Abstract
Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.
Highlights
Binding of Mycobacterial Glycoprotein Apa to hPSP-A ogous carbohydrate recognition domains (CRDs), which recognize and bind mannose-containing glycoconjugates exposed on the surface of numerous infectious pathogens, including mycobacteria [9]
Ferguson et al [6] identified mannosylated lipoarabinomannans (ManLAMs) as a potential ligand of human pulmonary surfactant protein (PSP)-D, whereas we found that purified human pulmonary surfactant protein A (PSP-A) exclusively binds to ManLAM and the structurally related lipomannan [11, 12]
Recent observations reported by some of us strongly support that ManLAM is a ligand of DC-SIGN, the major C-type lectins (C-TLs) class receptor for Mycobacterium tuberculosis (Mtb) found on the dendritic cell surface [8]
Summary
M. tuberculosis; C-TLs, C-type lectins; PSP, pulmonary surfactant protein; CRDs, carbohydrate recognition domains; ManLAMs, mannosylated lipoarabinomannans; BALF, bronchoalveolar lavage fluid; CSP, cell-surface protein; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid; h, human; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. In view of the great interest that such adhesins specific to the Mtb complex mycobacteria may have as potential targets for the development of new pharmacological competitors/inhibitors of the C-TL-mediated Mtb invasion [14], we reconsidered the presence in M. bovis BCG of still uncharacterized mycobacterial surface glycoproteins recognized by PSP-A With this aim, we developed an original functional proteomic strategy based on the use of crude human bronchoalveolar lavage fluid (BALF) from alveolar proteinosis patients (as a source of physiological native PSP-A) to detect and identify the putative mycobacterial surface ligand for PSP-A. Using this innovative lectin blot analysis approach combined with a specific extraction procedure for surface proteins, two-dimensional electrophoresis, and mass spectrometry peptide fingerprinting, we definitively confirmed the presence of such protein ligands that have been identified as the Apa protein complex
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