Identification of surface-exposed components of MOMP of Chlamydia trachomatis serovar F

Abstract

The identification of surface-exposed components of the major outer membrane protein (MOMP) of Chlamydia is critical for modeling its three-dimensional structure, as well as for understanding the role of MOMP in the pathogenesis of Chlamydia-related diseases. MOMP contains four variable domains (VDs). In this study, VDII and VDIV of Chlamydia trachomatis serovar F were proven to be surface-located by immuno-dot blot assay using monoclonal antibodies (MAbs). Two proteases, trypsin and endoproteinase Glu-C, were applied to digest the intact elementary body of serovar F under native conditions to reveal the surface-located amino acids. The resulting peptides were separated by SDS-PAGE and probed with MAbs against these VDs. N-terminal amino acid sequencing revealed: (1) The Glu-C cleavage sites were located within VDI (at Glu61) and VDIII (at Glu225); (2) the trypsin cleavage sites were found at Lys79 in VDI and at Lys224 in VDIII. The tryptic peptides were then isolated by HPLC and analyzed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and a quadrupole-orthogonal-TOF mass spectrometer coupled with a capillary liquid chromatograph. Masses and fragmentation patterns that correlated to the peptides cleaved from VDI and VDIII regions, and C-terminal peptides Ser333-Arg358 and Ser333-Lys350 were observed. This result demonstrated that these regions are surface-exposed. Data derived from comparison of nonreduced outer membrane complex proteolytic fragments with their reduced fractions revealed that Cys26, 29, 33, 116, 208, and 337 were involved in disulfide bonds, and Cys26 and 337, and 116 and 208 were paired. Based on these data, a new two-dimensional model is proposed.

Figure 1.

Immunoblot and Western blot analysis of intact EBs and endoproteinase-digested EBs of C. trachomatis serovar F. (A) Specificity of MAbs by immuno-dot blot. Viable EBs of each C. trachomatis serovar and guinea pig inclusion conjunctivitis (GPIC) were used as antigen(s) in the immuno-dot blot assay. The specificities of MAbs F-11, F-21, F-25, F-31, and GI-C3, which recognize different MOMP epitopes, were tested. MAb FI-A6, which recognizes lipopolysaccharide (LPS) epitope of all chlamydial serovars and strains, was used as a control. (B) The effect of heat and trypsin treatment on the antigenicity of immunoaccessible epitopes by immuno-dot blot. EBs of C. trachomatis serovar F were incubated at 4°C, heated for 30 min at 56°C, treated with trypsin for 30 min at 37°C or treated with trypsin plus PMSF for 30 min at 37°C, and then reacted with MAbs that recognize different MOMP determinants. (C) Western blot analysis of EBs digested with Glu-C; (D) Western blot analysis of EBs digested with trypsin. EBs of C. trachomatis serovar F digested with endoproteinase Glu-C or trypsin for 30 min at 37°C were used as the test antigens, and EBs incubated in PBS for 30 min at 37°C were used as a control. MAbs F-25, F-31, and GI-C3 were used as probes to detect the proteolytic peptides. Peptide bands corresponding to those recognized by MAbs are indicated by arrows, and the first five amino acids of the N terminus (from N-terminal sequencing) and the deduced C-terminal residues are annotated.

Figure 2.

Surface-exposed regions near the C terminus of MOMP, as detected by MALDI-TOF MS. Mass spectra of MOMP peptides, corresponding to residues 333–350 (A) and 333–358 (B) are shown. Reflectron MALDI-TOF MS spectra of individual HPLC fractions from the trypsin digest of intact EBs are shown over the mass ranges m/z 1885–2000 and m/z 2795–2930 Da; cysteines were reduced after digestion and pyridylethylated with 4-vinyl-pyridine. The assignments and monoisotopic masses of fragments are indicated above each peak.

Figure 3.

Cysteines involved in disulfide bridges, as shown by secondly modified with iodoacetamide. Reflectron MALDI-TOF MS spectrum of one HPLC fraction from the combination of trypsin, Asp-N, and Glu-C triple-enzyme digest of OMC demonstrates modified cysteine containing fragments. CAM indicates carboxymethylation of cysteine by iodoacetamide; PE, to pyridylethylation of cysteine by 4-vinyl-pyridine. The monoisotopic mass and sequence are indicated above each peak.

Figure 4.

A representative capillary LC MS/MS spectrum indicating that Cys208 is involved in disulfide bridges. MS/MS fragmentation of the precursor m/z 611.33 (labeled [M+3H]³⁺) produced the y- and b-ions series from which was determined the sequence of 16 amino acids, including iodoacetamide-alkylated Cys208. Nonreduced OMC was digested with trypsin, Asp-N, and Glu-C triple enzymes, followed by the double cysteine-modification (with intervening reduction) and HPLC separation steps, as detailed in Materials and Methods. CAM indicates carboxymethylation of cysteine by iodoacetamide. The deduced sequence is shown above the spectrum using the one-letter codes for amino acid residues.

Figure 5.

Cys26 and Cys337, Cys116 and Cys208 were found to be involved in disulfide bridges upon comparison of mass spectra of the nonreduced OMC and reduced OMC from the same fraction. Nonreduced OMC was digested with trypsin, Asp- N, and Glu-C triple enzymes, and subsequently was pyridylethylated with VP, followed by HPLC separation of the products (nonreduced fractions). The candidate fractions containing possible disulfide-bridged peptides were reduced and carboxymethylated with iodoacetamide (reduced fractions). (A,top) MALDI-TOF mass spectra from the nonreduced fraction over the mass ranges m/z 2200–2300 and 800–1600; cysteine 26 and cysteine 337 form one disulfide bridge to form the peptide with m/z 2271.3 of [M+H]⁺ ion. (Bottom) MALDI-TOF mass spectra from the reduced fraction over the mass ranges m/z 2200–2300 and 800– 1600; the two peptides were separated and appeared as iodoacetamide alkylated peptides with m/z 823.4 and m/z 1562.3 of [M+H]⁺ ions after the fraction was reduced. (B,top) ESI mass spectra from the nonreduced fraction over the mass ranges m/z 890–930 and 705–815. (Bottom) Two peptides, connected by one disulfide bond in the nonreduced fraction, were observed to be iodoacetamide alkylated. Peptides are marked with m/z value, charge state, and corresponding sequences.

Figure 6.

(A) Overview of the amino acid sequence of MOMP of C. trachomatis serovar F; (B)Topology sketch of MOMP as viewed from the barrel exterior. Residues of the β-strand are indicated by squares. Residues of the loops (L) and turns are in circles. Loops face the outside, while turns face the periplasm. VDI-IV represents the four variable domains of MOMP and is indicated in blue. Residues in the VDs are in bold. The cysteines that possibly form disulfide bonds are indicated in red and italics, and the potential disulfide bridges are shown with dotted lines. Each cystine carries a position number. One-letter codes of amino acids are used.