Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates

Parasitology ◽  
1999 ◽  
Vol 118 (5) ◽  
pp. 461-467 ◽  
Author(s):  
A. MONDRAGON ◽  
S. R. WILKINSON ◽  
M. C. TAYLOR ◽  
J. M. KELLY
Keyword(s):  
Melting Point ◽  
Trypanosoma Cruzi ◽  
Solid Medium ◽  
Logarithmic Phase ◽  
Wild Type ◽  
Experimental Procedure ◽  
Plating Method ◽  
Late Logarithmic Phase ◽  
Significant Step ◽  
Cell Inoculum

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0·65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28°C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0·6% agarose overlay containing G418 as the selective drug, on top of a 0·8% agar base. Transformation efficiencies were as high as 10−5 cells per μg of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of ‘shotgun transformation’ to generate libraries of T. cruzi recombinants.

scholarly journals Effects of cofD gene knock-out on the methanogenesis of Methanobrevibacter ruminantium

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jian Ma ◽  
Xueying Wang ◽  
Ting Zhou ◽  
Rui Hu ◽  
Huawei Zou ◽  
...  
Keyword(s):  
Growth Rate ◽  
Specific Growth ◽  
Production Capacity ◽  
Maximum Amount ◽  
Logarithmic Phase ◽  
Wild Type ◽  
Knock Out ◽  
Maximum Biomass ◽  
Reproductive Ability ◽  
Methanobrevibacter Ruminantium

AbstractThis study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

scholarly journals Inducible Nitric Oxide Synthase Is Not Essential for Control of Trypanosoma cruzi Infection in Mice

2004 ◽  
Vol 72 (7) ◽  
pp. 4081-4089 ◽  
Author(s):  
Kara L. Cummings ◽  
Rick L. Tarleton
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Trypanosoma Cruzi ◽  
Inducible Nitric Oxide Synthase ◽  
Interleukin 1 ◽  
Mouse Strains ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Immune control of many intracellular pathogens, including Trypanosoma cruzi, is reported to be dependent on the production of nitric oxide. In this study, we show that mice deficient in inducible nitric oxide synthase (iNOS or NOS2) exhibit resistance to T. cruzi infection that is comparable to that of wild-type mice. This is the case for two iNOS-deficient mouse strains, Nos2tm1Lau and Nos2 N5, infected with the Brazil or Tulahuen strain of T. cruzi. In all cases, blood parasitemia, tissue parasite load, and survival rates are similar between wild-type and iNOS-deficient mice. In contrast, both wild-type and Nos2tm1Lau mice died within 32 days postinfection when treated with the nitric oxide synthase inhibitor aminoguanidine. Increased transcription of NOS1 or NOS3 is not found in iNOS-knockout (KO) mice, indicating that the absence of nitric oxide production through iNOS is not compensated for by increased production of other NOS isoforms. However, Nos2tm1Lau mice exhibit enhanced expression of tumor necrosis factor alpha, interleukin-1, and macrophage inflammatory protein 1α compared to that of wild-type mice, and these alterations may in part compensate for the lack of iNOS. These results clearly show that iNOS is not required for control of T. cruzi infection in mice.

scholarly journals Light Gradient-Based Screening of Arabidopsis thaliana on a 384-Well Type Plant Array Chip

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 191
Author(s):  
Youn-Hee Park ◽  
Je-Kyun Park
Keyword(s):  
Arabidopsis Thaliana ◽  
White Light ◽  
Solid Medium ◽  
Red Light ◽  
Phytochrome B ◽  
Wild Type ◽  
Light Gradient ◽  
Gradient Based ◽  
Light Effects ◽  
Germination And Growth

Arabidopsis thaliana (Arabidopsis), as a model for plant research, is widely used for various aspects of plant science. To provide a more sophisticated and microscopic environment for the germination and growth of Arabidopsis, we report a 384-well type plant array chip in which each Arabidopsis seed is independently seeded in a solid medium. The plant array chip is made of a poly(methyl methacrylate) (PMMA) acrylic material and is assembled with a home-made light gradient module to investigate the light effects that significantly affect the germination and growth of Arabidopsis. The light gradient module was used to observe the growth pattern of seedlings according to the intensity of the white light and to efficiently screen for the influence of the white light. To investigate the response to red light (600 nm), which stimulates seed germination, the light gradient module was also applied to the germination test. As a result, the germination results showed that the plant array chip can be used to simultaneously screen wild type seeds and phytochrome B mutant seeds on a single array chip according to the eight red light intensities.

scholarly journals The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro

2020 ◽  
Vol 114 (7) ◽  
pp. 492-498 ◽  
Author(s):  
Gabriele Sass ◽  
Laura C Miller Conrad ◽  
Terrence-Thang H Nguyen ◽  
David A Stevens
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Iron Acquisition ◽  
Vero Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Current Standard ◽  
Cruzi Infection

Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

scholarly journals Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

2015 ◽  
Vol 59 (8) ◽  
pp. 4669-4679 ◽  
Author(s):  
Nilmar Silvio Moretti ◽  
Leonardo da Silva Augusto ◽  
Tatiana Mordente Clemente ◽  
Raysa Paes Pinto Antunes ◽  
Nobuko Yoshida ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Drug Targets ◽  
Wild Type ◽  
Content Type ◽  
Damage Repair ◽  
Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

scholarly journals Expression of a Mitochondrial Peroxiredoxin Prevents Programmed Cell Death in Leishmania donovani

2006 ◽  
Vol 5 (5) ◽  
pp. 861-870 ◽  
Author(s):  
Simone Harder ◽  
Meike Bente ◽  
Kerstin Isermann ◽  
Iris Bruchhaus
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Mitochondrial Membrane ◽  
Leishmania Donovani ◽  
Physiological Role ◽  
Logarithmic Phase ◽  
Insect Vector ◽  
Oxygen Species ◽  
Late Logarithmic Phase

ABSTRACT Leishmania promastigote cells transmitted by the insect vector get phagocytosed by macrophages and convert into the amastigote form. During development and transformation, the parasites are exposed to various concentrations of reactive oxygen species, which can induce programmed cell death (PCD). We show that a mitochondrial peroxiredoxin (LdmPrx) protects Leishmania donovani from PCD. Whereas this peroxiredoxin is restricted to the kinetoplast area in promastigotes, it covers the entire mitochondrion in amastigotes, accompanied by dramatically increased expression. A similar change in the expression pattern was observed during the growth of Leishmania from the early to the late logarithmic phase. Recombinant LdmPrx shows typical peroxiredoxin-like enzyme activity. It is able to detoxify organic and inorganic peroxides and prevents DNA from hydroxyl radical-induced damage. Most notably, Leishmania parasites overexpressing this peroxiredoxin are protected from hydrogen peroxide-induced PCD. This protection is also seen in promastigotes grown to the late logarithmic phase, also characterized by high expression of this peroxiredoxin. Apparently, the physiological role of this peroxiredoxin is stabilization of the mitochondrial membrane potential and, as a consequence, inhibition of PCD through removal of peroxides.

scholarly journals The ExsY Protein Is Required for Complete Formation of the Exosporium of Bacillus anthracis

2006 ◽  
Vol 188 (21) ◽  
pp. 7440-7448 ◽  
Author(s):  
Jeremy A. Boydston ◽  
Ling Yue ◽  
John F. Kearney ◽  
Charles L. Turnbough
Keyword(s):  
Bacillus Anthracis ◽  
Solid Medium ◽  
Basal Layer ◽  
Wild Type ◽  
Nucleoside Hydrolase ◽  
Spore Resistance ◽  
Normal Hair ◽  
Spore Outgrowth ◽  
Microscopic Inspection ◽  
Complete Formation

ABSTRACT The outermost layer of the Bacillus anthracis spore is the exosporium, which is composed of a paracrystalline basal layer and an external hair-like nap. The filaments of the nap are formed by a collagen-like glycoprotein called BclA, while the basal layer contains several different proteins. One of the putative basal layer proteins is ExsY. In this study, we constructed a ΔexsY mutant of B. anthracis, which is devoid of ExsY, and examined the assembly of the exosporium on spores produced by this strain. Our results show that exosporium assembly on ΔexsY spores is aberrant, with assembly arrested after the formation of a cap-like fragment that covers one end of the forespore—always the end near the middle of the mother cell. The cap contains a normal hair-like nap but an irregular basal layer. The cap is retained on spores prepared on solid medium, even after spore purification, but it is lost from spores prepared in liquid medium. Microscopic inspection of ΔexsY spores prepared on solid medium revealed a fragile sac-like sublayer of the exosporium basal layer, to which caps were attached. Examination of purified ΔexsY spores devoid of exosporium showed that they lacked detectable levels of BclA and the basal layer proteins BxpB, BxpC, CotY, and inosine-uridine-preferring nucleoside hydrolase; however, these spores retained half the amount of alanine racemase presumed to be associated with the exosporium of wild-type spores. The ΔexsY mutation did not affect spore production and germination efficiencies or spore resistance but did influence the course of spore outgrowth.

scholarly journals MpeR Regulates themtrEfflux Locus in Neisseria gonorrhoeae and Modulates Antimicrobial Resistance by an Iron-Responsive Mechanism

2012 ◽  
Vol 56 (3) ◽  
pp. 1491-1501 ◽  
Author(s):  
Alexandra Dubon Mercante ◽  
Lydgia Jackson ◽  
Paul J. T. Johnson ◽  
Virginia A. Stringer ◽  
David W. Dyer ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Efflux Pump ◽  
Regulatory System ◽  
Logarithmic Phase ◽  
Free Iron ◽  
Content Type ◽  
Protein Encoding ◽  
Late Logarithmic Phase ◽  
Physiologic Parameters ◽  
Inner Membrane Protein

ABSTRACTPrevious studies have shown that the MpeR transcriptional regulator produced byNeisseria gonorrhoeaerepresses the expression ofmtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation ofmpeRhas been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of themtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that themtrRgene, which encodes a direct repressor (MtrR) ofmtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of themtrCDEefflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression ofmtrRwas direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of themtrefflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.

scholarly journals Evidence for Contribution of Neutral Trehalase in Barotolerance of Saccharomyces cerevisiae

2000 ◽  
Vol 66 (12) ◽  
pp. 5182-5185 ◽  
Author(s):  
Hitoshi Iwahashi ◽  
Solomon Nwaka ◽  
Kaoru Obuchi
Keyword(s):  
Hydrostatic Pressure ◽  
Stationary Phase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Glucose Repression ◽  
Logarithmic Phase ◽  
Wild Type ◽  
Neutral Trehalase ◽  
I Gene

ABSTRACT In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression ofNTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.

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