Ph.D Scholar, School of Biotechnology, KIIT University
Mycobacterial lipoprotein, activator of innate and adaptive immune responese
Bacterial lipoproteins are known TLR-2 ligands, able to activate both innate and adaptive immune wings. Mtb genome codes for several such lipoproteins, yet to be studied. Here we establish the role of a previously uncharacterized mycobacterial lipoprotein in intracellular survival. Here we aim to establish the role of Mtb lipoprotein in modulating host immune response in human macrophages. To elucidate the mechanism behind increased bacterial survival we aim to study the signalling nexus regulating the immunomodulatory activity of VitD3 i.e. cathelicidin expression. Methods Wild type CDC155, MtbΔLpp and Complemented stains were used to infect human THP-1 cells. Intracellular survival in human macrophages would be checked by CFU assay. Antimicrobial responses were evaluated by checking expression of LL-37 and its upstream signalling molecules by qRT PCR and Western Blotting. Role of TLR-2 in innate response was ascertained by silencing TLR-2 in human macrophages. Effect on phagosomal maturation was studied by checking for expression of early and phagosomal markers such as EEA1, LAMP1 and Rab7 respectively. Decreased survival in MtbΔLpp and wild type levels of survival in complemented strains indicated at role played by Lpp in intracellular survival. Increased survival was due to TLR-2 mediated decreased expression of cathelicidin inside human macrophages. Furthermore inhibition of early endosomal (EEA1) and late endosomal (Rab7, LAMP1) markers showed that Mtb lipoprotein aids in blocking phagosomal maturation Collectively, our findings identified a new Mtb lipoprotein that could be used by pathogenic Mtb to alter host immune response and survive inside host cells.
Abstract: Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.
Pub.: 28 Jul '11, Pinned: 29 Jul '17
Abstract: Lipoproteins of Mycobacterium tuberculosis (M. tuberculosis) represent an important class of cell envelop proteins. On the whole, 99 putative lipoproteins have been identified in the genome of M. tuberculosis. Earlier investigations on individual mycobacterial lipoproteins demonstrate that some lipoproteins elicit a strong immune response, while others are virulence factors in different models of infection. LpqS is an uncharacterized lipoprotein encoded by the open reading frame Rv0847 of M. tuberculosis. In the present study, we have characterized this putative lipoprotein LpqS with respect to the virulence of M. tuberculosis. A mutant of the M. tuberculosis H37Rv strain not producing LpqS (ΔlpqS) was generated by specialized transduction. The deletion mutant showed reduced growth in Sauton's minimal media and was highly sensitive to SDS and copper, compared to the wild type when grown on solid media. In vitro infection studies showed that the mutant was attenuated for growth in PMA-activated THP-1 cells. Complementation of the mutant with a single copy of the gene cloned under the hsp60 promoter partially restored the phenotype of the wild type strain H37Rv. Thus lpqS plays an important role in sensing the host macrophage environment and might be required for the intracellular survival of M. tuberculosis. Cotranscription of lpqS with the genes downstream cysK2, Rv0849 and Rv0850 was also demonstrated by Reverse transcription PCR (RT-PCR) of intergenic regions.
Pub.: 09 Apr '13, Pinned: 29 Jul '17
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