Structural characterization of muropeptides from Chlamydia trachomatis peptidoglycan by mass spectrometry resolves "chlamydial anomaly".
The "chlamydial anomaly," first coined by James Moulder, describes the inability of researchers to detect or purify peptidoglycan (PG) from pathogenic Chlamydiae despite genetic and biochemical evidence and antibiotic susceptibility data that suggest its existence. We recently detected PG in Chlamydia trachomatis by a new metabolic cell wall labeling method, however efforts to purify PG from pathogenic Chlamydiae have remained unsuccessful. Pathogenic chlamydial species are known to activate nucleotide-binding oligomerization domain-containing protein 2 (NOD2) innate immune receptors by as yet uncharacterized ligands, which are presumed to be PG fragments (muramyl di- and tripeptides). We used the NOD2-dependent activation of NF-κB by C. trachomatis-infected cell lysates as a biomarker for the presence of PG fragments within specific lysate fractions. We designed a new method of muropeptide isolation consisting of a double filtration step coupled with reverse-phase HPLC fractionation of Chlamydia-infected HeLa cell lysates. Fractions that displayed NOD2 activity were analyzed by electrospray ionization mass spectrometry, confirming the presence of muramyl di- and tripeptides in Chlamydia-infected cell lysate fractions. Moreover, the mass spectrometry data of large muropeptide fragments provided evidence that transpeptidation and transglycosylation reactions occur in pathogenic Chlamydiae. These results reveal the composition of chlamydial PG and disprove the "glycanless peptidoglycan" hypothesis.
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Mathanraj
M
Packiam
Howard Hughes Medical Institute, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461; Department of Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461.
Brian
B
Weinrick
Howard Hughes Medical Institute, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461; Department of Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461 anthony.maurelli@usuhs.edu jacobsw@hhmi.org.
William R
WR
Jacobs
Department of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; anthony.maurelli@usuhs.edu jacobsw@hhmi.org.
Anthony T
AT
Maurelli
D015415:Biomarkers / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry; D002692:Chlamydia trachomatis / Q000737:chemistry*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D016328:NF-kappa B; D010455:Peptides; D010457:Peptidoglycan; D011134:Polysaccharides
NOD2 receptor; chlamydia; mass spectrometry; muropeptide; peptidoglycan