Another Look At The Cell Wall

A simple, high-throughput strategy to measure the stiffness of bacterial cells could be a boon in the hunt for new antibiotics, according to its developers (Mol. Microbiol., DOI: 10.1111/j.1365-2958.2012.08063.x).

Douglas B. Weibel of the University of Wisconsin, Madison, and KC Huang of Stanford University plan to use their technique to screen for proteins that pathogenic bacteria rely on to maintain their protective cell walls. Eventually Weibel hopes to develop small molecules that can shut down such potential antibacterial targets.

This wouldn’t be the first time antibiotic developers trained their arrows at the cell wall. It’s the target of the mother of all antibiotics, penicillin. This and other β-lactams interfere with the assembly of the peptidoglycan layer that makes cell walls stiff. But resistance to these molecules has driven many antibiotic developers to look at targets beyond the cell wall.

Weibel thinks the time is ripe to reevaluate the cell wall as an antibiotic target. “There are many components of the machinery involved in building the cell wall that have not been explored,” he says.

To find new antibiotic targets involved in building and maintaining this important barrier, Weibel wanted to screen libraries of bacteria containing mutations in cell wall synthesis machinery. He would look for mutations that affect the stiffness of the bacterial cell wall. But his lab found it difficult to make high-throughput stiffness measurements using existing methods, in particular atomic force microscopy, or AFM.

When he and Huang went back to the drawing board, the result was CLAMP, short for cell-length analysis of mechanical properties. The method relies on a standard phase-contrast microscope to measure how fast bacterial cells grow when encapsulated in agarose-based gels of defined stiffness. By fitting growth rates to a computational model, Weibel and Huang extract a quantitative measure of the bacteria’s cell wall stiffness.

“AFM is likely to be more accurate” than CLAMP, says Princeton University’s Joshua W. Shaevitz, who uses AFM and other techniques to study the bacterial cell wall. “However, AFM and other force microscopy techniques are low-throughput, which makes the screening of a large number of strains or drugs nearly impossible. Weibel and Huang’s method can be very high-throughput.”

CLAMP “is sufficiently simple that many labs can do it,” adds scanning probe expert Dmitri Vezenov of Lehigh University. Quantitative AFM requires expensive equipment and training, he notes.

Vezenov says that the method also “solves an important problem: consistency.” It’s notoriously difficult to make consistent quantitative measurements of stiffness on objects as small as cells, he says. CLAMP appears to provide the necessary reproducibility.

Antibiotic specialist Dewey G. McCafferty of Duke University suggests the method could be particularly useful for determining how individual components of the cell wall contribute to the barrier’s strength. Scientists could also use it to screen libraries of antibiotics for ones that kill cells by weakening their cell walls, he says.

The method could even help probe how organisms trapped in rock crevices or other tight spots change the stiffness of their cell walls to adapt to living in such environments, Weibel suggests. “It would be an unprecedented opportunity to look at individual cells’ physiology on a real-time basis.”