HETEROGENEITY OF INDIVIDUAL ADHESION BEHAVIOR DICTATES THE PROPERTIES OF THE EMERGENT MULTICELLULAR BACTERIAL STRUCTURE

CAMILA GENSKOWSKY; FERNANDO PERUANI; DARÍO CAPASSO; ARLINET KIERBEL; MARÍA VICTORIA PEPE; ADRIANA JAGER

Congreso; Congreso Conjunto SAIB-SAMIGE 2020; 2020

Formation of multicellular structures is an essential feature in the physiopathology of many disease-causing bacteria. This process involves a major change in bacterial behavior, i.e. the transition from a free-swimming, individual way of life to a multicellular, attached, and sessile lifestyle. However, little is known about this transition, especially when it takes place on biotic surfaces. This probably lies in the challenge to examine the dynamics of single cells on a very short timescale during the switch between these two states. In Pseudomonas aeruginosa interaction with the epithelial barrier, this transition requires the presence of an extruded apoptotic cell, where bacteria attach, as previously shown1. The apoptotic cell becomes the seed of a rounded bacterial aggregate whose size remains stable over several hours. Initial bacterial adhesion dynamics is strongly influenced by the properties of the substrate as well as by the orientation in which bacteria interact to it. The plasmatic membrane of apoptotic cells suffers dramatic changes as the apoptotic process evolves. Through confocal and electron microscopy studies, we found P. aeruginosa attaches to profuse vesiculated membranes where bacteria fit between surface protuberances. In addition, we determined that bacteria attach vertically, by the pole opposite the flagellum. To follow P. aeruginosa-aggregate formation dynamics, we used time-lapse confocal microscopy. MDCK cells were grown on micro-dishes with a cover glass bottom. Stack dimensions were set up throughout previously labeled extruded apoptotic cells. Samples were infected and immediately after image acquisition was started. This approach allowed to register aggregate formation in three dimensions plus time and tracking bacterial attachment at the single-cell level. We observed that once bacteria reach the apoptotic cell, remain in contact with cell membrane for a time period, to finally detaching and swimming away. To quantify this process, we measure the time each bacterium remains in contact with the cell membrane and refer to it as residence time. We found that the residence time distribution exhibits two distinct behaviors. To rationalize this finding, we derived a Markovian model with n states, and showed that the theoretical prediction for n: 2, corresponding to on-membrane and free-swimming bacterial states, is not consistent with the empirical data. However, the obtained empirical distribution is consistent with n: 3, a result that unveils the existence of two on-membrane states. By applying the Markovian model to experiments performed with a non-piliated mutant and by comparing with WT data, we revealed that one on-membrane state involves Type-IV pili attachment. Furthermore, according to the developed Markovian model, formed aggregates are not static, but dynamical structures involving a constant exchange of bacteria, a result confirmed by a series of independent experiments. 1 Capasso et al. PlosPathogen 2016