular trajectories trajectory, and ~Rcm is the centre-of-mass of all points. For a with well-defined radii. In both motility modes cells move in an perfect circle of radius, r, the Rgyr ¼ r. Rgyr gives accurate oblique direction that deviates strongly from the major cell axis information about the overall extent of the trajectory at the and have strong nutations along the trajectory. These motility longest observation time, whereas the MSD gives information behaviours are ablated in DmshA and DflaA mutants. We develop about displacements between points at fixed time intervals and is a hydrodynamic model to show that the bifurcation into these more suitable for short time lags. Qualitatively, two types of two surface motility phenotypes is a consequence of the highly trajectories are observed: one with tight near-circular orbits with nonlinear dependence of trajectory shape on frictional forces high curvatures (‘orbiting’, Fig. 1c) and one with long directional between MSHA pili and the surface: cells naturally loiter over persistence and small curvatures (‘roaming’, Fig. 1d). In both regions that interact more strongly with MSHA pili, due to cases, the direction of motion seems to be exclusively in the CW orbiting motility. This simple theoretical description agrees sense. From three separate trials, we observe that all cells orbit in remarkably well with the observed trajectories, including the the CW direction (N ¼ 255, 149 and 352). We search for S-shaped distribution of velocities, and the direction
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