Research
All cells must maintain their shape for normal function. While mammalian cells rely on an internal cytoskeleton of actin and microtubules to dictate shape and drive division, bacteria rely primarily on their cell envelope and its peptidoglycan-bearing cell wall.
Mycobacteria—the causative agents of tuberculosis, leprosy, and nontuberculous mycobacterial (NTM) infections—possess an exceptionally complex cell envelope that is highly divergent from other bacteria. Composed of the essential mycolyl-arabinogalactan-peptidoglycan (mAGP) complex, this structure is a primary determinant of intrinsic antibiotic resistance and pathogenesis.
Our laboratory investigates fundamental principles of mycobacterial physiology. We organize our research around three key areas centering on this unique cell envelope.
1. Mechanisms of cell envelope biogenesis
The mycobacterial cell envelope is assembled by specialized proteins that synthesize and incorporate its hallmark components: phospholipids, phosphatidylinositol mannosides (PIMs), lipomannan (LM), lipoarabinomannan (LAM), peptidoglycan, arabinogalactan, mycolic acids, and extractable surface lipids such as phthiocerol dimycocerosate (PDIM). This complex structure is essential for bacterial viability and virulence.
The inner membrane consists of a phospholipid bilayer containing major lipids such as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and cardiolipin (CL), all synthesized by dedicated enzymes. Lipidated precursors are translocated across the inner membrane by flippases and specialized transporters, delivering them to the periplasm for subsequent incorporation into the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex.
Peptidoglycan is synthesized via the Mur pathway and cross-linked by L,D- and D,D-transpeptidases. Arabinogalactan is polymerized by dedicated galactosyl- and arabinosyltransferases and subsequently covalently attached to the peptidoglycan layer. Mycolic acids, produced by the fatty acid synthase systems, are transported and transferred by mycolyltransferases to arabinogalactan or trehalose to form the outer membrane.
Our laboratory investigates how these proteins synthesize cell envelope precursors and transport them across distinct layers to reach their final sites of incorporation.
2. Coordination of cell elongation and division
In model rod-shaped bacteria, growth is mediated by the elongasome, a multi-protein complex that synthesizes cell wall along the lateral body. Division is driven by the divisome, which builds a septal wall at the mid-cell to separate daughter cells. While these mechanisms are well defined in E. coli and B. subtilis, mycobacteria employ fundamentally different strategies.
Mycobacteria grow via asymmetric polar extension, suggesting they have evolved novel mechanisms to spatially regulate cell wall synthesis. Unlike lateral growers, the mycobacterial elongasome must be recruited specifically to the poles, requiring unique scaffold proteins. Similarly, while the mycobacterial divisome builds a septal wall near the mid-cell, it lacks the canonical placement systems found in other bacteria. The last steps of division encompass in an asymmetric and dynamic process called “V-snapping,” The asymmetric growth of mycobacteria leads to cellular heterogeneity in the resulting daughter cells.
Our research focuses on the coordination of cell envelope biogenesis during these distinct growth phases. Specifically, we examine how the mycobacterial elongasome and divisome are assembled and targeted to active growth zones. We aim to define the regulatory signals that ensure the architectural fidelity of the envelope during replication.
3. Protein secretion
Beyond its unique cell wall synthesis machinery, mycobacteria lack many of the outer membrane markers found in other diderm bacteria. Instead, the mycobacterial outer membrane is thought to be populated by porins and two enigmatic families of proteins known as PE and PPE proteins. While non-pathogenic mycobacteria contain only a handful of genes encoding these proteins, pathogenic species possess a massive repertoire, accounting for a significant percentage of their coding genome. Despite this genomic expansion, the functions of these proteins remain poorly understood.
Both protein families are secreted by the specialized ESX (Type VII) secretion systems. They are highly polymorphic, featuring C-termini that vary extensively in sequence and size. Once secreted, PE/PPE proteins localize to the bacterial surface or are translocated into the host, where they mediate diverse functions ranging from immune evasion to nutrient acquisition.
Our laboratory seeks to define how ESX systems transport these substrates across the complex cell envelope and to elucidate the mechanistic roles these secreted proteins play within the host.
To address these questions, the Chen Lab combines bacterial genetics, biochemistry, and structural biology. Through this work, we aim to define the fundamental mechanisms of mycobacterial physiology. Our ultimate goal is to reveal new vulnerabilities that can be exploited for the development of novel treatments against this group of bacteria.