Expression and purification of recombinant methylated HBHA in<i>Mycobacterium smegmatis</i>

Delogu, Giovanni; Bua, Alessandra; Pusceddu, Cinzia; Parra, Marcela; Fadda, Giovanni; Brennan, Michael J.; Zanetti, Stefania Anna Lucia

doi:10.1016/j.femsle.2004.08.015

Cited by 52 publications

(61 citation statements)

References 23 publications

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“…Expression of Rv0805 in M. smegmatis-Rv0805 and its mutants were expressed under the Rv0805 gene promoter in M. smegmatis using derivatives of the pMV-10 -25 plasmid (23). Generation of plasmid constructs for expression of Rv0805, Rv0805N97A, Rv0805⌬40, and Rv0805⌬40N97A have been described earlier (19).…”

Section: Methodsmentioning

confidence: 99%

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Matange

Podobnik

Visweswariah

2014

Journal of Biological Chemistry

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Background: Metallophosphoesterases possess unique, poorly characterized cap domains located at the entrance to the active site. Results: The C terminus of the cap domain of the Rv0805 metallophosphoesterase modulates its expression, cell envelope localization, and biological functions. Conclusion:The functions of the Rv0805 metallophosphoesterase are regulated by the cap domain. Significance: Cap domains may provide catalysis-independent features to metallophosphoesterases that shape their cellular functions.

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Section: Methodsmentioning

confidence: 99%

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Matange

Podobnik

Visweswariah

2014

Journal of Biological Chemistry

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“…Dose-response experiments revealed that optimum IFN-c secretion occurred at a HBHA dose of 100 mg?mL -1 using peripheral mononuclear cells. Methylated HBHA antigen was produced in cultures of Mycobacterium smegmatis 3.38 as previously described [20]. 4) IFN-c ELISPOT responses to purified protein derivative (PPD; 100 mg?mL -1 ; Staten Serum Institute, Copenhagen, Denmark) were performed using IFN-c pre-coated ELISPOT plates.…”

Section: Methodsmentioning

confidence: 99%

Utility of quantitative T-cell responsesversusunstimulated interferon-γ for the diagnosis of pleural tuberculosis

Dheda

Zyl-Smit²,

Sechi³

et al. 2009

Eur Respir J

110

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The clinical utility of antigen-specific interferon (IFN)-c release assays (IGRAs) using pleural mononuclear cells, for the diagnosis of tuberculosis (TB), requires clarification.We compared the diagnostic utility of unstimulated pleural IFN-c levels with several pleural antigen-specific T-cell IGRAs (early secretory antigenic target-6 and culture filtrate protein-10 (T-SPOT.1TB, QuantiFERON1-TB Gold In-tube), purified protein derivative (PPD) and heparinbinding haemagglutinin (HBHA)) in 78 South African TB suspects. Test results were compared against a clinical score and a reference standard.Out of 74 evaluable subjects 48, seven and 19 had definite, probable and no TB, respectively. 11 (15%) out of 74 pleural samples (nine (19%) out of 48 of the definite TB cases) had total cell counts that were inadequate for T-cell processing. In the remaining 63 samples, the sensitivity, specificity, positive predictive value and negative predictive value of different diagnostic methods were as follows. Maximal bioclinical score: 54, 89, 92 and 43%, respectively; T-SPOT.1TB: 86, 60, 84 and 64%, respectively; QuantiFERON1-TB Gold In-tube: 57, 80, 87 and 44%, respectively; HBHA-specific IGRA: 59, 31, 64 and 27%, respectively; PPD-specific IGRA: 81, 40, 76 and 46%, respectively; and pleural fluid unstimulated IFN-c: 97, 100, 100 and 94%, respectively. Unstimulated IFN-c was the most accurate test for distinguishing TB from non-TB effusions in a high-burden setting. The antigen-specific T-cell IGRAs were limited by suboptimal accuracy and the inability to isolate sufficient mononuclear cells to perform the assay.

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“…1-318 and Rv0805 1-278 coding sequences were amplified as described earlier (6), and cloned into pMV10 -25 (22), to allow expression of Rv0805 under the mycobacterial hsp60 gene promoter. A control vector (pMV vector control) was generated by digesting pMV10 -25 with NheI and XbaI and religating the vector backbone.…”

Section: ϩmentioning

confidence: 99%

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Podobnik

Tyagi

Matange

et al. 2009

Journal of Biological Chemistry

110

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Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.Mycobacterium tuberculosis is probably one of the most successful human pathogens known so far, being singly responsible for the largest number of deaths worldwide due to an infectious disease. M. tuberculosis is phagocytosed by the macrophage and is able to subvert the defenses of the host by a number of mechanisms. These include the presence of a complex cell wall that prevents free passage of potentially toxic material, the ability of the bacteria to withstand the acidic environment of the phagolysosome, and to neutralize reactive oxygen and nitrogen species produced by the activated macrophage (1). An increased understanding of mechanisms utilized by this pathogen to evade the host immune system and continue to reside in the hostile environment of the macrophage, would no doubt provide avenues for the development of drugs to novel targets in the bacterium.Cross-communication between the pathogen and host could involve the utilization of signaling molecules that are conserved evolutionarily. Cyclic AMP is found in all kingdoms of life, and proteins that synthesize and degrade the cyclic nucleotide have been well characterized. The genome of M. tuberculosis H37Rv encodes 16 mammalian-like nucleotide cyclase-like genes (2), and intracellular and extracellular levels of cAMP are very high in both pathogenic and non-pathogenic (e.g. Mycobacterium smegmatis) mycobacteria (2, 3). Recent evidence has highlighted the importance of cAMP in modulating the host macrophage response to M. tuberculosis infection (4). A single adenylyl cyclase was shown to be responsible for the burst of cAMP that is seen in the macrophage following phagocytosis of M. tuberculosis, and this bacterially derived increase in cAMP was essential to attenuate the response of the macrophage to the pathogen.The regulated degradation of cAMP is as important as its synthesi...

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Expression and purification of recombinant methylated HBHA inMycobacterium smegmatis

Cited by 52 publications

References 23 publications

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

The Non-catalytic “Cap Domain” of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Utility of quantitative T-cell responsesversusunstimulated interferon-γ for the diagnosis of pleural tuberculosis

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

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