Search tips
Search criteria

Results 1-7 (7)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs 
PLoS ONE  2013;8(10):e77930.
The failure of Mycobacterium bovis Bacille Calmette-Guérin to impart satisfactory protection against adult pulmonary tuberculosis has necessitated the development of more effective TB vaccines. The assumption that the vaccine strain should be antigenically as similar as possible to the disease causing pathogen has led to the evaluation of M.tuberculosis mutants as candidate tuberculosis vaccines.
Methods/Principal Findings
In this study, we have generated a mutant of M.tuberculosis (Mtb∆mms) by disrupting 3 virulence genes encoding a mycobacterial secretory acid phosphatase (sapM) and two phosphotyrosine protein phosphatases (mptpA and mptpB) and have evaluated its protective efficacy in guinea pigs. We observed that Mtb∆mms was highly attenuated in THP-1 macrophages. Moreover, no bacilli were recovered from the lungs and spleens of guinea pigs after 10 weeks of Mtb∆mms inoculation, although, initially, the mutant exhibited some growth in the spleens. Subsequently, when Mtb∆mms was evaluated for its protective efficacy, we observed that similar to BCG vaccination, Mtb∆mms exhibited a significantly reduced CFU in the lungs of guinea pigs when compared with the unvaccinated animals at 4 weeks after challenge. In addition, our observations at 12 weeks post challenge demonstrated that Mtb∆mms exhibited a more sustainable and superior protection in lungs as compared to BCG. However, the mutant failed to control the hematogenous spread as the splenic bacillary load between Mtb∆mms vaccinated and sham immunized animals was not significantly different. The gross pathological observations and histopathological observations corroborated the bacterial findings. Inspite of disruption of phosphatase genes in MtbΔmms, the lipid profiles of M.tuberculosis and MtbΔmms were identical indicating thereby that the phenotype of the mutant was ascribed to the loss of phosphatase genes and the influence was not related to any alteration in the lipid composition.
This study highlights the importance of M.tuberculosis mutants in imparting protection against pulmonary TB.
PMCID: PMC3799640  PMID: 24205032
2.  Perilipin Deficiency and Autosomal Dominant Partial Lipodystrophy 
The New England journal of medicine  2011;364(8):740-748.
Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.
PMCID: PMC3773916  PMID: 21345103
3.  In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for persistence in mice 
Nature medicine  2007;13(12):1515-1520.
The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system of healthy individuals. Identification of mechanisms that enable Mtb to persist is key to limiting latent tuberculosis, which affects one third of the world’s population. Here we demonstrate that conditional gene silencing makes it possible to determine if an Mtb gene that is required for optimal growth in vitro is also important for virulence, and during which phase of an infection it is required. Application of this approach to prcBA, which encode the core of the mycobacterial proteasome, reveals an unpredicted essentiality of the core proteasome for persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-γ-deficient mice, pointing to a function of the proteasome beyond defence against the adaptive immune response. Genes that are essential for growth in vitro and/or in vivo account for approximately 20% of Mtb’s genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.
PMCID: PMC3174471  PMID: 18059281
4.  Human Frame Shift Mutations Affecting the Carboxyl Terminus of Perilipin Increase Lipolysis by Failing to Sequester the Adipose Triglyceride Lipase (ATGL) Coactivator AB-hydrolase-containing 5 (ABHD5)* 
The Journal of Biological Chemistry  2011;286(40):34998-35006.
Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740–748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.
PMCID: PMC3186430  PMID: 21757733
Cell Biology; Lipase; Lipid Droplets; Lipid Metabolism; Lipodystrophy
5.  The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide 
PLoS Pathogens  2010;6(8):e1001040.
Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function.
Author Summary
The eukaryotic proteasome is ubiquitous and essential for many basic cellular processes. In contrast to most bacteria, which do not express a proteasome, Mycobacterium tuberculosis encodes a proteasome predicted to be essential or required for optimal growth of the pathogen. Genetic silencing of the proteasome core genes further suggested that the M. tuberculosis proteasome plays an important role in defense against nitric oxide and in persistence of the pathogen during chronic mouse infections. In this manuscript we generated a genetic deletion mutant of the proteasome core genes proving that the 20S proteasome is not essential for growth of M. tuberculosis. We complemented the proteasome knockout with a proteolytically active and a mutated, proteolysis defective proteasome. This revealed that proteasomal proteolysis is dispensable for in vitro and in vivo growth and nitric oxide resistance of M. tuberculosis and suggests that the proteasome core serves a proteolysis-independent function. In contrast, long-term survival of the pathogen in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. We further provide evidence that nitric oxide is not responsible for killing of the proteasome knockout during chronic mouse infections. Thus, proteasomal proteolysis facilitates mycobacterial persistence independently of defense against nitric oxide. We propose that the failure to survive starvation contributes to the impaired persistence of M. tuberculosis lacking a proteolytically active proteasome during chronic infections.
PMCID: PMC2920845  PMID: 20711362
6.  Nucleotide-Binding Oligomerization Domain Protein 2-Deficient Mice Control Infection with Mycobacterium tuberculosis▿  
Infection and Immunity  2007;75(11):5127-5134.
Nucleotide-binding oligomerization domain proteins (NODs) are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. NOD2 has recently been shown to be important for host cell cytokine responses to Mycobacterium tuberculosis, to synergize with Toll-like receptor 2 (TLR2) in mediating these responses, and thus to serve as a nonredundant recognition receptor for M. tuberculosis. Here, we demonstrate that macrophages and dendritic cells from NOD2-deficient mice were impaired in the production of proinflammatory cytokines and nitric oxide following infection with live, virulent M. tuberculosis. Mycolylarabinogalactan peptidoglycan (PGN), the cell wall core of M. tuberculosis, stimulated macrophages to release tumor necrosis factor (TNF) and interleukin-12p40 in a partially NOD2-dependent manner, and M. tuberculosis PGN required NOD2 for the optimal induction of TNF. However, NOD2-deficient mice were no more susceptible to infection with virulent M. tuberculosis than wild-type mice: they controlled the replication of M. tuberculosis in lung, spleen, and liver as well as wild-type mice, and both genotypes displayed similar lung pathologies. In addition, mice doubly deficient for NOD2 and TLR2 were similarly able to control an M. tuberculosis infection. Thus, NOD2 appears to participate in the recognition of M. tuberculosis by antigen-presenting cells in vitro yet is dispensable for the control of the pathogen during in vivo infection.
PMCID: PMC2168277  PMID: 17709422
7.  The Functionally Conserved Nucleoporins Nup124p from Fission Yeast and the Human Nup153 Mediate Nuclear Import and Activity of the Tf1 Retrotransposon and HIV-1 VprV⃞ 
Molecular Biology of the Cell  2005;16(4):1823-1838.
We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro–translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124pAA264–454 and Nup153AA448–634 that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124+) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.
PMCID: PMC1073664  PMID: 15659641

Results 1-7 (7)