ABSTRACT
BACKGROUND: Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene. RESULTS: Herein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF. CONCLUSION: These results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis.
Subject(s)
Bacterial Proteins/chemistry , Cysteine/chemistry , Isomerases/chemistry , Isomerases/metabolism , Mycobacterium tuberculosis/chemistry , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Cysteine/metabolism , Disulfides/chemistry , Disulfides/metabolism , Genes, Bacterial , Isomerases/genetics , Models, Molecular , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oxidoreductases/genetics , Protein Refolding , Protein Structure, Secondary , Protein Structure, Tertiary , Proteome , Sequence Homology, Amino Acid , Substrate Specificity , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
While the majority of medical inpatients in Uganda are assumed to be HIV-positive, HIV testing is limited in inpatient settings. This study describes HIV testing practices and risk behavior among medical inpatients at an urban hospital in Uganda. We interviewed 395 adults on the day of discharge. Overall, 46% tested for HIV before or during admission. Of the 20% tested during hospitalization, 64% were HIV-positive. Among 47% who had sex in the previous year, only 14% used condoms consistently and only 20% knew the HIV status of their sexual partner, indicating that participants would benefit from risk-reduction counseling. Yet, only 26% of participants tested during hospitalization received post-test counseling. Half of the participants with HIV-related illnesses left the hospital without being offered the test, a missed opportunity for HIV prevention counseling and care. The findings indicate that hospitals are important venues for HIV counseling and testing.