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To initiate biofilm formation it is critical for bacteria to sense a surface and respond precisely. Type 4 pili (T4P) have been shown to be important in surface sensing, however, mechanism(s) driving downstream changes important for the switch to biofilm growth have not been clearly defined. Here, using macroscopic bulk assays and single cell tracking analyses ofPseudomonas aeruginosa, we uncover a new role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two hybrid and bimolecular fluorescence complementation assays show that PilO physically interacts with SadC and that the PilO-SadC interaction inhibits SadC’s activity resulting in decreased biofilm formation and increased motility. We show that disrupting the PilO-SadC interaction contributes to greater variation of cyclic-di-GMP levels among cells, thereby increasing cell-to-cell heterogeneity in the levels of this signal. Thus, this work shows thatP. aeruginosauses a component of the T4P scaffold to fine-tune the levels of this nucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.Significance StatementT4P ofP. aeruginosaare important for surface sensing and regulating intracellular cyclic-di-GMP levels. This work identifies a new role for the T4P alignment complex, previously known for its role in supporting pili biogenesis, in surface-dependent signaling. Furthermore, our findings indicate thatP. aeruginosauses a single DGC, via a complex web of protein-protein interactions, to integrate signaling through the T4P and the flagellar motor to fine-tune cyclic-di-GMP levels. A key implication of this work is that more than just regulating signal levels, cells must modulate the dynamic range of cyclic-di-GMP to precisely control the transition to a biofilm lifestyle.

Despite possessing the machinery to sense, adhere to, and proliferate on surfaces, it is commonly observed that bacteria initially have a difficult time attaching to a surface. Before forming a bacterial biofilm, planktonic bacteria exhibit a random period of transient surface attachment known as “reversible attachment” which is poorly understood. Using community tracking methods at single-cell resolution, we examine how reversible attachment progresses during initial stages of surface sensing.Pseudomonas aeruginosastrains PAO1 and PA14, which exhibit similar exponential trends of surface cell population increase, show unanticipated differences when the behavior of each cell was considered at the full lineage level and interpreted using the unifying quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two complementary surface colonization strategies, which are roughly analogous to “immediate-” vs “deferred-gratification” in a prototypical cognitive-affective processing system. PAO1 lineages commit relatively quickly to a surface compared to PA14 lineages. PA14 lineages allow detaching cells to retain memory of the surface so that they are primed for improved subsequent surface attachment. In fact, it is possible to identify motility suppression events in PA14 lineages in the process of surface commitment. We hypothesize that these contrasting strategies are rooted in downstream differences between Wsp-based and Pil-Chp-based surface sensing systems.

Combined immunotherapy with anti-PD-1/PD-L1 and anti-CTLA4 has resulted in superior clinical responses compared to single agent therapy. The underlying mechanisms for this synergy have yet to be elucidated and investigations have largely focused on cellular interactions. Herein, we report a molecular crosstalk in which the PD-1 ligand PD-L1 and the CTLA4 ligand CD80 heterodimerize in cis. This heterodimerization blocks both PD-L1:PD-1 and CD80:CTLA4 interactions, but preserves the ability of CD80 to activate the T cell costimulatory receptor CD28. Remarkably, PD-L1 expression on antigen presenting cells (APCs) protects CD80 from CTLA4 mediated trans-endocytosis, and the therapeutic PD-L1 blockade antibody atezolizumab paradoxically downregulates CD80 on APCs, presumably reducing its co-stimulatory ability. Importantly, this effect can be negated by co-blockade of CTLA4 with ipilimumab. Our study reveals an unexpected immune stimulatory role of cis-acting PD-L1 and a mechanism of anti-PD-L1/anti-CTLA4 crosstalk, providing a therapeutic rationale for combination blockade of PD-L1 and CTLA4.

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition from planktonic to biofilm growth in many bacterial species.Pseudomonas aeruginosahas two surface sensing systems that produce c-di-GMP in response to surface adherence. The current thinking in the field is that once cells attach to a surface, they uniformly respond with elevated c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment,P. aeruginosaengages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.