scholarly journals Role of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Visceral Organ Infection by Leishmania donovani

2012 ◽  
Vol 12 (1) ◽  
pp. 70-77 ◽  
Author(s):  
Wen-Wei Zhang ◽  
Laura-Isobel McCall ◽  
Greg Matlashewski
Keyword(s):  
Leishmania Donovani ◽  
Null Mutant ◽  
Wild Type ◽  
Visceral Organ ◽  
Content Type ◽  
Visceral Organs ◽  
Deficient Medium ◽  
Visceral Disease ◽  
Organ Infection

ABSTRACTThe initial 7 steps of the glycolytic pathway from glucose to 3-phosphoglycerate are localized in the glycosomes inLeishmania, including step 6, catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). InL. donovaniandL. mexicana, there exists a second GAPDH enzyme present in the cytosol that is absent inL. braziliensisand that has become a pseudogene inL. major.To investigate the role of the cytosolic GAPDH (cGAPDH), anL. donovanicGAPDH-null mutant was generated, and conversely, the functionalL. donovanicGAPDH was introduced intoL. majorand the resulting engineered parasites were characterized. TheL. donovanicGAPDH-null mutant was able to proliferate at the same rate as the wild-type parasite in glucose-deficient medium. However, in the presence of glucose, theL. donovanicGAPDH-null mutant consumed less glucose and proliferated more slowly than the wild-type parasite and displayed reduced infectivity in visceral organs of experimentally infected mice. This demonstrates that cGAPDH is functional inL. donovaniand is required for survival in visceral organs. Restoration of cGAPDH activity inL. major, in contrast, had an adverse effect onL. majorproliferation in glucose-containing medium, providing a possible explanation of why it has evolved into a pseudogene inL. major. This study indicates that there is a difference in glucose metabolism betweenL. donovaniandL. major, and this may represent an important factor in the ability ofL. donovanito cause visceral disease.

scholarly journals Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

2014 ◽  
Vol 58 (8) ◽  
pp. 4682-4689 ◽  
Author(s):  
Trupti Pandharkar ◽  
Xiaohua Zhu ◽  
Radhika Mathur ◽  
Jinmai Jiang ◽  
Thomas D. Schmittgen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Mechanism Of Action ◽  
Leishmania Donovani ◽  
Synergistic Activity ◽  
Wild Type ◽  
Content Type ◽  
Axenic Amastigotes ◽  
Intracellular Parasites

ABSTRACTArylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellularLeishmaniaparasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raisedLeishmania donovaniaxenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism inLeishmania, is dramatically reduced in DB766R parasites.In vitrosusceptibility assays demonstrated that CYP5122A1 half-knockoutL. donovanipromastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellularL. donovaniamastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

scholarly journals WhiB5, a Transcriptional Regulator That Contributes to Mycobacterium tuberculosis Virulence and Reactivation

2012 ◽  
Vol 80 (9) ◽  
pp. 3132-3144 ◽  
Author(s):  
Stefano Casonato ◽  
Axel Cervantes Sánchez ◽  
Hirohito Haruki ◽  
Monica Rengifo González ◽  
Roberta Provvedi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Chronic Infection ◽  
Null Mutant ◽  
Wild Type ◽  
Secretion Systems ◽  
Content Type ◽  
Type Vii Secretion ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACTThe proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, aMycobacterium tuberculosisprotein belonging to this superfamily. A null mutant was constructed inM. tuberculosisH37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, thewhiB5mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain toS-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, includingsigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

scholarly journals CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jihong Li ◽  
Menglin Ma ◽  
Mahfuzur R. Sarker ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Null Mutant ◽  
Intestinal Infection ◽  
Wild Type ◽  
Global Regulator ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Type D ◽  
Epsilon Toxin ◽  
Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

scholarly journals Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists?

2011 ◽  
Vol 77 (20) ◽  
pp. 7227-7235 ◽  
Author(s):  
William W. Driscoll ◽  
John W. Pepper ◽  
Leland S. Pierson ◽  
Elizabeth A. Pierson
Keyword(s):  
Alternative Hypothesis ◽  
Natural Populations ◽  
Regulatory System ◽  
Wild Type ◽  
Content Type ◽  
The Public ◽  
Extracellular Metabolites ◽  
Mutualistic Interaction ◽  
Alternative Hypotheses

ABSTRACTBacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacteriumPseudomonas chlororaphisstrain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

scholarly journals Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Adria Carbo ◽  
Danyvid Olivares-Villagómez ◽  
Raquel Hontecillas ◽  
Josep Bassaganya-Riera ◽  
Rupesh Chaturvedi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
T Cell ◽  
Wild Type ◽  
Content Type ◽  
Interleukin 21 ◽  
H Pylori ◽  
Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

scholarly journals Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

2014 ◽  
Vol 58 (6) ◽  
pp. 2997-3007 ◽  
Author(s):  
Rati Tandon ◽  
Sharat Chandra ◽  
Rajendra Kumar Baharia ◽  
Sanchita Das ◽  
Pragya Misra ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Proliferating Cell Nuclear Antigen ◽  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Isolate ◽  
Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

scholarly journals Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

2012 ◽  
Vol 56 (4) ◽  
pp. 1990-1996 ◽  
Author(s):  
Alix Pantel ◽  
Stéphanie Petrella ◽  
Nicolas Veziris ◽  
Florence Brossier ◽  
Sylvaine Bastian ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hot Spot ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Content Type ◽  
Highly Active ◽  
Definition Of

ABSTRACTFluoroquinolone (FQ) resistance is emerging inMycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target inM. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified inM. tuberculosisclinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

Identification of Crp as a novel regulator of the Std fimbrial expression in Salmonella

Microbiology ◽  
2021 ◽  
Author(s):  
Karine Dufresne ◽  
France Daigle
Keyword(s):  
Wild Type Strain ◽  
Distal Region ◽  
Transcriptional Factor ◽  
Major Influence ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Serovar Typhimurium ◽  
Metabolic Regulators

The Salmonella enterica serovar Typhi genome contains 14 putative fimbrial systems. The Std fimbriae belong to the chaperone-usher family and its regulation has not been investigated in S. Typhi. Several regulators of Std were previously identified in the closely related serovar Typhimurium. We hypothesize that regulators of S. Typhimurium may be shared with S. Typhi, but that several other regulators remain to be discovered. Here, we describe the role of more than 50 different candidate regulators on std expression. Three types of regulators were investigated: known regulators in S. Typhimurium, in silico predicted regulators and virulence/metabolic regulators. Expression of std was determined in the regulator mutants and compared with the wild-type strain. Overall, 21 regulator mutations affect std promoter expression. The role of Crp, a newly identified factor for std expression, was further investigated. Crp acted as an activator of std expression on a distal region of the std promoter region. Together, our results demonstrate the major influence of Crp as a novel transcriptional factor on std promoter expression and later production of Std fimbriae in Salmonella .

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