scholarly journals Leishmania major LACK Antigen Is Required for Efficient Vertebrate Parasitization

2003 ◽  
Vol 198 (11) ◽  
pp. 1689-1698 ◽  
Author(s):  
Ben L. Kelly ◽  
Daniel B. Stetson ◽  
Richard M. Locksley
Keyword(s):  
Drug Target ◽  
Leishmania Major ◽  
Genomic Organization ◽  
Vaccine Candidate ◽  
Single Copy ◽  
Wild Type ◽  
Immunodeficient Mice ◽  
Human Leishmaniasis ◽  
Parasite Viability

The Leishmania major LACK antigen is a key target of the immune response in susceptible BALB/c mice and remains a viable vaccine candidate for human leishmaniasis. We describe the genomic organization of the four lack genes in the L. major diploid genome together with results of selected lack gene targeting. Parasites containing a single lack gene in either the upstream or downstream locus grew comparably to wild-type promastigotes in vitro, but failed to parasitize BALB/c mice efficiently, even in a T cell–deficient environment. The replication of single copy lack mutants as amastigotes was attenuated in macrophages in vitro, and parasites failed to increase in numbers in immunodeficient mice, despite their persistence over months. Complementation with an additional lack copy was sufficient to induce robust lesion development, which also occurred using parasites with two lack genes. Conversely, attempts to generate lack-null parasites failed, suggesting that LACK is required for parasite viability. These data suggest that LACK is critical for effective mammalian parasitization and thus represents a potential drug target for leishmaniasis.

scholarly journals Farnesyl Diphosphate Synthase Is a Cytosolic Enzyme in Leishmania major Promastigotes and Its Overexpression Confers Resistance to Risedronate

2006 ◽  
Vol 5 (7) ◽  
pp. 1057-1064 ◽  
Author(s):  
Aurora Ortiz-Gómez ◽  
Carmen Jiménez ◽  
Antonio M. Estévez ◽  
Juana Carrero-Lérida ◽  
Luis M. Ruiz-Pérez ◽  
...  
Keyword(s):  
Drug Target ◽  
Leishmania Major ◽  
Intracellular Localization ◽  
Initial Step ◽  
Isoprenoid Biosynthesis ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type

ABSTRACT Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.

scholarly journals In Vitro Antileishmanial Activity of Nicotinamide

2005 ◽  
Vol 49 (2) ◽  
pp. 808-812 ◽  
Author(s):  
D. Sereno ◽  
A. Monte Alegre ◽  
R. Silvestre ◽  
B. Vergnes ◽  
A. Ouaissi
Keyword(s):  
Leishmania Major ◽  
Growth Arrest ◽  
Antileishmanial Activity ◽  
Vitamin B ◽  
Wild Type ◽  
Intracellular Growth ◽  
Cell Growth Arrest ◽  
Transgenic Parasites

ABSTRACT Our study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B3. A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmania amastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania major SIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.

scholarly journals Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain

Molecules ◽  
2018 ◽  
Vol 23 (3) ◽  
pp. 696 ◽  
Author(s):  
Masoud Keighobadi ◽  
Saeed Emami ◽  
Milad Lagzian ◽  
Mahdi Fakhar ◽  
Alireza Rafiei ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Structural Stability ◽  
In Silico ◽  
Leishmania Major ◽  
In Silico Analysis ◽  
Laboratory Strain ◽  
Wild Type ◽  
Silico Analysis

scholarly journals Migration-Inhibitory Factor Gene-Deficient Mice Are Susceptible to Cutaneous Leishmania major Infection

2001 ◽  
Vol 69 (2) ◽  
pp. 906-911 ◽  
Author(s):  
Abhay R. Satoskar ◽  
Marcelo Bozza ◽  
Miriam Rodriguez Sosa ◽  
Guoshing Lin ◽  
John R. David
Keyword(s):  
Migration Inhibitory Factor ◽  
Protective Immunity ◽  
Leishmania Major ◽  
Inhibitory Factor ◽  
Interleukin 4 ◽  
Wild Type ◽  
Leishmanicidal Activity ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT To determine the role of endogenous migration-inhibitory factor (MIF) in the development of protective immunity against cutaneous leishmaniasis, we analyzed the course of cutaneous Leishmania major infection in MIF gene-deficient mice (MIF−/−) and wild-type (MIF+/+) mice. Following cutaneous L. major infection, MIF−/− mice were susceptible to disease and developed significantly larger lesions and greater parasite burdens than MIF+/+ mice. Interestingly, antigen-stimulated lymph node cells from MIF−/− mice produced more interleukin-4 (IL-4) and gamma interferon (IFN-γ) than those from MIF+/+ mice, although the differences were statistically not significant. IFN-γ-activated resting peritoneal macrophages from MIF−/− mice showed impaired macrophage leishmanicidal activity and produced significantly lower levels of nitric oxide and superoxide in vitro. The macrophages from MIF−/− mice, however, produced much more IL-6 than macrophages from wild-type mice. These findings demonstrate that endogenous MIF plays an important role in the development of protective immunity against L. majorin vivo. Furthermore, they indicate that the susceptibility of MIF−/− mice to L. major infection is due to impaired macrophage leishmanicidal activity rather than dysregulation of Th1 and Th2 responses.

scholarly journals Disruption of the ech42 (Endochitinase-Encoding) Gene Affects Biocontrol Activity in Trichoderma harzianum P1

1999 ◽  
Vol 12 (5) ◽  
pp. 419-429 ◽  
Author(s):  
S. L. Woo ◽  
B. Donzelli ◽  
F. Scala ◽  
R. Mach ◽  
G. E. Harman ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Trichoderma Harzianum ◽  
Pythium Ultimum ◽  
Single Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
Culture Filtrates ◽  
In Vitro Antifungal Activity ◽  
Better Than

The biocontrol strain P1 of Trichoderma harzianum was genetically modified by targeted disruption of the single-copy ech42 gene encoding for the secreted 42-kDa endochitinase (CHIT42). Stable mutants in which ech42 was interrupted, and unable to produce CHIT42, were obtained and characterized. These mutants lacked the ech42 transcript, the protein, and endochitinase activity in culture filtrates, and they were unable to clear a medium containing colloidal chitin. Other chitinolytic and glucanolytic enzymes expressed during mycoparasitism were not affected by the disruption of ech42. The disrupted mutant D11 grew and sporulated similarly to the wild type. In vitro antifungal activity of the ech42 disruptant culture filtrates against Botrytis cinerea and Rhizoctonia solani was reduced about 40%, compared with wild type; antifungal activity was fully restored by adding an equivalent amount of CHIT42 as secreted by P1. The mutant exhibited the same biocontrol effect against Pythium ultimum as strain P1, but the antagonism against B. cinerea on bean leaves by the mutant was significantly reduced (33% less biocontrol), compared with strain P1. Conversely, the endochitinase-deficient mutant performed better than the wild type (16% improvement of survival) in biocontrol experiments in soil infested with the soilborne fungus R. solani. These results indicate that the antagonistic interaction between the T. harzianum strain and various fungal hosts is based on different mechanisms.

scholarly journals The Burkholderia pseudomallei ΔasdMutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice

2011 ◽  
Vol 79 (10) ◽  
pp. 4010-4018 ◽  
Author(s):  
Michael H. Norris ◽  
Katie L. Propst ◽  
Yun Kang ◽  
Steven W. Dow ◽  
Herbert P. Schweizer ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Cell Model ◽  
Single Copy ◽  
Wild Type ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Life Threatening ◽  
A Cell ◽  
Bioterrorism Agent

ABSTRACTBurkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently createdB. pseudomallei1026b Δasdmutantin vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of theasdgene, growth was restored to wild-type levels. Further characterization of theB. pseudomalleiΔasdmutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δasdmutant. RAW264.7 cells infected by the Δasdmutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-typeB. pseudomalleicell infections. The Δasdmutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, theB. pseudomalleiΔasdmutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list.

scholarly journals Chemokine CCL28 Is a Potent Therapeutic Agent for Oropharyngeal Candidiasis

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Jie He ◽  
Monica A. Thomas ◽  
Jaime de Anda ◽  
Michelle W. Lee ◽  
Emma Van Why ◽  
...  
Keyword(s):  
Therapeutic Agent ◽  
Opportunistic Infections ◽  
Membrane Disruption ◽  
Oropharyngeal Candidiasis ◽  
Wild Type ◽  
Content Type ◽  
Immunodeficient Mice ◽  
Drug Classes

ABSTRACT Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of Candida infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti-Candida activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of C. albicans. Polyamine protein transporters are not required for CCL28 anti-Candida activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to C. albicans results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.

scholarly journals Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates

2000 ◽  
Vol 74 (14) ◽  
pp. 6448-6458 ◽  
Author(s):  
Tao Tao ◽  
Mario H. Skiadopoulos ◽  
Fatemeh Davoodi ◽  
Jeffrey M. Riggs ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virus Type ◽  
Vaccine Candidate ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Parainfluenza Virus ◽  
Tail Domain ◽  
Wild Type ◽  
Vaccine Candidates

ABSTRACT We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuatedcp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.

scholarly journals Regulation of Mycobacterium tuberculosis whiB3 in the Mouse Lung and Macrophages

2006 ◽  
Vol 74 (11) ◽  
pp. 6449-6457 ◽  
Author(s):  
N. Banaiee ◽  
W. R. Jacobs ◽  
J. D. Ernst
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mouse Lung ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Environmental Signals ◽  
Immunodeficient Mice ◽  
Reverse Transcription Pcr ◽  
Factor Alpha

ABSTRACT Mycobacterium tuberculosis is a highly successful human pathogen, with ∼2 × 109 individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor−/−, Rag1−/−, and tumor necrosis factor alpha−/−) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.

scholarly journals Increased Glycolytic ATP Synthesis Is Associated with Tafenoquine Resistance inLeishmania major

2011 ◽  
Vol 55 (3) ◽  
pp. 1045-1052 ◽  
Author(s):  
José Ignacio Manzano ◽  
Luis Carvalho ◽  
José M. Pérez-Victoria ◽  
Santiago Castanys ◽  
Francisco Gamarro
Keyword(s):  
Leishmania Major ◽  
Alternative Therapy ◽  
Atp Synthesis ◽  
Drug Efflux ◽  
Wild Type ◽  
Mechanisms Of Resistance ◽  
Glycolytic Atp ◽  
Drug Free

ABSTRACTTafenoquine (TFQ), an 8-aminoquinoline used to treat and preventPlasmodiuminfections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity bothin vitroandin vivo, where it targetsLeishmaniamitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood thatLeishmaniawill develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistantLeishmania majorpromastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in allLeishmanialines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance inLeishmania.

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