microbioreactor-based-production-anchorage-dependent-mesenchymal

Microbioreactor-Based Production of Anchorage-Dependent Mesenchymal Stromal Cells Primed for Acute Respiratory Distress Syndrome

Cell-based therapy is an emerging therapeutic approach for a range of health conditions. However, producing sufficient quantities of quality cells remains ...

Cornell University

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, direct compression and hydrostatic pressure. In all cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of  range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of  and of the pathogens  and . An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for , 0.84 ± 0.02 MPa for  treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for , and 1.52 ± 0.06 MPa for  (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

Determining the Young's Modulus of the Bacterial Cell Envelope.

Multidrug efflux pumps confer not only antibiotic resistance to bacteria but also cell proliferation. In gram-negative bacteria, the ATP-binding cassette (ABC)-family transporter MacB, the adaptor protein MacA, and the outer membrane protein TolC form the MacA:MacB:TolC assembly to extrude antibiotics and virulence factors. Here, using quantitative single-molecule single-cell imaging, we uncover that, in  cells, there is a large excess of MacB (and TolC) driving the limiting adaptor protein MacA mostly into the MacAB-TolC assembly. Moreover, the excess MacB transporters can dynamically cluster around the assembly, and MacA can dynamically disassemble from the MacAB-TolC assembly, leading to an adaptor protein shuttling mechanism for efficient substrate sequestration from the periplasm toward efflux. We further show that both MacB clustering and MacAB-TolC assembly can be perturbed chemically or physically via microfluidics-based extrusion loading for compromised antibiotic tolerance. These insights may provide opportunities for countering the activities of multidrug efflux systems for antimicrobial treatments.

Article	Year
Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway. Scientific reports\| PubMed ID: 37633922	2023
Determining the Young's Modulus of the Bacterial Cell Envelope. ACS biomaterials science & engineering\| PubMed ID: 38593061	2024
Transporter excess and clustering facilitate adaptor protein shuttling for bacterial efflux. Cell reports. Physical science\| PubMed ID: 40083904	2025

Junsung Lee

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