CHEMBIOCHEM HIGHLIGHTS DOI: 10.1002/cbic.201402143
The Chlamydial Anomaly Clarified? Tamimount Mohammadi and Eefjan Breukink*[a]
Chlamydia trachomatis belongs to the family of Chlamydiaceae. validated in diverse bacterial species, and their specificity to These obligate intracellular pathogens are responsible for a label (sites of newly assembled) chlamydial peptidoglycan variety of diseases in humans and animals, including sexually was demonstrated. Moreover, applying the probes to cells transmitted diseases, respiratory infections, and blindness.[1] infected with C. trachomatis yielded a ring-like structure exclusively at the division site of RBs; this indicated labeling of The lifecycle of chlamydiae comprises two alternating forms: the infectious form or elementary body (EB), which is extracellular and metabolically inactive, and the noninfectious form or reticulate body (RB), which is intracellular, metabolically active, and divides by binary fission.[2] The transition from EB to RB takes place inside the host cells where the pathogen is surrounded by a membrane-bound vacuole known as inclusion. As chlamydiae resemble Gram-negative bacteria in their morphology, they are expected to possess a cell wall. However, no discernible peptidoglycan—the essential constituent of bacterial cell walls—has ever been detected in chlamydiae. On the other hand, chlamydiae have been shown to be sensitive to antibiotics that target cell-wall biosynthesis.[3] Exposure to penicillin led to aberrant morphology, inhibition of RB division and prevention of its differentiation into EB.[3, 4] This sensitivity to antibiotics and the lack of a visible peptidoglycan cell wall is known as the “chlamydial anomaly”.[2–5] Efforts to decode this paradox have been hampered by the unavailability of sensitive methods to detect (small amounts of) peptidoglycan in chlamydiae. However, a recent study published in Nature by Liechti, Kuru et al.[6] seems to resolve this biologically interesting phenomenon. In this study, the authors used a new technique based on modified d-amino acid probes to visualize sites of peptidoglycan syn- Figure 1. Labeling of peptidoglycan with modified dipeptide probes. The dipeptide analogue, d-alanine-d-alanine modified with alkyne or azide functional groups, is incorporatthesis during the developmental cycle of C. tracho- ed into the terminal stem peptide of the peptidoglycan precursor by the MurF enzyme matis. These probes consisted of a dipeptide that to form UDP-MurNAc-pentapeptide. After transfer to a lipid carrier (undecaprenyl phosmimicked the terminal d-alanine-d-alanine in the phate) and subsequent addition of GlcNAc, this cell-wall building block is transported peptide side chain of cell-wall building blocks. One of across the cytoplasmic membrane to the periplasm where it is processed and then incorporated into the pre-existing cell wall. After incorporation, the functional group in the dithe d-amino acids in this dipeptide was an alanine peptide is captured by a conventional fluorescent dye by using click chemistry. GlcNAc, variant that contained either an azide or an alkyne in N-acetylglucosamine; MurNAc, N-acetylmuramic acid. its side chain. This made it possible to selectively functionalize the peptidoglycan through click chemisnewly synthesized peptidoglycan. A time-course experiment try with conventional fluorescent dyes after incorporation of with C. trachomatis-infected cells was then conducted to deterthe dipeptide analogue into chlamydial cell wall (Figure 1). mine at which time of infection the dipeptide probe is incorThe incorporation of the modified dipeptide analogues was porated. Labeled peptidoglycan was discernible as early as [a] Dr. T. Mohammadi, Dr. E. Breukink eight hours post infection; that is, during the early stages of Membrane Biochemistry and Biophysics, Department of Chemistry EB-to-RB transition. Together, the findings provide evidence for Faculty of Science, Utrecht University the presence of detectable peptidoglycan in the chlamydial Padualaan 8, 3584 CH Utrecht (The Netherlands) species. E-mail: [email protected] 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 3
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These are not the final page numbers! ÞÞ
CHEMBIOCHEM HIGHLIGHTS Just prior to this study,[6] the existence of peptidoglycan was revealed in the sacculus of Protochlamydia amoebophila,[7] which has a profile resembling that of other Gram-negative bacteria. However, the unusual fluorescent pattern detected in C. trachomatis poses a new question: whether peptidoglycan differs in structure from other free-living bacteria and consequently serves a different purpose(s) in this genus in particular and in Chlamydiaceae in general. The synthesis of peptidoglycan at low amounts in chlamydiae, which could not be detected with conventional methods used to detect peptidoglycan in other bacterial species, would suggest that it is not essential for survival and might have an alternative function. In view of this and the fact that exposure to penicillin has no bactericidal effect on Chlamydia, but rather induces persistence (noninfectious, enlarged RBs in inclusion that are no more able to divide), an optional role in division is conceivable. The potential involvement of chlamydial peptidoglycan in cell division is supported by the findings published earlier by Henrichfreise et al.[8] and Vollmer et al.[9] These authors demonstrated that Chlamydia and Wolbachia (endosymbionts of arthropods and filarial nematodes) maintained a nearly complete, functional lipid II biosynthesis pathway despite the absence of a detectable peptidoglycan cell wall (at the time). The chlamydial cell was shown to synthesize enzymes involved in the biosynthesis of cell-wall precursors, MurA–MurF, MraY, and MurG (essential for the synthesis of lipid II), FtsW (for the transport of lipid II across the cytoplasmic membrane), penicillin-binding proteins PBP2 and PBP3 (for the processing of lipid II), and the amidase AmiA.[2, 5, 8, 9] Although FtsZ, the key protein in the assembly of the division machinery (divisome) is lacking, the genomes of Chlamydiae encode essential division proteins including FtsW (the lipid II flippase during division),[10] FtsI, FtsK and MreB (the homologue of the eukaryotic actin). Similar to other free-living bacterial species, the chlamydial cytoskeletal protein MreB was shown to form a complex with MurF, MraY and MurG that directs lipid II synthesis to the division site and therefore was proposed to compensate for the absence of FtsZ in preserving a functional divisome.[11] Based on this, a crucial role of lipid II
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org in chlamydial cell division was hypothesized.[8, 9, 11] The molecular details lying behind this process, however, have yet to be elucidated. Other important questions regarding how peptidoglycan synthesis is regulated during the chlamydial lifecycle also still need to be answered. Overall, the ability to visualize peptidoglycan in chlamydiae will have significant implications for future studies of the biology of intracellular bacteria. Combined with other established (microscopy) methods, the new labeling technique could be of value in studying the structural details of the bacterial cell wall and in understanding its role during interaction with the host cell. Furthermore, it might be employed to assess the effect of antibiotics used to treat chlamydial infections and could ultimately uncover new strategies for therapeutic targets. Keywords: cell walls · Chlamydia · labeling · peptidoglycans [1] [2] [3] [4] [5] [6] [7]
[8]
[9]
[10]
[11]
R. W. Peeling, R. C. Brunham, Emerging Infect. Dis. 1996, 2, 307 – 319. A. J. McCoy, A. T. Maurelli, Trends Microbiol. 2006, 14, 70 – 77. J. W. Moulder, Infect. Agents Dis. 1993, 3, 87 – 99. I. Chopra, Microbiology 1998, 144, 2673 – 2678. J. M. Ghuysen, C. Goffin, Antimicrob. Agents Chemother. 1999, 43, 2339 – 2344. G. W. Liechti, E. Kuru, E. Hall, A. Kalinda, Y. V. Brun, M. VanNieuwenhze, A. T. Maurelli, Nature 2014, 506, 507 – 510. M. Pilhofer, K. Aistleitner, J. Biboy, J. Gray, E. Kuru, E. Hall, Y. V. Brun, M. S. VanNieuwenhze, W. Vollmer, M. Horn, G. J. Jensen, Nat. Commun. 2013, 4, 2856. B. Henrichfreise, A. Schiefer, T. Schneider, E. Nzukou, C. Poellinger, T. J. Hoffmann, K. L. Johnston, K. Moelleken, I. Wiedemann, K. Pfarr, A. Hoerauf, H. G. Sahl, Mol. Microbiol. 2009, 73, 913 – 923. J. Vollmer, A. Schiefer, T. Schneider, K. Jlicher, K. L. Johnston, M. J. Taylor, H. G. Sahl, A. Hoerauf, K. Pfarr, Int. J. Med. Microbiol. 2013, 303, 140 – 149. T. Mohammadi, V. van Dam, R. Sijbrandi, T. Vernet, A. Zapun, A. Bouhss, M. Diepeveen-de Bruin, M. Nguyen-distche, B. de Kruijff, E. Breukink, EMBO J. 2011, 30, 1425 – 1432. A. Gaballah, A. Kloeckner, C. Otten, H. G. Sahl, B. Henrichfreise, PLoS One 2011, 6, e25129.
Received: March 31, 2014 Published online on && &&, 0000
ChemBioChem 0000, 00, 1 – 3
&2&
These are not the final page numbers! ÞÞ
HIGHLIGHTS Getting visible: A new method to label bacterial cell walls shows the presence of functional peptidoglycan in the important pathogen Chlamydia trachomatis. This might clarify the long-standing paradox of the “chlamydial anomaly”.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
T. Mohammadi, E. Breukink* && – && The Chlamydial Anomaly Clarified?
ChemBioChem 0000, 00, 1 – 3
&3&
These are not the final page numbers! ÞÞ
The Chlamydial Anomaly Clarified? Tamimount Mohammadi and Eefjan Breukink*[a]
Chlamydia trachomatis belongs to the family of Chlamydiaceae. validated in diverse bacterial species, and their specificity to These obligate intracellular pathogens are responsible for a label (sites of newly assembled) chlamydial peptidoglycan variety of diseases in humans and animals, including sexually was demonstrated. Moreover, applying the probes to cells transmitted diseases, respiratory infections, and blindness.[1] infected with C. trachomatis yielded a ring-like structure exclusively at the division site of RBs; this indicated labeling of The lifecycle of chlamydiae comprises two alternating forms: the infectious form or elementary body (EB), which is extracellular and metabolically inactive, and the noninfectious form or reticulate body (RB), which is intracellular, metabolically active, and divides by binary fission.[2] The transition from EB to RB takes place inside the host cells where the pathogen is surrounded by a membrane-bound vacuole known as inclusion. As chlamydiae resemble Gram-negative bacteria in their morphology, they are expected to possess a cell wall. However, no discernible peptidoglycan—the essential constituent of bacterial cell walls—has ever been detected in chlamydiae. On the other hand, chlamydiae have been shown to be sensitive to antibiotics that target cell-wall biosynthesis.[3] Exposure to penicillin led to aberrant morphology, inhibition of RB division and prevention of its differentiation into EB.[3, 4] This sensitivity to antibiotics and the lack of a visible peptidoglycan cell wall is known as the “chlamydial anomaly”.[2–5] Efforts to decode this paradox have been hampered by the unavailability of sensitive methods to detect (small amounts of) peptidoglycan in chlamydiae. However, a recent study published in Nature by Liechti, Kuru et al.[6] seems to resolve this biologically interesting phenomenon. In this study, the authors used a new technique based on modified d-amino acid probes to visualize sites of peptidoglycan syn- Figure 1. Labeling of peptidoglycan with modified dipeptide probes. The dipeptide analogue, d-alanine-d-alanine modified with alkyne or azide functional groups, is incorporatthesis during the developmental cycle of C. tracho- ed into the terminal stem peptide of the peptidoglycan precursor by the MurF enzyme matis. These probes consisted of a dipeptide that to form UDP-MurNAc-pentapeptide. After transfer to a lipid carrier (undecaprenyl phosmimicked the terminal d-alanine-d-alanine in the phate) and subsequent addition of GlcNAc, this cell-wall building block is transported peptide side chain of cell-wall building blocks. One of across the cytoplasmic membrane to the periplasm where it is processed and then incorporated into the pre-existing cell wall. After incorporation, the functional group in the dithe d-amino acids in this dipeptide was an alanine peptide is captured by a conventional fluorescent dye by using click chemistry. GlcNAc, variant that contained either an azide or an alkyne in N-acetylglucosamine; MurNAc, N-acetylmuramic acid. its side chain. This made it possible to selectively functionalize the peptidoglycan through click chemisnewly synthesized peptidoglycan. A time-course experiment try with conventional fluorescent dyes after incorporation of with C. trachomatis-infected cells was then conducted to deterthe dipeptide analogue into chlamydial cell wall (Figure 1). mine at which time of infection the dipeptide probe is incorThe incorporation of the modified dipeptide analogues was porated. Labeled peptidoglycan was discernible as early as [a] Dr. T. Mohammadi, Dr. E. Breukink eight hours post infection; that is, during the early stages of Membrane Biochemistry and Biophysics, Department of Chemistry EB-to-RB transition. Together, the findings provide evidence for Faculty of Science, Utrecht University the presence of detectable peptidoglycan in the chlamydial Padualaan 8, 3584 CH Utrecht (The Netherlands) species. E-mail: [email protected] 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 3
&1&
These are not the final page numbers! ÞÞ
CHEMBIOCHEM HIGHLIGHTS Just prior to this study,[6] the existence of peptidoglycan was revealed in the sacculus of Protochlamydia amoebophila,[7] which has a profile resembling that of other Gram-negative bacteria. However, the unusual fluorescent pattern detected in C. trachomatis poses a new question: whether peptidoglycan differs in structure from other free-living bacteria and consequently serves a different purpose(s) in this genus in particular and in Chlamydiaceae in general. The synthesis of peptidoglycan at low amounts in chlamydiae, which could not be detected with conventional methods used to detect peptidoglycan in other bacterial species, would suggest that it is not essential for survival and might have an alternative function. In view of this and the fact that exposure to penicillin has no bactericidal effect on Chlamydia, but rather induces persistence (noninfectious, enlarged RBs in inclusion that are no more able to divide), an optional role in division is conceivable. The potential involvement of chlamydial peptidoglycan in cell division is supported by the findings published earlier by Henrichfreise et al.[8] and Vollmer et al.[9] These authors demonstrated that Chlamydia and Wolbachia (endosymbionts of arthropods and filarial nematodes) maintained a nearly complete, functional lipid II biosynthesis pathway despite the absence of a detectable peptidoglycan cell wall (at the time). The chlamydial cell was shown to synthesize enzymes involved in the biosynthesis of cell-wall precursors, MurA–MurF, MraY, and MurG (essential for the synthesis of lipid II), FtsW (for the transport of lipid II across the cytoplasmic membrane), penicillin-binding proteins PBP2 and PBP3 (for the processing of lipid II), and the amidase AmiA.[2, 5, 8, 9] Although FtsZ, the key protein in the assembly of the division machinery (divisome) is lacking, the genomes of Chlamydiae encode essential division proteins including FtsW (the lipid II flippase during division),[10] FtsI, FtsK and MreB (the homologue of the eukaryotic actin). Similar to other free-living bacterial species, the chlamydial cytoskeletal protein MreB was shown to form a complex with MurF, MraY and MurG that directs lipid II synthesis to the division site and therefore was proposed to compensate for the absence of FtsZ in preserving a functional divisome.[11] Based on this, a crucial role of lipid II
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org in chlamydial cell division was hypothesized.[8, 9, 11] The molecular details lying behind this process, however, have yet to be elucidated. Other important questions regarding how peptidoglycan synthesis is regulated during the chlamydial lifecycle also still need to be answered. Overall, the ability to visualize peptidoglycan in chlamydiae will have significant implications for future studies of the biology of intracellular bacteria. Combined with other established (microscopy) methods, the new labeling technique could be of value in studying the structural details of the bacterial cell wall and in understanding its role during interaction with the host cell. Furthermore, it might be employed to assess the effect of antibiotics used to treat chlamydial infections and could ultimately uncover new strategies for therapeutic targets. Keywords: cell walls · Chlamydia · labeling · peptidoglycans [1] [2] [3] [4] [5] [6] [7]
[8]
[9]
[10]
[11]
R. W. Peeling, R. C. Brunham, Emerging Infect. Dis. 1996, 2, 307 – 319. A. J. McCoy, A. T. Maurelli, Trends Microbiol. 2006, 14, 70 – 77. J. W. Moulder, Infect. Agents Dis. 1993, 3, 87 – 99. I. Chopra, Microbiology 1998, 144, 2673 – 2678. J. M. Ghuysen, C. Goffin, Antimicrob. Agents Chemother. 1999, 43, 2339 – 2344. G. W. Liechti, E. Kuru, E. Hall, A. Kalinda, Y. V. Brun, M. VanNieuwenhze, A. T. Maurelli, Nature 2014, 506, 507 – 510. M. Pilhofer, K. Aistleitner, J. Biboy, J. Gray, E. Kuru, E. Hall, Y. V. Brun, M. S. VanNieuwenhze, W. Vollmer, M. Horn, G. J. Jensen, Nat. Commun. 2013, 4, 2856. B. Henrichfreise, A. Schiefer, T. Schneider, E. Nzukou, C. Poellinger, T. J. Hoffmann, K. L. Johnston, K. Moelleken, I. Wiedemann, K. Pfarr, A. Hoerauf, H. G. Sahl, Mol. Microbiol. 2009, 73, 913 – 923. J. Vollmer, A. Schiefer, T. Schneider, K. Jlicher, K. L. Johnston, M. J. Taylor, H. G. Sahl, A. Hoerauf, K. Pfarr, Int. J. Med. Microbiol. 2013, 303, 140 – 149. T. Mohammadi, V. van Dam, R. Sijbrandi, T. Vernet, A. Zapun, A. Bouhss, M. Diepeveen-de Bruin, M. Nguyen-distche, B. de Kruijff, E. Breukink, EMBO J. 2011, 30, 1425 – 1432. A. Gaballah, A. Kloeckner, C. Otten, H. G. Sahl, B. Henrichfreise, PLoS One 2011, 6, e25129.
Received: March 31, 2014 Published online on && &&, 0000
ChemBioChem 0000, 00, 1 – 3
&2&
These are not the final page numbers! ÞÞ
HIGHLIGHTS Getting visible: A new method to label bacterial cell walls shows the presence of functional peptidoglycan in the important pathogen Chlamydia trachomatis. This might clarify the long-standing paradox of the “chlamydial anomaly”.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
T. Mohammadi, E. Breukink* && – && The Chlamydial Anomaly Clarified?
ChemBioChem 0000, 00, 1 – 3
&3&
These are not the final page numbers! ÞÞ
