Fig 1.
Schematic presentation of MUL_3720.
(A) The MUL_3720 protein consists of a N-terminal B-lectin domain and a C-terminal LysM domain linked by a proline rich sequence. MUL_3720 was recombinantly expressed in E. coli either as full length (aa 1–207) or truncated protein (aa 115–207) with a N-terminal hexa-histidine-tag. (B) The predicted amino acid sequence is shown with residues of the B-lectin and the LysM domain marked in red and green, respectively. The consensus sequence motifs QXDXNXVXY involved in alpha-D-mannose recognition are indicated in bold. Strands (arrows), helixes (bars), exposed (underlined) and buried (dotted underlined) regions were predicted with PredictProtein.
Fig 2.
Reactivity of MUL_3720-specific antibodies.
Mouse mAbs JD3.2 (1), JD3.3 (2), JD3.4 (3), JD3.6 (4) and JD3.7 (5) and rabbit polyclonal IgG SZ3398 (6) and SZ3403 (7) were tested for their reactivity with MUL_3720 (aa 1–207) (A), truncated MUL_3720 (aa 115–207) (B) and the endogenous MUL_3720 in a total protein lysate of M. ulcerans strain NM20/02 (C) by Western Blot analysis.
Fig 3.
MUL_3720 is expressed by M. ulcerans strains of various geographic origin.
Total protein lysates of M. ulcerans strains from Ghana, Côte d’Ivoire, Togo, China, Japan and Australia were analyzed by Western Blot. Detection with mAbs JD3.2 (A) and JD3.4 (B) demonstrates expression of MUL_3720 in strains from different geographical regions, while staining with mAb DD3.7 served as a loading control. MUL_3720 expression levels are lower in M. ulcerans strains belonging to the ancestral (China, Japan) as compared to those of the classical lineage (Africa and Australia).
Fig 4.
Analysis of mycobacterial lysates for the presence of MUL_3720 orthologs.
Total protein lysates of different mycobacterial strains were analyzed by Western Blot. MAb DD3.7 served as a loading control. (A) Detection with pAb SZ3398 demonstrates expression of MUL_3720 orthologs in some of the mycobacteria tested, but not in M. tuberculosis and M. bovis. (B) MUL_3720 orthologs in additional mycobacterial species were detected with JD3.4, indicating the recognition of different protein epitopes. (C) Analysis with mAb JD3.2 led to the detection of MUL_3720 orthologs in M. malmoense and M. marinum.
Fig 5.
Immunohistochemical analysis of M. ulcerans using anti-MUL_3720 antibodies.
Histological sections were either stained with carbolfuchsin (counterstain methylenblue) (A, C, E) or with specific monoclonal antibodies (JD3.2) against MUL_3720 (B, D, F). (A) A punch biopsy of a BU patient showing the typical histopathological hallmarks of an active lesion and harbouring large clusters of AFBs inside the subcutaneous tissue (purple clumps) is depicted. (B) Staining of the same tissue specimen with anti-MUL_3720 antibodies. Serial sections were stained by ZN (C) and anti-MUL_3720 antibodies (D). While the whole bacterium is stained by ZN (E) single dots are detected by using anti-MUL_3720 antibodies (F).
Fig 6.
Localization of MUL_3720 in the wildtype or overexpressing M. ulcerans strain. (A) IF analysis (magnification 1000x) shows that MUL_3720 is homogeneously distributed at the cell surface. Primary JD3.2 anti-MUL_3720 antibody was used at a concentration of 0.1 μg/ml. Bars: 2 μm (B) Western Blot analysis of M. ulcerans lysates underlines the high expression of MUL_3720 by the mutant strain. As for the IFA analysis, JD3.2 anti-MUL_3720 antibody was used at a concentration of 0.1 μg/ml. A GroEL-antibody was included as loading control.
Table 1.
List of proteins interacting with the N-terminus of MUL_3720 as determined by M-PFC assay.
Fig 7.
Sandwich ELISA capturing MUL_3720.
Antigen capture sandwich ELISAs using mAb JD3.4 in combination with polyclonal anti-MUL_3720 rabbit IgG as capturing and detecting antibodies, respectively, were carried out to detect serial dilutions of recombinant MUL_3720 (A) and the endogenous MUL_3720 present in M. ulcerans lysates (B).
Fig 8.
Detection of MUL_3720 in M. ulcerans infected mouse foot pads.
Antigen capture sandwich ELISAs using mAb JD3.4 in combination with polyclonal anti-MUL_3720 rabbit IgG as capturing and detecting antibodies, respectively, were carried out to detect MUL_3720 in lysates of mouse foot pads infected with M. ulcerans. Lysates of non-infected mouse foot pads served as negative control. Purified recombinant MUL_3720 was included as positive control. Asterisks indicate significant differences (P < 0.0001) between mean ODs of infected and non-infected mouse foot pad lysates using a two-tailed Student’s t test.