Labeling and Detection of Peptidoglycan in Chlamydia and Chlamydia-Related Organisms Utilizing D-Alanine Analogs

The incorporation and visualization of modified D-amino acids into the peptidoglycan (PG) of actively replicating microbes have made a tremendous impact on the field of bacterial physiology. Here, we describe the optimization of this technique for labeling PG in obligate, intracellular Chlamydia species as well as Chlamydia-like organisms. Fluorescent D-amino acids (FDAAs) are D-alanine analogs that readily incorporate into the stem peptides of living bacteria and can be visualized rapidly with a fluorescence microscope without the need for laborious postfixation procedures. By comparison, D-amino acid analogs (DAAAs) and D-amino acid dipeptides (DAADs) similarly incorporate into newly synthesized peptidoglycan but require postfixation steps to effectively visualize PG. These probes were foundational in demonstrating the presence of PG in Protochlamydia and Chlamydia species, and PG labeling is a convenient tool that can augment other fluorescence imaging approaches. Here, we provide a protocol for the incorporation of D-alanine analogs into the PG of intracellular bacteria and the subsequent steps enabling these amino acids to be visualized via fluorescence microscopy. We also describe the use of an analysis tool that enables the mapping of PG-labeled objects in three-dimensional space and a protocol for confirming that modified amino acids are incorporating into bacterial PG.