Dodecameric structure of a small heat shock protein from Mycobacterium marinum M.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones present ubiquitously in all kingdoms of life. Their low molecular weight subunits associate to form higher order structures. Under conditions of stress, sHSPs prevent aggregation of substrate proteins by undergoing rapid changes in their conformation or stoichiometry. Polydispersity and dynamic nature of these proteins have made structural investigations through crystallography a daunting task. In pathogens like Mycobacteria, sHSPs are immuno-dominant antigens, enabling survival of the pathogen within the host and contributing to disease persistence. We characterized sHSPs from Mycobacterium marinum M and determined the crystal structure of one of these. The protein crystallized in three different conditions as dodecamers, with dimers arranged in a tetrahedral fashion to form a closed cage-like architecture. Interestingly, we found a pentapeptide bound to the dodecamers revealing one of the modes of sHSP-substrate interaction. Further, we have observed that ATP inhibits the chaperoning activity of the protein.

Enhanced Permeability and Retention-like Extravasation of Nanoparticles from the Vasculature into Tuberculosis Granulomas in Zebrafish and Mouse Models.

The enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NPs) flowing in blood to reach tumors by a passive targeting mechanism. Here, using the transparent zebrafish model infected with Mycobacterium marinum we show that an EPR-like process also occurs allowing different types of NPs to extravasate from the vasculature to reach granulomas that assemble during tuberculosis (TB) infection. PEGylated liposomes and other NP types cross endothelial barriers near infection sites within minutes after injection and accumulate close to granulomas. Although ∼100 and 190 nm NPs concentrated most in granulomas, even ∼700 nm liposomes reached these infection sites in significant numbers. We show by confocal microscopy that NPs can concentrate in small aggregates in foci on the luminal side of the endothelium adjacent to the granulomas. These spots are connected to larger foci of NPs on the ablumenal side of these blood vessels. EM analysis suggests that NPs cross the endothelium via the paracellular route. PEGylated NPs also accumulated efficiently in granulomas in a mouse model of TB infection with Mycobacterium tuberculosis, arguing that the zebrafish embryo model can be used to predict NP behavior in mammalian hosts. In earlier studies we and others showed that uptake of NPs by macrophages that are attracted to infection foci is one pathway for NPs to reach TB granulomas. This study reveals that when NPs are designed to avoid macrophage uptake, they can also efficiently target granulomas via an alternative mechanism that resembles EPR.

Quantitative N-Terminal Footprinting of Pathogenic Mycobacteria Reveals Differential Protein Acetylation.

N-terminal acetylation (NTA) is a post-transcriptional modification of proteins that is conserved from bacteria to humans. In bacteria, the enzymes that mediate protein NTA also promote antimicrobial resistance. In pathogenic mycobacteria, which cause human tuberculosis and other chronic infections, NTA has been linked to pathogenesis and stress response, yet the fundamental biology underlying NTA of mycobacterial proteins remains unclear. We enriched, defined, and quantified the NT-acetylated populations of both cell-associated and secreted proteins from both the human pathogen, Mycobacterium tuberculosis, and the nontuberculous opportunistic pathogen, Mycobacterium marinum. We used a parallel N-terminal enrichment strategy from proteolytic digests coupled to charge-based selection and stable isotope ratio mass spectrometry. We show that NTA of the mycobacterial proteome is abundant, diverse, and primarily on Thr residues, which is unique compared with other bacteria. We isolated both the acetylated and unacetylated forms of 256 proteins, indicating that NTA of mycobacterial proteins is homeostatic. We identified 16 mycobacterial proteins with differential levels of NTA on the cytoplasmic and secreted forms, linking protein modification and localization. Our findings reveal novel biology underlying the NTA of mycobacterial proteins, which may provide a basis to understand NTA in mycobacterial physiology, pathogenesis, and antimicrobial resistance.

Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of Mycobacterium marinum from THP1-derived macrophages.

BACKGROUND: Although mycobacterial glycolipids are among the first-line molecules involved in host-pathogen interactions, their contribution in virulence remains incomplete. Mycobacterium marinum is a waterborne pathogen of fish and other ectotherms, closely related to Mycobacterium tuberculosis. Since it causes tuberculosis-like systemic infection it is widely used as a model organism for studying the pathogenesis of tuberculosis. It is also an occasional opportunistic human pathogen. The M. marinum surface-exposed lipooligosaccharides (LOS) are immunogenic molecules that participate in the early interactions with macrophages and modulate the host immune system. Four major LOS species, designated LOS-I to LOS-IV, have been identified and characterized in M. marinum. Herein, we investigated the interactions between a panel of defined M. marinum LOS mutants that exhibited various degrees of truncation in the LOS structure, and human-derived THP-1 macrophages to address the potential of LOSs to act as pro- or avirulence factors. RESULTS: A moderately truncated LOS structure did not interfere with M. marinum invasion. However, a deeper shortening of the LOS structure was associated with increased entry of M. marinum into host cells and increased elimination of the bacilli by the macrophages. These effects were dependent on Toll-like receptor 2. CONCLUSION: We provide the first evidence that LOSs inhibit the interaction between mycobacterial cell wall ligands and appropriate macrophage pattern recognition receptors, affecting uptake and elimination of the bacteria by host phagocytes.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for top-down characterization of the Mycobacterium marinum secretome.

Capillary zone electrophoresis (CZE) with an electrokinetically pumped sheath-flow nanospray interface was coupled with a high-resolution Q-Exactive mass spectrometer for the analysis of culture filtrates from Mycobacterium marinum. We confidently identified 22 gene products from the wildtype M. marinum secretome in a single CZE-tandem mass spectrometry (MS/MS) run. A total of 58 proteoforms were observed with post-translational modifications including signal peptide removal, N-terminal methionine excision, and acetylation. The conductivities of aqueous acetic acid and formic acid solutions were measured from 0.1% to 100% concentration (v/v). Acetic acid (70%) provided lower conductivity than 0.25% formic acid and was evaluated as low ionic-strength and a CZE-MS compatible sample buffer with good protein solubility.

Structural determination and Toll-like receptor 2-dependent proinflammatory activity of dimycolyl-diarabino-glycerol from Mycobacterium marinum.

Although it was identified in the cell wall of several pathogenic mycobacteria, the biological properties of dimycolyl-diarabino-glycerol have not been documented yet. In this study an apolar glycolipid, presumably corresponding to dimycolyl-diarabino-glycerol, was purified from Mycobacterium marinum and subsequently identified as a 5-O-mycolyl-ß-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1')-glycerol (designated Mma_DMAG) using a combination of nuclear magnetic resonance spectroscopy and mass spectrometry analyses. Lipid composition analysis revealed that mycolic acids were dominated by oxygenated mycolates over α-mycolates and devoid of trans-cyclopropane functions. Highly purified Mma_DMAG was used to demonstrate its immunomodulatory activity. Mma_DMAG was found to induce the secretion of proinflammatory cytokines (TNF-α, IL-8, IL-1ß) in human macrophage THP-1 cells and to trigger the expression of ICAM-1 and CD40 cell surface antigens. This activation mechanism was dependent on TLR2, but not on TLR4, as demonstrated by (i) the use of neutralizing anti-TLR2 and -TLR4 antibodies and by (ii) the detection of secreted alkaline phosphatase in HEK293 cells co-transfected with the human TLR2 and secreted embryonic alkaline phosphatase reporter genes. In addition, transcriptomic analyses indicated that various genes encoding proinflammatory factors were up-regulated after exposure of THP-1 cells to Mma_DMAG. Importantly, a wealth of other regulated genes related to immune and inflammatory responses, including chemokines/cytokines and their respective receptors, adhesion molecules, and metalloproteinases, were found to be modulated by Mma_DMAG. Overall, this study suggests that DMAG may be an active cell wall glycoconjugate driving host-pathogen interactions and participating in the immunopathogenesis of mycobacterial infections.