Engineering nanowires in bacteria to elucidate electron transport structural-functional relationships.

Bacterial pilin nanowires are protein complexes, suggested to possess electroactive capabilities forming part of the cells' bioenergetic programming. Their role is thought to be linked to facilitating electron transfer between cells and the external environment to permit metabolism and cell-to-cell communication. There is a significant debate, with varying hypotheses as to the nature of the proteins currently lying between type-IV pilin-based nanowires and polymerised cytochrome-based filaments. Importantly, to date, there is a very limited structure-function analysis of these structures within whole bacteria. In this work, we engineered Cupriavidus necator H16, a model autotrophic organism to express differing aromatic modifications of type-IV pilus proteins to establish structure-function relationships on conductivity and the effects this has on pili structure. This was achieved via a combination of high-resolution PeakForce tunnelling atomic force microscopy (PeakForce TUNA™) technology, alongside conventional electrochemical approaches enabling the elucidation of conductive nanowires emanating from whole bacterial cells. This work is the first example of functional type-IV pili protein nanowires produced under aerobic conditions using a Cupriavidus necator chassis. This work has far-reaching consequences in understanding the basis of bio-electrical communication between cells and with their external environment.

Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria.

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.

Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer.

The use of therapeutic monoclonal antibodies (mAbs) has revolutionized cancer treatment. The conjugation of mAbs to nanoparticles has been broadly exploited to improve the targeting efficiency of drug nanocarriers taking advantage of high binding efficacy and target selectivity of antibodies for specific cell receptors. However, the therapeutic implications of nanoconjugation have been poorly considered. In this study, half-chain fragments of the anti-EGFR mAb cetuximab were conjugated to colloidal nanoparticles originating stable nanoconjugates that were investigated as surrogates of therapeutic mAbs in triple negative breast cancer (TNBC). Three TNBC cell lines were selected according to EGFR expression, which regulates activation of MAPK/ERK and PI3K/Akt pathways, and to distinctive molecular profiling including KRAS, PTEN, and BRCA1 mutations normally associated with diverse sensitivity to treatment with cetuximab. The molecular mechanisms of action of nanoconjugated half-chain mAb, including cell targeting, interference with downstream signaling pathways, proliferation, cell cycle, and apoptosis, along with triggering of ADCC response, were investigated in detail in sensitive and resistant TNBC cells. We found that half-chain mAb nanoconjugation was able to enhance the therapeutic efficacy and improve the target selectivity against sensitive, but unexpectedly also resistant, TNBC cells. Viability assays and signaling transduction modulation suggested a role of BRCA1 mutation in TNBC resistance to cetuximab alone, whereas its effect could be circumvented using half-chain cetuximab nanoconjugates, suggesting that nanoconjugation not only improved the antibody activity but also exerted different mechanisms of action. Our results provide robust evidence of the potential of half-chain antibody nanoconjugates in the treatment of TNBC, which could offer a new paradigm for therapeutic antibody administration, potentially allowing improved curative efficiency and reduced minimal effective dosages in both sensitive and resistant tumors.