(4db) Connecting Individual-Cell Regulation to Bacterial Biofilm Development to Advance Treatment and Engineering Solutions

A. Introduction

My research interest lies in understanding bacterial biofilms using interdisciplinary approaches. Biofilm is an important lifestyle of bacteria in nature and host habitats, where cells form structured communities embedded in an extracellular matrix. Biofilms play a critical role in health and engineering settings, as they are often involved in chronic infections and biofouling. However, they can also be useful in bioremediation and beneficial in our microbiota. Understanding bacterial biofilms present opportunities for therapeutic strategies that target biofilm formation and engineering solutions that make use of biofilms. Centuries of dedicated research have provided a wealth of knowledge in bacteria, yet relatively little is known about biofilms. A major obstacle to traversing scales is the need for multidisciplinary expertise and tools. My interdisciplinary training background in soft-matter physics, microscopy, microbiology, and ecology uniquely positions me to pursue unanswered questions at such scientific interface. The overarching goal for my independent research career is to connect individual-cell regulation in bacteria to biofilm development to advance treatment and engineering solutions.

B. Previous Accomplishments

PhD Research:

1. Discovery of a new class of topological solitons. Topological solitons are quasi-particles of field deformations that are topologically nontrivial. They are of great fundamental interest and promise technological applications because of their unique particle-like properties and the capability of encoding information in topology. During PhD, I studied the properties of different 2D and 3D topological solitons in liquid crystals (LCs), including their stability, soliton-soliton interaction, self-assembly, stimuli-induced dynamics, etc. I also discovered and characterized a new class of stable solitons. My knowledge in LC topological solitons also allowed me to model the stability of similar solitonic structures in other material systems such as solid-state chiral magnets, which precedes the recently published experimental realization.
2. Super-resolution imaging of orientationally-ordered soft materials. I developed and characterized a super-resolution stimulated emission depletion polarizing microscopy (STED-PM) technique with sensitivity in molecular orientation in LCs and applied it to reveal nm-scale director structures of solitons and defects. The technique can be extended to polymers, active matters, colloids, membranes, and various other soft matter and biological systems with orientational ordering.

Postdoctoral Research:

1. Studying the social evolution of sharable biofilm matrix components. Central to the evolutionary advantage of biofilm formation is cell-cell and cell-surface adhesion achieved by extracellular matrix components. The diffusible nature of the extracellular matrix raises the question: How can diffusible matrix production be stable against exploitation? I first establish that diffusible matrix proteins in Vibrio cholerae biofilms are indeed exploitable public goods. However, exploitation is localized within a quantifiable spatial range around the producer cell clusters. Based on the exploitation range and the length scale of cell-group structures, I developed a spatial ecological model that reveals stable conditions for diffusible matrix production and quantitatively reproduces data from ecological competition assays. The stable conditions, including sparse distribution of cell groups and presence of environmental flows, are consistent with those in the host and natural habitats and provide an explanation for the evolutionary stability of diffusible matrix production. The concept of exploitation range and the associated analysis tools developed in this work are generally applicable to relevant social evolution studies.
2. Heterogeneity in developing biofilms. The regulation of biofilm lifecycle involves cell-to-cell communication through chemical signals that enables synchronous decisions. However, phenotypic heterogeneity is widely observed in clonal communities. Quantification of phenotypic heterogeneity in biofilms has been lacking and it is unknown if heterogeneity plays a significant role in biofilm development. To this aim, I performed single-cell time-lapse imaging of biofilms in V. cholerae with fluorescent reporters for cyclic diguanylate (c-di-GMP), a conserved second messenger that controls motile-to-sessile lifestyle switching, and biofilm-related genes. I found that high levels of heterogeneity prevail at the individual-cell level in both intracellular c-di-GMP concentration and matrix production. Moreover, high- and low-c-di-GMP cells spatially segregate in a way analogous to pattern formation in multicellular tissue and organisms. Using matrix mutants, numerical modeling, and a partial lineage-tracking technique I developed, we showed that such pattern formation depends on the presence of specific matrix components and reveal the physical principles behind phenotypic segregation in developing biofilms. Our results revealed heterogeneity as a strategy for bet-hedging and improving colonization efficiency, and provided renewed insights into the picture of biofilm lifecycle

C. Future Directions

Building upon my strong interdisciplinary background, my lab will focus on studying the development and emergent properties of bacterial biofilms with a multidisciplinary approach. The overarching scheme is to connect individual-cell regulation in bacteria, such as cell-to-cell variation and morphological regulation, to emergent properties and the developmental program of biofilms at the community level. I expect to not only gain fundamental insights into the complex biological system, but also inspire effective therapeutic treatments, engineering solutions, and materials applications.

1. Explore the origin of heterogeneity in bacterial communities: In my postdoctoral research, I revealed that significant heterogeneity prevails in clonal populations of V. cholerae. Understanding the source of cell-to-cell variation in bacterial communities is critical to treating infections or repurposing bacteria for therapeutics and engineering. In my lab, I will embark on deciphering the origin of heterogeneity by employing high throughput assays to measure gene-expression dynamics and heterogeneity in various mutants. Preliminary results hint at certain positive and negative feedback loops in the regulatory circuitry that could contribute to heterogeneity. I will also develop dynamic system models to gain insights into the nature of gene-expression dynamics and heterogeneity from the perspective of systems biology. I expect to develop a wholistic picture of regulation strategies generalizable to other biological systems that leads to heterogeneity in a microbial communities.
2. Explore the evolutionary advantage of morphological regulation in biofilms. Cell shape is a tightly regulated phenotype in many bacteria. In Vibrio spp., the curvature of the characteristic comma shape is the result of a periplasmic protein CrvA. The evolutionary advantage of curved cell shape and its coregulation with biofilm formation is largely unknown. I will design ecological competition assays between strains with different cell curvature. The fitness of strains with different curvatures under different developmental conditions and environmental challenges will help pinpoint how morphological regulation facilitates biofilm development and reveal the advantages of cell-shape plasticity. Additionally, I will develop agent-based simulations to model the biofilm formation process where the shape of each individual cell can be modified. I expect to establish functional connection between cell-shape regulation and biofilm development, as well as the underlying biophysical principles.
3. Investigate the connection between biofilm architecture and biological functions. Mature biofilms of rod-like cells often display liquid-crystal order and defects, where cells align along a preferred direction but are disordered in certain regions. Cell-ordering defects have been shown to harbor concentrated metabolic and developmental functions in tissues, such as cell apoptosis and cell differentiation. However, it is unknown if a correlation between structure and function is present in biofilms. To this aim, I will combine imaging-based ordering analysis with spatial quantification of gene expression, such as single-molecule fluorescence in situ hybridization (smFISH) and fluorescence reporters in 3D biofilms. This will elucidate if multicellular communities employ emergent structural properties to concentrate specific biological activities in defined locations in biofilms and reveal treatment targets.
4. Bacterial biofilms as living liquid-crystal (LC) materials. Bacterial biofilms present several advantages for studying novel phases in LCs, such as accessibility by optical microscopy, cell morphology that can be engineered by genetics or drugs, and cell-cell interaction that can be modified by matrix production. The versatility and dynamic range of controls can give rise to rich phase behavior. The phase diagram obtained in the engineered system can also inform behavior of cells in biofilm under physiologically relevant conditions. From the application perspective, biological LCs can be excellent biomaterials due to alignment and response to ordering modulation, and may find applications in 3D scaffold for tissue engineering, cell-cultured meat, artificial muscles, etc.

D. Teaching Interests

My diverse background in physics, quantitative biology, microbiology, and imaging allows me to teach a wide range of courses. I am interested in teaching introductory and intermediate undergraduate-level courses such as Engineering Physics, Thermodynamics, Statistical Mechanics, Biological Physics, etc. I am also open to courses that I am less familiar with. Additionally, I am eager to develop a new course, Soft Matter in Biology, focusing on biological systems that can be understood using soft-matter physics and engineering tools, and the ensuing biological function. The target audience will be engineering and physics majors who are interested in applying physical sciences in living systems, as well as biology majors who seek engineering and quantitative understanding of biological phenomena. I envisage a diverse composition of students in the class would promote mutual enrichment in the understanding of the interdisciplinary subject.