Metabolic and Bactericidal Effects of Targeted Suppression of NadD and NadE Enzymes in Mycobacteria

ABSTRACT Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by 13C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets

Plasmodium falciparum merozoite surface and rhoptry proteins as malaria vaccine candidates

Thesis (Ph. D.)--University of Hawaii at Manoa, 1992.Includes bibliographical references (leaves 126-137)Microfiche.x, 137 leaves, bound ill. (some col.) 29 cmPlasmodium falciparum merozoite surface proteins were isolated from in vitro cultured parasites using monoclonal antibody (mAb) 5.2. Serum samples from rabbits immunized with the native merozoite surface precursor glycoprotein (gp195) had a mean EUSA titer of 1/560,000 against native gp195 but only 1/890 to denatured, reduced and alkylated (dR/A) gp195, while rabbits immunized with dR/A gp195 had mean ELISA titers of 1/23,100 and 1/14,650 to native gpI95 and dR/A gp195, respectively. Serum samples from rabbits immunized with native gp195 had a mean parasite growth inhibition of 87% and recognized the 42 and 19 kDa C-terminal processing fragments. Serum samples from rabbits immunized with dR/A gp195 did not inhibit parasite growth in vitro and poorly recognized the native C-terminal processing fragment. This study suggests that recombinant or synthetic peptide gp195 subunit vaccines must contain the native secondary and/or tertiary protein structures. Forty-five gp195-specific mAbs were produced in BALB/c, C57/B1.10, or Swiss-Webster mice using Freund's complete adjuvant (FCA) or Lipid A-15 PH. Of 26 mAbs tested for inhibition of parasite growth in vitro, two mAbs (CE2 and EB2) inhibited parasite growth partially (59% and 52%, respectively) at high concentrations (500 µg/ml). These antibodies recognized linear, group specific epitope(s) on the 83 kDa N-terminal processing fragment. All other mAb, including mAb 5.2 and two others to the C-terminal 19 kDa processing fragment, were weakly or non-inhibitory. Combinations of N-terminal or C-terminal mAb were also not inhibitory. The inability to produce a mAb which inhibits parasite growth at low concentration (1-10 µg/ml) is discussed. MAb AC9 was produced and used to isolate the 80, 70 and 40 kDa rhoptry associated protein-1 (RAP-1) complex. The isolated RAP-1 proteins reacted with mAb 30c13, previously used by Perrin to isolate a protective 42 kDa protein and by Braun-Breton to isolate a rhoptry-associated serine protease, as well as mAb 2.13, previously used by Ridley to demonstrate induction of protective immunity by RAP-1. In addition, mAb 219.5 was used to isolate the 140, 130, and 105 kDa RAP-3 complex. Both RAP-1 and RAP-3, along with gpI95 as a control, were used to immunize rabbits. Serum samples from rabbits immunized with gp195, RAP-1, and RAP-3 inhibited parasite growth in vitro 87%, 89%, and 89%, respectively. These results suggest that RAP-3 as well as RAP-I, should be investigated as a possible blood-stage malaria vaccine candidates. Three matrix metalloproteinases (MMP's) having relative molecular weight (Mr) 220, 95 and 75K in gelatin zymograms were found to be associated with RAP-1, but not RAP-3 or gp195. The MMP activity was inhibited by EDTA but not PMSF and was restored by addition of calcium. Of eight divalent metal cations tested, 0.1 mM cobalt optimized gelatinolytic activity when combined with 1.0 mM calcium. Gelatinases having the same M, were also precipitated by antiserums to normal human macrophage and fibroblast MMP's. Therefore, these MMP's are believed to be host enzymes non-covalently associated with RAP-1 proteins

Suppression of human immunodeficiency virus type 1 replication by a soluble factor produced by CD8+ lymphocytes from HIV-2-infected baboons

Human immunodeficiency virus type 2 (HIV-2)-infected baboons (Papio cynocephalus) provide a valuable animal model for the study of acquired immunodefidency syndrome (AIDS) pathogenesis since many features of disease progression resemble HIV-1-infection of humans. In some HIV-2-infected baboons that are clinically healthy, a CD8+ cell antiviral response, that is partly mediated by a soluble factor, controls viral replication in vitro. In the present study, we demonstrate that CD8+ cells derived from HIV-2-infected baboon peripheral blood, lymph nodes, adenoids and tonsils had antiviral activity in co-cultures of CD8+ and CD4+ cells that inversely correlates with viral load. A soluble factor was found to be active against the chemokine-resistant, syncytium-inducing HIV-1 SF2 and HIV-1 SF33 isolates and was relatively heat stable at 100°C for 10 min. Moreover, inhibition of the transcription from the long terminal repeat of HIV-1 was observed in 1G5 cells after activation with phorbol 12-myristate 13-acetate. Therefore, the soluble suppressing activity of CD8+ cells in HIV-2-infected baboons may be analogous to the CD8+ cell antiviral factor described in human HIV-infected asymptomatic people

The CD8+ T Cell Noncytotoxic Antiviral Responses.

The CD8+ T cell noncytotoxic antiviral response (CNAR) was discovered during studies of asymptomatic HIV-infected subjects more than 30 years ago. In contrast to CD8+ T cell cytotoxic lymphocyte (CTL) activity, CNAR suppresses HIV replication without target cell killing. This activity has characteristics of innate immunity: it acts on all retroviruses and thus is neither epitope specific nor HLA restricted. The HIV-associated CNAR does not affect other virus families. It is mediated, at least in part, by a CD8+ T cell antiviral factor (CAF) that blocks HIV transcription. A variety of assays used to measure CNAR/CAF and the effects on other retrovirus infections are described. Notably, CD8+ T cell noncytotoxic antiviral responses have now been observed with other virus families but are mediated by different cytokines. Characterizing the protein structure of CAF has been challenging despite many biologic, immunologic, and molecular studies. It represents a low-abundance protein that may be identified by future next-generation sequencing approaches. Since CNAR/CAF is a natural noncytotoxic activity, it could provide promising strategies for HIV/AIDS therapy, cure, and prevention

The CD8+ T Cell Noncytotoxic Antiviral Responses.

The CD8+ T cell noncytotoxic antiviral response (CNAR) was discovered during studies of asymptomatic HIV-infected subjects more than 30 years ago. In contrast to CD8+ T cell cytotoxic lymphocyte (CTL) activity, CNAR suppresses HIV replication without target cell killing. This activity has characteristics of innate immunity: it acts on all retroviruses and thus is neither epitope specific nor HLA restricted. The HIV-associated CNAR does not affect other virus families. It is mediated, at least in part, by a CD8+ T cell antiviral factor (CAF) that blocks HIV transcription. A variety of assays used to measure CNAR/CAF and the effects on other retrovirus infections are described. Notably, CD8+ T cell noncytotoxic antiviral responses have now been observed with other virus families but are mediated by different cytokines. Characterizing the protein structure of CAF has been challenging despite many biologic, immunologic, and molecular studies. It represents a low-abundance protein that may be identified by future next-generation sequencing approaches. Since CNAR/CAF is a natural noncytotoxic activity, it could provide promising strategies for HIV/AIDS therapy, cure, and prevention