Tuning cellular response by modular design of bioactive domains in collagen.

Collagen's ability to direct cellular behavior suggests that redesigning it at the molecular level could enable manipulation of cells residing in an engineered microenvironment. However, the fabrication of full-length collagen mimics of specified sequence de novo has been elusive, and applications still rely on material from native tissues. Using a bottom-up strategy, we synthesized modular genes and expressed recombinant human collagen variants in Saccharomyces cerevisiae. The resulting biopolymers contained prescribed cell-interaction sites that can direct and tune cellular responses, with retention of the important triple-helical self-assembled structure. Removal of the native integrin-binding sites GROGER, GAOGER, GLOGEN, GLKGEN, and GMOGER in human collagen III yielded collagen that did not support adhesion of mammalian cells. Introduction of GFOGER sequences to this scaffold at specified locations and densities resulted in varying degrees of cellular attachment. The recruitment of focal adhesion complexes on the different collagens ranged from a 96% reduction to a 56% increase over native collagen I. Adhesion to the GFOGER-containing variants was entirely dependent and partially dependent on the ß1 and α2 subunits of integrin, respectively, with cell adhesion on average reduced by 86% with anti-ß1 and 38% with anti-α2 integrin antibody incubation. Results support the importance of local context in collagen-cell interactions. The investigation demonstrates the flexibility of this approach to introduce targeted changes throughout the collagen polymer for producing fully-prescribed variants with tailored properties.

Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production.

Among melons, cantaloupes are most frequently implicated in outbreaks and surveillance-based recalls due to Salmonella enterica. There is limited but compelling evidence that associates irrigation water quality as a significant risk of preharvest contamination of melons. However, the potential for root uptake from water and soil and subsequent systemic transport of Salmonella into melon fruit is uncharacterized. The aim of this work was to determine whether root uptake of S. enterica results in systemic transport to fruit at high doses of applied inoculum through sub-surface drip and furrow irrigation during field production of melons. Cantaloupe and honeydew were grown under field conditions, in a silt clay loam soil using standard agronomic practices for California. An attenuated S. enterica sv. Typhimurium strain was applied during furrow irrigation and, in separate plots, buried drip-emitter lines delivered the inoculum directly into the established root zone. Contamination of the water resulted in soil contamination within furrows however Salmonella was not detected on top of the beds or around melon roots of furrow-irrigated rows demonstrating absence of detectable lateral transfer across the soil profile. In contrast, positive detection of the applied isolate occurred in soil and the rhizosphere in drip injected plots; survival of Salmonella was at least 41 days. Despite high populations of the applied bacteria in the rhizosphere, after surface disinfection, internalized Salmonella was not detected in mature melon fruit (n=485). Contamination of the applied Salmonella was detected on the rind surface of melons if fruit developed in contact with soil on the sides of the inoculated furrows. Following an unusual and heavy rain event during fruit maturation, melons collected from the central area of the beds, were shown to harbor the furrow-applied Salmonella. Delivery of Salmonella directly into the peduncle, after minor puncture wounding, resulted in detection of applied Salmonella in the sub-rind tissue below the fruit abscission zone. Results indicate that Salmonella internalization from soil and vascular systemic transport to fruit is unlikely to occur from irrigation water in CA production regions, even if substantially above normal presumptive levels of contamination. Although contaminated irrigation water and subsequently soil in contact with fruit remains a concern for contamination of the external rind, results suggest an acceptable microbial indicator threshold and critical limit for the presence of Salmonella in applied water may be possible by defining appropriate microbiological standards for melon irrigation in California and regions with similar climate, soil texture, and crop management practices.

Recombinant human collagen and biomimetic variants using a de novo gene optimized for modular assembly.

A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy that yields full-length human collagen III and specifically defined variants. We used a computational algorithm that applies codon degeneracy to design oligonucleotides that favor correct hybridizations while disrupting incorrect ones for gene synthesis. The resulting recombinant polymers were expressed in Saccharomyces cerevisiae engineered with prolyl-4-hydroxylase. Our modular approach enabled mixing-and-matching domains to fabricate different combinations of collagen variants that contained different secretion signals at the N-terminus and cysteine residues imbedded within the triple-helical domain at precisely defined locations. This work shows the flexibility of our strategy for designing and assembling specifically tailored biomimetic collagen polymers with re-engineered properties.