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Chlamydial Anomaly” Structural characterization of muropeptides from Chlamydia trachomatis peptidoglycan by mass spectrometry resolves “chlamydial anomaly” Mathanraj Packiama,1, Brian Weinrickb,c, William R. Jacobs Jr.b,c,2, and Anthony T. Maurellia,2 aDepartment of Microbiology and Immunology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; bHoward Hughes Medical Institute, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461; and cDepartment of Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461 Contributed by William R. Jacobs Jr., July 20, 2015 (sent for review June 18, 2015; reviewed by Harlan D. Caldwell and Joseph P. Dillard) The “chlamydial anomaly,” first coined by James Moulder, describes In a similar fashion, variations in the stem peptide sequence would the inability of researchers to detect or purify peptidoglycan (PG) from alter NOD1 receptor recognition and the host inflammatory re- pathogenic Chlamydiae despite genetic and biochemical evidence and sponse (15). Because two serious inflammatory consequences of antibiotic susceptibility data that suggest its existence. We recently Chlamydia trachomatis infection are pelvic inflammatory disease detected PG in Chlamydia trachomatis by a new metabolic cell wall and trachoma, the exact molecular identity of chlamydial PG has labeling method, however efforts to purify PG from pathogenic Chla- high biological relevance and is essential to identifying the role PG mydiae have remained unsuccessful. Pathogenic chlamydial spe- plays in the induction of inflammation in these disease states. cies are known to activate nucleotide-binding oligomerization Pilhofer et al. (16) recently succeeded in purifying PG sacculi domain-containing protein 2 (NOD2) innate immune receptors from an environmental Protochlamydia species but were unable to by as yet uncharacterized ligands, which are presumed to be PG detect or purify PG from Simkania, a distinct environmental spe- fragments (muramyl di- and tripeptides). We used the NOD2-depen- cies, by standard PG sacculi purification protocols. Because path- dent activation of NF-κBbyC. trachomatis-infected cell lysates as a ogenic Chlamydiae are more similar to Simkania (17) than to biomarker for the presence of PG fragments within specific lysate Protochlamydia in the content of PG biosynthetic pathway genes, fractions. We designed a new method of muropeptide isolation con- Pilhofer et al. speculated that pathogenic Chlamydiae could be sisting of a double filtration step coupled with reverse-phase HPLC devoid of PG sacculi similar to Simkania (16). We recently dem- fractionation of Chlamydia-infected HeLa cell lysates. Fractions onstrated the presence of PG in C. trachomatis by a new metabolic that displayed NOD2 activity were analyzed by electrospray ion- cell wall labeling method (18) and observed that chlamydial PG ization mass spectrometry, confirming the presence of muramyl does not appear to form a sacculus but is present in a ring-like di- and tripeptides in Chlamydia-infected cell lysate fractions. structure at the apparent cell division plane. Given the absence of a Moreover, the mass spectrometry data of large muropeptide frag- PG sacculus, we hypothesized that purification of PG from path- ments provided evidence that transpeptidation and transglycosy- ogenic Chlamydiae required a technique other than the standard lation reactions occur in pathogenic Chlamydiae. These results PG sacculi isolation method to succeed. In addition, previous un- reveal the composition of chlamydial PG and disprove the “glycan- successful searches for PG in pathogenic Chlamydiae focused on less peptidoglycan” hypothesis. Significance chlamydia | peptidoglycan | mass spectrometry | NOD2 receptor | muropeptide The existence of peptidoglycan (PG) in pathogenic Chlamydiae is supported by genetic data and antibiotic susceptibility, but the he existence of peptidoglycan (PG) in pathogenic Chlamy- failure to isolate PG from pathogenic Chlamydiae has led to the Tdiae has long been debated. Although genetic analysis and an- “chlamydial anomaly.” Moreover, the lack of a transglycosylase tibiotic susceptibility indicate the presence of PG in Chlamydia (1–3), domain in some Chlamydia penicillin-binding proteins suggests all attempts to detect and purify PG have been unsuccessful (4–7), that Chlamydiae may possess a “glycanless PG.” We successfully resulting in the paradox known as the “chlamydial anomaly” (8). enriched Chlamydia muropeptides from Chlamydia-infected cell Ghuysen and Goffin (9) hypothesized that Chlamydia might syn- lysates using nucleotide-binding oligomerization domain-con- thesize a “glycanless PG” based on the observation that the Chla- taining protein 2 (NOD2)-dependent NF-κB activation as a bio- mydia genome encodes two high molecular mass penicillin-binding marker for the presence of PG fragments in specific fractions. proteins (PBPs) that are devoid of transglycosylase activity, which is Mass spectrometry analysis indicated the presence of chlamydial essential for elongation of the glycan chain of classical PG. Besides muropeptides and classified chlamydial PG as type A1γ in the doubts about the presence of a glycan backbone in chlamydial PG, Schleifer and Kandler classification. This study disproves the gly- there are ambiguities about the sequence of the stem peptide. Patin canless PG hypothesis and is, to our knowledge, the first structural et al. (10) suggested glycine, L-serine, or L-alanine as the possible confirmation of chlamydial PG in pathogenic species. first amino acid residue of the chlamydial PG stem. Resolution of the chlamydial anomaly by purification and structural characteriza- Author contributions: M.P. and A.T.M. designed research; M.P. and B.W. performed re- tion of chlamydial PG will have a profound impact not only on search; W.R.J. contributed new reagents/analytic tools; M.P., B.W., and A.T.M. analyzed data; and M.P., B.W., W.R.J., and A.T.M. wrote the paper. chlamydial biology but also on shaping our understanding of the host Reviewers: H.D.C., NIH National Institute of Allergy and Infectious Diseases; and J.P.D., innate immune responses. Many pathogenic Chlamydia species ac- University of Wisconsin-Madison School of Medicine and Public Health. tivate the nucleotide-binding oligomerization domain-containing The authors declare no conflict of interest. protein (NOD) family of innate receptors during infection (11–14). 1Present address: Biological Research Laboratory, Eminent Services Corporation, Frederick, The inflammatory potential of chlamydial PG would vary depending MD 21703. on the composition of both the sugar backbone as well as the stem 2To whom correspondence may be addressed. Email: [email protected] or peptide sequence. NOD2 receptors recognize the sugar backbone of [email protected]. “ ” PG, and therefore, a glycanless chlamydial PG may not activate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NOD2 receptors and fail to induce an inflammatory response (15). 1073/pnas.1514026112/-/DCSupplemental. 11660–11665 | PNAS | September 15, 2015 | vol. 112 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1514026112 Downloaded by guest on September 23, 2021 A Larger PG fragments present in the activating fractions provided 0.4 evidence for cross-links formed between PG stem peptide chains and for glycan chains composed of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) saccharides. Our molecular 0.3 detection of chlamydial PG fragments resolves the much-debated chlamydial anomaly, disproves the glycanless PG hypothesis re- 0.2 garding pathogenic Chlamydiae, and identifies chlamydial MDP as a ligand for the host innate immune receptor NOD2. 0.1 Results NF-κB activation (OD 650) 0.0 Chlamydia-Infected HeLa Cell Lysates Specifically Induce NOD2-Dependent NF-κBActivity.Multiple studies have shown that intracellular patho- Mock genic Chlamydiae induce NF-κB signaling by activating NOD2 re- Inf 2 hrsInf 8 hrs Inf 18 hrsInf 28 hrsInf 38 hrs ceptors (11–14). To isolate chlamydial ligand(s) that activate NOD2 Time post-infection (i.e., MDP), HeLa cells were infected with C. trachomatis L2 serovar for 2 h and cell lysates were harvested at various time points *** throughout the developmental cycle (2, 8, 18, 28, and 38 h) and B filtered first through a 0.2-μm filter and then through a 3-kDa 0.8 *** centrifugal filter. When added (extracellularly) to HEK-Blue NOD2 secreted alkaline phosphatase (SEAP) reporter cell lines, the 3-kDa 0.6 NOD2 centrifugal filter flow-through from Chlamydia-infected cells at 18 h *** ** Null2 κ *** postinfection (PI) induced maximum NF- B activity compared with 0.4 earlier or later time points (Fig. 1A). Because peak induction of *** ns ns ns NOD2 activity was found at 18 h PI, all experiments were per- 0.2 formed with cell lysates harvested at 18 h PI. NF-κB induction from infected cell lysates was significantly higher than mock-infected controls (P < 0.005 unpaired t test), and the activity was NOD2- NF-κB activation (OD 650) 0.0 p mA dependent in HEK-Blue NOD2 cells as NF-κB induction by in- Mock k + fected cell lysates was minimal in HEK-Blue Null2 cells that are Infected coMM Inf + Amp Inf + DCS M Mock + DCS devoid of the NOD2 receptor (Fig. 1B). Heat and RNase A treatments of cell lysates had minimal effect on the infected Fig. 1. Kinetics of Chlamydia NOD2 ligand production during the develop- sample’s ability
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