Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response.

Protein O-mannosylation is crucial for the biology of Mycobacterium tuberculosis but the key mannosylated protein(s) involved and its(their) underlying function(s) remain unknown. Here, we demonstrated that the M. tuberculosis mutant (Δpmt) deficient for protein O-mannosylation exhibits enhanced release of lipoarabinomannan (LAM) in a complex with LprG, a lipoprotein required for LAM translocation to the cell surface. We determined that LprG is O-mannosylated at a unique threonine position by mass spectrometry analyses of the purified protein. However, although replacement of this amino acid by an alanine residue completely abolished LprG O-mannosylation, the increased release of the LAM/LprG complex was preserved. We found that the increased secretion of this complex is due to enhanced LAM production in the Δpmt M. tuberculosis and M. smegmatis mutants relative to their wild-type counterparts. This abnormal release of LAM/LprG has functional consequences on the induction of inflammatory responses and provides a possible explanation for the reduced virulence of the M. tuberculosis Δpmt mutant.

Innate immune response of alveolar macrophage to house dust mite allergen is mediated through TLR2/-4 co-activation.

House dust mite, Dermatophagoides pteronyssinus (Der p), is one of the major allergens responsible for allergic asthma. However, the putative receptors involved in the signalization of Der p to the innate immune cells are still poorly defined as well as the impact of their activation on the outcome of the allergen-induced cell response. We previously reported that the HDM activation of mouse alveolar macrophages (AM) involves the TLR4/CD14 cell surface receptor complex. Here using a TLR ligand screening essay, we demonstrate that HDM protein extract engages the TLR2, in addition to the TLR4, in engineered TLR-transfected HEK cells but also in the MH-S mouse alveolar macrophage cell line model. Moreover we found that the concomitant recruitment of the MH-S cell's TLR2 and TLR4 receptors by the HDM extract activates the MyD88-dependent signaling pathway and leads to the secretion of the NF-κB regulated pro-inflammatory factors NO and TNF-α. However unlike with the canonical TLR4 ligand (i.e. the bacterial LPS) mobilization of TLR4 by the HDM extract induces a reduced production of the IL-12 pro-inflammatory cytokine and fails to trigger the expression of the T-bet transcription factor. Finally we demonstrated that HDM extract down-regulates LPS induced IL-12 and T-bet expression through a TLR2 dependent mechanism. Therefore, we propose that the simultaneous engagement of the TLR2 and TLR4 receptors by the HDM extract results in a cross regulated original activation pattern of the AM which may contribute to the Th2 polarization of the allergen-induced immune response. The deciphering of these cross-regulation networks is of prime importance to open the way for original therapeutic strategies taking advantage of these receptors and their associated signaling pathways to treat allergic asthma.

Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis.

A posttranslational protein O-mannosylation process resembling that found in fungi and animals has been reported in the major human pathogen Mycobacterium tuberculosis (Mtb) and related actinobacteria. However, the role and incidence of this process, which is essential in eukaryotes, have never been explored in Mtb. We thus analyzed the impact of interrupting O-mannosylation in the nonpathogenic saprophyte Mycobacterium smegmatis and in the human pathogen Mtb by inactivating the respective putative protein mannosyl transferase genes Msmeg_5447 and Rv1002c. Loss of protein O-mannosylation in both mutant strains was unambiguously demonstrated by efficient mass spectrometry-based glycoproteomics analysis. Unexpectedly, although the M. smegmatis phenotype was unaffected by the lack of manno-proteins, the Mtb mutant had severely impacted growth in vitro and in cellulo associated with a strong attenuation of its pathogenicity in immunocompromised mice. These data are unique in providing evidence of the biological significance of protein O-mannosylation in mycobacteria and demonstrate the crucial contribution of this protein posttranslational modification to Mtb virulence in the host.

The effect of water-soluble chitosan on macrophage activation and the attenuation of mite allergen-induced airway inflammation.

Chitin and chitosan have versatile anti-tumor, anti-fungal, and antimicrobial biological properties. Oral intakes and intranasal administration of chitin attenuated allergen-induced airway inflammation in sensitized mice, which may be due to its Th1 adjuvant properties. However, their mechanism of action is not entirely clear. In this report, we demonstrate that water-soluble chitosan (WSC) has specific immunomodulatory effects on dust mite allergen Dermatophagoides farinae (Der f)-stimulated, monocyte-derived macrophages (MDM). These effects include polarizing the cytokine balance towards Th1 cytokines, decreasing the production of the inflammatory cytokines IL-6 and TNF-alpha, down-regulating CD44 and TLR4 receptor expression, and inhibiting T cell proliferation. Scanning electron microscope (SEM) examination found that WSC reduced the rate of pseudopodia formation in Der f-stimulated MDM from allergic asthma patients. The effect of WSC on allergen-stimulated MDM may be mediated via inhibition of PKCzeta phosphorylation and NF-kappaB pathway activation. In a murine model of asthma, we found that intranasal application of WSC attenuates Der f-induced lung inflammation by reducing infiltration of inflammatory cells, epithelial damage, and goblet cell hyperplasia and markedly decreasing production of Arg I, iNOs, and thymic stromal lymphopoietin (TSLP) in the bronchial epithelium. Therefore, we believe that WSC may provide a new therapeutic modality for allergic asthma.