Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.

Detection of biological features at the cellular level with sufficient sensitivity in complex tissue remains a major challenge. To appreciate this challenge, this would require finding tens to hundreds of cells (a 0.1 mm tumor has ~125 cells), out of ~37 trillion cells in the human body. Near-infrared optical imaging holds promise for high-resolution, deep-tissue imaging, but is limited by autofluorescence and scattering. To date, the maximum reported depth using second-window near-infrared (NIR-II: 1000–1700 nm) fluorophores is 3.2 cm through tissue. Here, we design an NIR-II imaging system, “Detection of Optically Luminescent Probes using Hyperspectral and diffuse Imaging in Near-infrared” (DOLPHIN), that resolves these challenges. DOLPHIN achieves the following: (i) resolution of probes through up to 8 cm of tissue phantom; (ii) identification of spectral and scattering signatures of tissues without a priori knowledge of background or autofluorescence; and (iii) 3D reconstruction of live whole animals. Notably, we demonstrate noninvasive real-time tracking of a 0.1 mm-sized fluorophore through the gastrointestinal tract of a living mouse, which is beyond the detection limit of current imaging modalities.

Oligonucleotides hold great promise as a recognition-based biomaterials assembly and disassembly tool. Chemically modified oligonucleotides such as locked nucleic acids (LNA) provide the added advantage of nuclease resistance. In the current study, we focus on programming the assembly and disassembly of LNA-linked colloidal particles as a function of sequence composition. We find that incorporation of LNA residues ($30%) into either one or both primary hybridization partner strands results in a higher duplex density than for isosequential DNA strands. Mismatched primary hybridization partners with sequence length of 11-15 bases have similar initial primary duplex densities. The displacement of mismatched strands by 15 base-long, perfectly matched competitive target strands, however, does depend on the base length of the original mismatched partner strand. Confocal microscopy confirms that substantial colloidal assembly occurs for both perfectly matched and mismatched LNA sequences that are 9 bases in length. Extensive disassembly for the mismatched case is then triggered through the introduction of 15 base-long competitive target strands. Our work demonstrates that LNA can be used to programmatically assemble and disassemble colloidal particles.

The hybridization activity of single-stranded DNA and locked nucleic acid (LNA) sequences on microspheres is quantified in situ using flow cytometry. In contrast to conventional sample preparation for flow cytometry that involves several wash steps for posthybridization analysis, the current work entails directly monitoring hybridization events as they occur between oligonucleotide-functionalized microspheres and fluorescently tagged 9 or 15 base-long targets. We find that the extent of hybridization between single-stranded, immobilized probes and soluble targets generally increases with target sequence length or with the incorporation of LNA nucleotides in one or both oligonucleotide strands involved in duplex formation. The rate constants for duplex formation, on the other hand, remain nearly identical for all but one probe-target sequence combination. The exception to this trend involves the LNA probe and shortest perfectly matched DNA target, which exhibit a rate constant that is an order of magnitude lower than any other probe-target pair, including a mismatched duplex case. Separate studies entailing brief heat treatments to suspensions generally do not consistently yield appreciable differences in associated target densities to probe-functionalized microspheres.