A Search for the Bacterial Mucopeptide Component, Muramic Acid, in Chlamydia

Garrett, Andrea; Harrison, Margaret J.; Manire, G. P.

doi:10.1099/00221287-80-1-315

Cited by 65 publications

(45 citation statements)

References 17 publications

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“…These differences might be explained by the presence of peptidoglycan in EBs, which would confer mechanical stability. However, numerous attempts to detect peptidoglycan in EBs have consistently failed (8,9,12) and the rigidity of EBs is probably conferred by two cross-linked cysteine-rich envelope proteins whose synthesis is initiated during differentiation of RBs to EBs (7,22).…”

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Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP- N -Acetylmuramate:Amino Acid Ligase Activity

et al. 2003

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Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable V max /K m values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.Chlamydiae are an important group of obligate intracellular pathogens that cause serious infections in humans (25). They are distinguished from other eubacteria by a unique development cycle involving two morphological forms, one adapted to extracellular survival (the infectious elementary body [EB]) and the other adapted to intracellular multiplication (the reticulate body [RB]) (22,25). In the development cycle, entry of EBs into host cells is followed by transformation to RBs, RB division, and expansion of the chlamydial microcolony, followed by differentiation back to EBs and release from the infected cell (22,25). The fragility and pleomorphism of the RB contrasts with the rigidity and stability of the EB (7, 22). These differences might be explained by the presence of peptidoglycan in EBs, which would confer mechanical stability.

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Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP- N -Acetylmuramate:Amino Acid Ligase Activity

et al. 2003

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“…This cycle is characterized at one extreme by the elementary body (EB) form, which is adapted for extracellular survival and infection of new hosts, and at the other extreme by the reticulate body (RB) form, which is adapted for intracellular survival and multiplication. Both life cycle forms possess a cell envelope which consists of an outer membrane and an inner membrane (5,19) but lacks or is deficient in peptidoglycan (1,6,13,18). Despite this deficiency, EBs are osmotically stable, although RBs are readily lysed by ultrasonic treatment (18).…”

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Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis

Hatch¹,

Miceli²,

Sublett³

1986

J Bacteriol

131

135

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The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extraceilular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psiftaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psiftaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.

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“…Characteristics that distinguish these bacteria from other obligate intracellular parasites are (i) a complex life cycle that includes an infectious extracellular cell type, the elementary body (EB), and a noninfectious, intracellular, and replicating cell type, the reticulate body (RB) (5,14,35); (ii) disulfide bonding of outer membrane proteins as a mechanism of maintaining structural stability (4,18,19,30) in the absence of peptidoglycan (2,16,27,40); and (iii) replication of the parasite within phagosomes apparently modified by the bacteria to inhibit fusion with lysosomes (5,14,35).…”

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Identification and properties of chlamydial polypeptides that bind eucaryotic cell surface components

Hackstadt¹

1986

J Bacteriol

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An electroblotting technique was used to identify proteins of Chiamydia that bound surface-radioiodinated and Triton X-100-solubilized HeLa cell extracts. Two proteins, with apparent molecular masses of 18 and 32 kilodaltons (kDa), that bound HeLa cell surface components were identified on Chlamydia trachomatis L2 elementary bodies (EBs). Radioiodinated heparin, which disrupts chlamydial association with cultured cells, was also bound by these proteins. These two proteins were found on EBs but were absent or were present in reduced amounts on the noninfectious reticulate bodies. All C. trachomatis strains tested displayed two such proteins, although the apparent molecular weight of the larger protein varied with serotype in correlation with biotype and the disease that it caused. Two Chlamydia psittaci strains examined displayed only a single binding protein in the range of 17 to 19 kDa. All of the binding proteins stained intensely and distinctively on silver-stained sodium dodecyl sulfate-polyacrylamide gels and displayed an unusual sensitivity to reducing agents. The 32-kDa protein was not seen and did not bind 12'I-labeled HeLa cell components if the EBs were solubilized in the presence of 2-mercaptoethanol. The 32-kDa protein was not affected by dithiothreitol, however. Similar to the effect of 2-mercaptoethanol, the 32-kDa protein was not visualized after treatment of EBs with the protease inhibitors tosyl-phenylalanine chloromethyl ketone (TPCK) or tosyl-lysine chloromethyl ketone (TLCK). TPCK and TLCK also abolished infectivity as did the alkylating agents N-ethylmaleimide and iodoacetamide, yet the latter two agents did not affect the appearance of the 32-kDa protein. These proteins were not detected in immunoblots with either rabbit antisera to C. trachomatis L2 EBs or by serum from a patient with lymphogranuloma venereum. The role of these proteins in the interaction of chlamydiae with host cells is not clear, but the binding of eucaryotic cell surface components and heparin, presence only during the infectious stage of the life cycle, variation between serotypes in correlation with disease, and sensitivity to reducing agents or protease inhibitors, collectively, suggest a role for these proteins in parasite-host interactions.Chlamydia species are procaryotic obligate intracellular parasites of eucaryotes. Characteristics that distinguish these bacteria from other obligate intracellular parasites are (i) a complex life cycle that includes an infectious extracellular cell type, the elementary body (EB), and a noninfectious, intracellular, and replicating cell type, the reticulate body (RB) (5, 14, 35); (ii) disulfide bonding of outer membrane proteins as a mechanism of maintaining structural stability (4,18,19,30) in the absence of peptidoglycan (2, 16, 27, 40); and (iii) replication of the parasite within phagosomes apparently modified by the bacteria to inhibit fusion with lysosomes (5,14,35).There are two species of Chlamydia, C. trachomatis and C. psittaci. The two species share a number of biologi...

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A Search for the Bacterial Mucopeptide Component, Muramic Acid, in Chlamydia

Cited by 65 publications

References 17 publications

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP- N -Acetylmuramate:Amino Acid Ligase Activity

Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP- N -Acetylmuramate:Amino Acid Ligase Activity

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis

Identification and properties of chlamydial polypeptides that bind eucaryotic cell surface components

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