Neisseria gonorrhoeae segregate cells lacking Type IV Pilus retractive forces during microcolony development K. B. Eckenrode1,2, I. Spielman1, K. Alzurqa1, C.A, Weber3, W. Poenisch3, V. Zaburdaev3, N. Biais1,2 1Brooklyn College, Brooklyn, NY,  2CUNY Graduate Center, NY, NY,  3Max-Planck-Institute for the Physics of Complex Systems, Dresden, Germany MOTIVATION PILUS AS A FORCE MODEL CELLS WITHOUT FORCE ARE OUTCASTS Neisseria gonorrhoeae (Ng), a human pathogen, colonize urogenital epithelial cells, which produces a threatening infection. During colonization, Neisseria gonorrhea's extracellular protein “sticky” filament named type IV pilus (Tfp) attract gonococci together to assemble microcolonies. The dynamics of microcolony formation are still unclear. Specifically, we are interested in the role of forces at single cell resolution during microcolony formation. Tfp is sufficient to produce loose microcolonies in Neisseria gonorrhoeae; however, to assemble dense microcolonies an ATPase motor named pilT is required. We aim to investigate a heterogeneous mixture of bacteria: 50% with pilT and 50% without pilT. How will a heterogeneous population mix to form an microcolony aggregate? We see spatiotemporal arrangements during bacterial microcolony development that segregates cells lacking retractive force along the perimeter of the colony. This work will advance our knowledge of microcolony architecture and the initial stages of Neisseria gonorrhoeae infection. A C B D We mixed 50% WT (fluorescently labeled tdTomato) and 50% ∆pilT fluorescently labeled GFP) planktonic cells, and after 3 hours we observed the above microcolonies. Our results indicate that cells without pilT ATPase motor proteins are segregated to the outside perimeter of the microcolony. Our model simulation (A) and our microscopy images (B) below. Captured with Nikon Eclipse Ti at 60X magnification. Pilus dynamics have proven to be a robust model for studying how mechanical force influences microorganisms. When pilus retract, they can produce dense microcolonies (A). When pilus cannot retract, the colony morphology is much looser (B) than the WT strain. In previous work, WT pilus bundles were measured to pull at forces greater than 200pN for short (seconds) to long (hours) periods of time (C, D) (Biais, 2008). TYPE IV PILUS DYNAMICS CELLS ACTIVELY DIFFUSE IN MICROCOLONY Planktonic cells Microcolony Movement of 2 fluorescent cells in colony To study how single cells move within a microcolony, we tracked live fluorescent cells within microcolonies to measure the total distance travelled over short time intervals (schematic above). Our model simulation shows cells diffuse in the microcolony because velocity (µm2 / s) decreases as distance (µm) increases, which is representative of diffusion. FUTURE DIRECTIONS Eukaryotes produce varieties of heterogeneous populations during development. By creating spatiotemporal patterns, animals can create complex and evolving body systems. We are interested in creating prokaryotic heterogeneous populations by altering forces from Tfp. This model will allow us to investigate the role of colony development and survival in bacterial heterogeneous populations. D (Diffusion coefficient) www.mechano-micro-biology.org BIAIS LAB WEBSITE v Type IV pilus (Tfp) dynamics play a central role in prokaryotic survival and pathogenesis. Tfp filament construction relies on a set of conserved proteins to orchestrate polymerization and depolymerization of monomers that create an active appendage. The figure above is a simplified version of Tfp dynamics; therefore, many key proteins are not displayed for clarity.