Fig 1.
Immunoblot detection of antigen in the four major life cycle stages of T. congolense IL3000 using mAb Tc6/42.6.4.
BSF: bloodstream forms; PCF: procyclic culture forms; EMF: epimastigote forms; MCF: metacyclic forms. Modified with permission from [1].
Fig 2.
MALDI-TOF mass spectrum of the trypsin-digested ~26 kDa gel band recognized by mAb Tc6/42.6.4.
The peak at 1227.72 m/z corresponds to the peptide VLQMHELTTR and the peak at 1457.51 m/z corresponds to the peptide LSFNEVCSGCER (with two carbamidomethylations).
Fig 3.
Multiple sequence alignment of the T. congolense calflagins.
The positions of the two MS-identified tryptic peptides identified by mass spectrometry are highlighted in yellow and red boxes. The lower case, white highlighted v represent amino acid (valine-isoleucine) differences in two of the ORFs.
Fig 4.
Primary sequence alignment of T. congolense calflagin with those of T. cruzi FCaBP and T. brucei Tb24.
The secondary structural elements (α-helices and β-strands) are depicted as cones and arrows, respectively, and were derived from the I-TASSER based structure prediction for T. congolense calflagin, the x-ray crystal structure of T. cruzi FCaBP (3CS1) and the NMR data for T. brucei Tb24 (2LVV). The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted in green, salmon, cyan, and yellow, respectively. Residues in the 12-residue Ca2+ binding loops at position 1, 3, 5 and 12 are underlined. Invariant basic residues on the protein surface that are associated with membrane binding are colored blue. Non-conserved surface-exposed residues are highlighted using bold print.
Fig 5.
Confocal immunofluorescence microscopy showing localization of T. congolense calflagin in the presence and absence of calcium.
Maximum intensity projections of fixed T. congolense PCF were probed with mAb Tc6/42.6.4 and anti-para-flagellar rod protein in the presence (A, B, and C) and absence (D, E, and F) of calcium. Green: calflagin; Red: para-flagellar rod protein; Blue: DAPI/DNA.
Fig 6.
Structural characterization and surface analysis of T. congolense calflagin Modeled T. congolense calflagin (middle panel) was compared to the crystal structure of T. cruzi FCaBP (left vertical column) and to the NMR structure of T. brucei Tb24 (right vertical column).
(A) the predicted model of T. congolense calflagin (middle vertical column) exhibits four EF-hands motifs. The EF-hands (EF1, EF2, EF3 and EF4) are colored green, salmon, cyan, yellow, accordingly. Left panel: structural alignment of T. congolense (grey) over T. cruzi FCaBP (green). Right panel: T. congolense calflagin structure aligned over T. brucei Tb24 (magenta). (B) Surface representation of the three calflagin models overlapping panel A, showing exposed hydrophobic (gray), basic (blue) and acidic (red) respectively. (C) 180° rotation of B in the y-axis. Black arrows pointing towards putative epitopes and numbered according to their respective α-helix location.
Fig 7.
Calflagin-based serodiagnosis of trypanosome infections in Ugandan cattle.
(A) ELISA signal intensities resulting from bovine IgG and IgM antibodies binding to solid-phase adsorbed recombiant T. congolense calflagin. All values were normalized against signals elicited from plasma of laboratory raised, trypanosome negative calves. (B) ROC curves generated from the serodiagnostic ELISA results. Areas under curve for IgG and IgM were 0.623 and 0.709 respectively. For reference, an area under the curve of 1.00 equates to a test with 100% sensitivity and specificity, whereas an area under the curve of 0.500 indicates that a test has no value in differentiating between the binary population.