Robert James Citorik

Bacteriophage-based synthetic biology for the study of infectious diseases

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome.

Microbiome therapeutics — Advances and challenges☆

The microbial community that lives on and in the human body exerts a major impact on human health, from metabolism to immunity. In order to leverage the close associations between microbes and their host, development of therapeutics targeting the microbiota has surged in recent years. Here, we discuss current additive and subtractive strategies to manipulate the microbiota, focusing on bacteria engineered to produce therapeutic payloads, consortia of natural organisms and selective antimicrobials. Further, we present challenges faced by the community in the development of microbiome therapeutics, including designing microbial therapies that are adapted for specific geographies in the body, stable colonization with microbial therapies, discovery of clinically relevant biosensors, robustness of engineered synthetic gene circuits and addressing safety and biocontainment concerns. Moving forward, collaboration between basic and applied researchers and clinicians to address these challenges will poise the field to herald an age of next-generation, cellular therapies that draw on novel findings in basic research to inform directed augmentation of the human microbiota.

LPLUNC1 Modulates Innate Immune Responses to Vibrio cholerae

BACKGROUND Recent studies demonstrate that long palate, lung, and nasal epithelium clone 1 protein (LPLUNC1) is involved in immune responses to Vibrio cholerae, and that variations in the LPLUNC1 promoter influence susceptibility to severe cholera in humans. However, no functional role for LPLUNC1 has been identified. METHODS We investigated the role of LPLUNC1 in immune responses to V. cholerae, assessing its affect on bacterial growth and killing and on innate inflammatory responses to bacterial outer membrane components, including purified lipopolysaccharide (LPS) and outer membrane vesicles. We performed immunostaining for LPLUNC1 in duodenal biopsies from cholera patients and uninfected controls. RESULTS LPLUNC1 decreased proinflammatory innate immune responses to V. cholerae and Escherichia coli LPS. The effect of LPLUNC1 was dose-dependent and occurred in a TLR4-dependent manner. LPLUNC1 did not affect lipoprotein-mediated TLR2 activation. Immunostaining demonstrated expression of LPLUNC1 in Paneth cells in cholera patients and controls. CONCLUSIONS Our results demonstrate that LPLUNC1 is expressed in Paneth cells and likely plays a role in modulating host inflammatory responses to V. cholerae infection. Attenuation of innate immune responses to LPS by LPLUNC1 may have implications for the maintenance of immune homeostasis in the intestine.

A Systems Biology Approach To Modeling Vibrio cholerae Gene Expression under Virulence-Inducing Conditions

Vibrio cholerae is a Gram-negative bacillus that is the causative agent of cholera. Pathogenesis in vivo occurs through a series of spatiotemporally controlled events under the control of a gene cascade termed the ToxR regulon. Major genes in the ToxR regulon include the master regulators toxRS and tcpPH, the downstream regulator toxT, and virulence factors, the ctxAB and tcpA operons. Our current understanding of the dynamics of virulence gene expression is limited to microarray analyses of expression at selected time points. To better understand this process, we utilized a systems biology approach to examine the temporal regulation of gene expression in El Tor V. cholerae grown under virulence-inducing conditions in vitro (AKI medium), using high-resolution time series genomic profiling. Results showed that overall gene expression in AKI medium mimics that of in vivo studies but with less clear temporal separation between upstream regulators and downstream targets. Expression of toxRS was unaffected by growth under virulence-inducing conditions, but expression of toxT was activated shortly after switching from stationary to aerating conditions. The tcpA operon was also activated early during mid-exponential-phase growth, while the ctxAB operon was turned on later, after the rise in toxT expression. Expression of ctxAB continued to rise despite an eventual decrease in toxT. Cluster analysis of gene expression highlighted 15 hypothetical genes and six genes related to environmental information processing that represent potential new members of the ToxR regulon. This study applies systems biology tools to analysis of gene expression of V. cholerae in vitro and provides an important comparator for future studies done in vivo.

Synthetic Biogenesis of Bacterial Amyloid Nanomaterials with Tunable Inorganic-Organic Interfaces and Electrical Conductivity.

Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires. Specifically, we used cell-synthesized curli fibers as templates for nucleating and growing gold nanoparticles and showed that nanoparticle size could be modulated as a function of curli fiber gold-binding affinity. Furthermore, we demonstrated that gold nanoparticles can be preseeded onto curli fibers and followed by gold enhancement to form nanowires. Using these two approaches, we created artificial cellular systems that integrate inorganic-organic materials to achieve tunable electrical conductivity. We envision that cell-synthesized amyloid nanofibers will be useful for interfacing abiotic and biotic systems to create living functional materials..

An ingestible bacterial-electronic system to monitor gastrointestinal health

Using bugs in the gut to detect blood Bacteria are environmentally resilient and can be engineered to sense various biomolecules. Mimee et al. combined biosensor bacteria with a miniaturized wireless readout capsule to produce a minimally invasive device capable of in vivo biosensing in harsh, difficult-to-access environments (see the Perspective by Gibson and Burgell). The device successfully measured gastrointestinal bleeding in pigs. Science, this issue p. 915; see also p. 856 An ingestible device for sensing gut biomarkers is created by combining biological and electrical engineering approaches. Biomolecular monitoring in the gastrointestinal tract could offer rapid, precise disease detection and management but is impeded by access to the remote and complex environment. Here, we present an ingestible micro-bio-electronic device (IMBED) for in situ biomolecular detection based on environmentally resilient biosensor bacteria and miniaturized luminescence readout electronics that wirelessly communicate with an external device. As a proof of concept, we engineer heme-sensitive probiotic biosensors and demonstrate accurate diagnosis of gastrointestinal bleeding in swine. Additionally, we integrate alternative biosensors to demonstrate modularity and extensibility of the detection platform. IMBEDs enable new opportunities for gastrointestinal biomarker discovery and could transform the management and diagnosis of gastrointestinal disease.

Synthesizing and Patterning Tunable Multiscale Materials with Engineered Biofilms

A major challenge in materials science is to create self-assembling, functional, and environmentally responsive materials which can be patterned across multiple length scales. Natural biological systems, such as biofilms, shells, and skeletal tissues, implement dynamic regulatory programs to assemble complex multiscale materials comprised of living and non-living components1–9. Such systems can provide inspiration for the design of heterogeneous functional systems which integrate biotic and abiotic materials via hierarchical self-assembly. Here, we present a synthetic-biology platform for synthesizing and patterning self-assembled functional amyloid materials across multiple length scales with bacterial biofilms. We engineered Escherichia coli curli amyloid production under the tight control of synthetic regulatory circuits and interfaced amyloids with inorganic materials to create a biofilm-based electrical switch whose conductance can be selectively toggled by specific environmental signals. Furthermore, we externally tuned synthetic biofilms to build nanoscale amyloid biomaterials with different structure and composition through the controlled expression of their constituent subunits with artificial gene circuits. By using synthetic cell-cell communication, our engineered biofilms can also autonomously manufacture dynamic materials whose structure and composition change with time. In addition, we show that by combining subunit-level protein engineering, controlled genetic expression of self-assembling subunit proteins, and macroscale spatial gradients, synthetic biofilms can pattern protein biomaterials across multiple length scales. This work lays a foundation for synthesizing, patterning, and controlling composite materials with engineered biological systems. We envision that this approach can be expanded to other cellular and biomaterials contexts for the construction of self-organizing, environmentally responsive, and tunable multiscale composite materials with heterogeneous functionalities.