Analysis of Peptidoglycan Fragment Release.
Most bacteria break down a significant portion of their cell wall peptidoglycan during each round of growth and cell division. This process generates peptidoglycan fragments of various sizes that can either be imported back into the cytoplasm for recycling or released from the cell. Released fragments have been shown to act as microbe-associated molecular patterns for the initiation of immune responses, as triggers for the initiation of mutualistic host-microbe relationships, and as signals for cell-cell communication in bacteria. Characterizing these released peptidoglycan fragments can, therefore, be considered an important step in understanding how microbes communicate with other organisms in their environments. In this chapter, we describe methods for labeling cell wall peptidoglycan, calculating the rate at which peptidoglycan is turned over, and collecting released peptidoglycan to determine the abundance and species of released fragments. Methods are described for both the separation of peptidoglycan fragments by size-exclusion chromatography and further detailed analysis by HPLC.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 4157 Microbial Science Building, 1550 Linden Drive, Madison, WI, 53706, USA.
Ryan E
RE
Schaub
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 4157 Microbial Science Building, 1550 Linden Drive, Madison, WI, 53706, USA.
Jonathan D
JD
Lenz
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 4157 Microbial Science Building, 1550 Linden Drive, Madison, WI, 53706, USA. jpdillard@wisc.edu.
Joseph P
JP
Dillard
D001426:Bacterial Proteins / Q000737:chemistry; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D056148:Chromatography, Reverse-Phase; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D009344:Neisseria gonorrhoeae / Q000737:chemistry / Q000378:metabolism; D010457:Peptidoglycan / Q000737:chemistry*
D001426:Bacterial Proteins; D010457:Peptidoglycan
HPLC; Murein; PG; Peptidoglycan; Peptidoglycan fragments; Peptidoglycan turnover; Pulse-chase; Size-exclusion chromatography