Xiaolong Luo, Ph.D. - US grants

technology /technique development; statistics /biometry; computers; biomedical resource;

1264509
Sintim, Herman O.

This award is supporting the research of Professors Herman O. Sintim, William Bentley and Gary Rubloff of the University of Maryland at College Park. The team will develop multimodal and multifunctional microfluidic systems for studying cell signaling by integrating stimuli-responsive biofabrication with device-imposed, complex gradient generation of bacterial signaling molecules. This device will then be used to systematically investigate the response of bacterial cells to quorum sensing molecules and environmental cues. Additionally, the device will be used to identify new molecules that inhibit bacterial chemotaxis.

Due to the central role that bacterial cell signaling plays in bacterial physiology, there is a high interest in understanding or unraveling the various factors that control bacterial response to signaling molecules. Transformative technologies that can aid the screening of molecules that inhibit bacterial communication would have potential applications in medicine, agriculture and industry. The broader impact of this project includes development of a new technological platform to study how bacteria interact with each other and the environment. This project is highly multidisciplinary, involving chemical biologists, bioengineers and biosystems engineers. Therefore, the students who will be involved with the project will be trained to solve scientific problems using diverse approaches.

This award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.

PI: Luo, Xiaolong
Proposal Number: 1553330

Natural cell membranes are composed of a thin lipid bilayer (LB) that forms a continuous barrier around the cell, and abundant membrane proteins that play key functions of the cell and stabilize LB. The cell membrane sits on a supporting intracellular matrix called cytoskeleton layer that defines cell shape and further stabilizes LB. Ion channels and molecular receptors of membrane proteins on LB are the main targets of fundamental research and pharmaceutical drugs. As such, model LBs have been the crucial platform to study transport and signaling processes of membrane proteins. However, current model LBs suffer from notorious limitations including (1) short LB lifetime, (2) fluidic and/or electrical inaccessibility to both sides of the membranes, and (3) lack of the rich constituents of natural cell membrane. Addressing these limitations of model LB systems should significantly expedite both fundamental biological studies and pharmaceutical drug screenings.

This proposal outlines a five-year program of integrated research and educational activities focusing on the development of highly stable lipid bilayers (HSLB) in microfluidic networks. The investigator proposes to fabricate LB on a freestanding, semi-permeable and mechanically robust biopolymer membrane, the first time such a configuration being pursued. The work will assess the hypothesis that the supporting membrane can serve as a model cytoskeleton layer for the lipid bilayer with high stability that presents in natural cell membranes. The fabricated HSLB will be characterized and compared with current model LBs, applied to study ion channel activities and the virus-cell membrane fusion process, and scaled up for other research and industrial users. Compared to current suspended and supported LBs, the developed HSLB system will provide long-term stability, better replication of cell membranes and ease of scaling-up, as well as enabling simultaneous fluidic, electrical and optical measurements and manipulations. When fully established, the HSLB platform can be a game-changer for studying fundamental membrane biology and identifying membrane-associated novel drug targets, which are greatly limited by the current model LB systems. Besides bioengineers developing microfluidic and Lab-on-a-Chip devices, this research will be of interest to scientists studying biopolymer materials and membrane protein activities, industrial researchers investigating drug targets and high throughput screening, and educators teaching biomaterials and biomicrosystems.

Summary: The overall objective of this proposal is to expand from co-culture to tri-culture of the pathogenic yeast, Candida albicans, and bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, with unprecedented spatiotemporal resolution and the ability to meet different culture requirements in an effort to model the complexity observed in microbiome. In nature, microbial life occurs in a concourse where interactions such as cell-cell signaling or metabolite trading between the same species and often across kingdoms are key to their survival. In human respiratory and gastrointestinal tracts, the balance between competition and cooperation between fungi and bacteria is of particular importance as these interactions can determine the outcome of highly invasive infections. Microbial communities often grow in matrices called biofilms with intricate spatial structure, and many coexist as micro-colonies separated by a few hundred micrometers. This spatial structure has been hypothesized to be important in microbial ecology. Modeling the microbial interactions in a well-controlled and spatially analogous manner is of great interest in microbiome engineering and developing new biological technologies. The vast majority of microbial models or the microbe-host systems, however, are limited to mixed or binary cultures that either are challenging to track changes occurring in individual populations, or lack the compatibility to support different nutrient and environmental requirements for different species. In particular, co-culturing anaerobic and aerobic bacteria is impossible with current technologies. Here, the PI proposes a unique ?fluitrode? platform that circumvents the aforementioned bottlenecks. The research will test four hypotheses: (1) Different microbe species cultured in a spatially controlled manner have significant advantages over standard mixed cultures; (2) Effect of spatial resolution on the communication between C. albicans and P. aeruginosa can be revealed using the fluitrode platform; (3) Differences in microbial communication between aerobic and anaerobic states can be revealed with the heterogeneous culturing platform; and (4) The expansion to tri-culture C. albicans, P. aeruginosa and S. aureus can lead to the construction of a synthetic microbiome of many more species with individual culturing media, optimal spatial resolution, and heterogeneous oxygen requirements. Successful culturing of the three species will lead to establishing synthetic microbiome with more complexity as well as more controllability that is impossible with other approaches. In the short term, the synthetic microbiome will expedite scientific communities to understand the intricate cell-cell interactions in native microbiome. In the long term, better models of polymicrobial interactions will pave the way to developing better treatments for microbial-based diseases, which is a major public health concern.