Dalleska, N. | Search bioRxiv

bioRxiv2026

In vivo spatial coordination with synthetic paracrine signaling

The immune system uses paracrine signaling to spatially confine potent responses such as inflammation. A bio-orthogonal synthetic paracrine system could enable engineering of analogous multicellular circuits in which different cell types coordinate their functions in a spatially organized fashion. Here, using the plant hormone auxin as a bio-orthogonal chemical signal, we introduce programmable paracrine circuits that distribute sensing and effector functions to different cell types to spatially restrict responses in mouse xenografts. Cells engineered to express auxin biosynthetic genes generated auxin-dense regions with tunable length scales in vivo. This localized signaling ability enabled design of a multicellular sentinel-effector system, in which THP-1 sentinel cells conditionally produce auxin in regions expressing the tumor-specific antigen EGFRvIII, and Jurkat effector cells respond by locally modulating the activity of a chimeric antigen receptor (CAR). This two-cell type system was able to achieve localized activation of engineered effector cells in vivo. These results establish a foundation for engineering multicellular therapeutic systems that focus responses in specific tissue contexts or disease sites.

Synthetic Biology↗

bioRxiv2025

Phosphorus stress and spatial confinement lower the threshold for quorum-sensing activation of redox-active metabolite production in Pseudomonas synxantha

Bacteria often coordinate collective behaviors such as biofilm formation and secondary metabolite production through quorum sensing (QS), a regulatory system traditionally linked to high cell density. However, in environments like soil, where microbial populations are spatially fragmented, sparse, nutrient-limited, and subject to mass transport, the mechanisms that enable QS-dependent processes remain incompletely understood. Here, we investigate the regulation of a secreted redox-active metabolite, phenazine-1-carboxylic acid (PCA), in Pseudomonas synxantha 2-79, a model rhizobacterium, under phosphorus (P) limitation, a persistent stress in many soils. Using a combination of microscopy and molecular genetic approaches, we show that P limitation sensitizes the QS activation threshold by an order of magnitude, enabling phenazine induction at relatively low population densities in comparison to P replete conditions. This induction is abolished in QS-deficient mutants and restored by the addition of exogenous acyl-homoserine lactone (AHL), demonstrating that quorum sensing remains essential but its threshold becomes environmentally tuned. Under P limitation, spatial confinement and pore saturation levels further shape the timing and location of induction, illustrating how physical structure and nutrient stress can modulate bacterial activities. Moreover, phosphorus stress confers both collaborative and competitive advantages, enabling P. synxantha to undergo low-cell density AHL cross-talk between related Pseudomonas spp. and to suppress other rhizobacteria. Lastly, on plant roots, PCA genes are more predominantly induced under P limitation. These findings illustrate how the nutrient status of an environment can modulate the onset of quorum sensing, enabling quorum-regulated behaviors to activate at lower thresholds.

microbiology↗