scholarly journals A Single Amino Acid at Residue 188 of Hexon Protein is Responsible for the Pathogenicity of the Emerging Novel Fowl Adenovirus 4

2021 ◽  
Author(s):  
Yu Zhang ◽  
Aijing Liu ◽  
Yanan Wang ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Molecular Basis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Live Attenuated Vaccine ◽  
Hexon Protein

Since 2015, severe hydropericardium-hepatitis syndrome (HHS) associated with a novel fowl adenovirus 4 (FAdV-4) has emerged in China, representing a new challenge for the poultry industry. Although various highly pathogenic FAdV-4 strains have been isolated, the virulence factor and the pathogenesis of novel FAdV-4 are unclear. In our previous studies, we reported that a large genomic deletion (1966 bp) is not related to increased virulence. In this study, two recombinant chimeric viruses, rHN20 strain and rFB2 strain, were generated from a highly pathogenic FAdV-4 strain by replacing hexon or fiber-2 gene of a non-pathogenic FAdV-4, respectively. Both chimeric strains showed similar titers to the wild type strain in vitro . Notably, rFB2 and the wild type strain induced 100% mortality, while no mortality or clinical signs appeared in chickens inoculated with rHN20, indicating that hexon, but not fiber-2, determines the novel FAdV-4 virulence. Furthermore, an R188I mutation in the hexon protein identified residue 188 as the key amino acid for the reduced pathogenicity. The rR188I mutant strain was significantly neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was able to replicate efficiently. Finally, the immunogenicity of the rescued rR188I was investigated. Non-pathogenic rR188I provided full protection against lethal FAdV-4 challenge. Collectively, these findings provide an in-depth understanding of the molecular basis of novel FAdV-4 pathogenicity and present rR188I as a potential live attenuated vaccine candidate or a novel vaccine vector for HHS vaccines. Importance HHS associated with a novel FAdV-4 infection in chickens has caused huge economic losses to the poultry industry in China since 2015. The molecular basis for the increased virulence remains largely unknown. Here, we demonstrate that the hexon gene is vital for FAdV-4 pathogenicity. Furthermore, we show that the amino acid residue at position 188 of the hexon protein is responsible for pathogenicity. Importantly, the rR188I mutant strain was neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was not. Further, the rR188I mutant strain provided complete protection against FAdV-4 challenge. Our results provide a molecular basis of the increased virulence of novel FAdV-4. We propose that the rR188I mutant is a potential live attenuated vaccine against HHS and a new vaccine vector for HHS-combined vaccines.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

2003 ◽  
Vol 185 (16) ◽  
pp. 4672-4682 ◽  
Author(s):  
Shruti Haralalka ◽  
Suvobroto Nandi ◽  
Rupak K. Bhadra
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Mutant Strain ◽  
Virulence Factors ◽  
Antibiotic Production ◽  
Wild Type ◽  
Altered Expression ◽  
Transcript Levels

ABSTRACT The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA VCH) was cloned and sequenced. The relA VCH gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A ΔrelA::kan allele was generated by replacing ∼31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

scholarly journals Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Monchaya Rattanaprasert ◽  
Jan-Peter van Pijkeren ◽  
Amanda E. Ramer-Tait ◽  
Maria Quintero ◽  
Car Reen Kok ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Lactobacillus Reuteri ◽  
Selective Advantage ◽  
Wild Type ◽  
Carbohydrate Source ◽  
Content Type ◽  
Gnotobiotic Mice

ABSTRACT Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo. In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two β-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway. IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

scholarly journals Bacillus subtilis Phosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-Responsive phoB-PS+V Promoters during Pho Response

2005 ◽  
Vol 187 (15) ◽  
pp. 5166-5178 ◽  
Author(s):  
Wael R. Abdel-Fattah ◽  
Yinghua Chen ◽  
Amr Eldakak ◽  
F. Marion Hulett
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphate Deprivation

ABSTRACT The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-PS, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-PV. EσE was necessary and sufficient for PS expression in vitro. PS expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σE is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed PS in vitro via direct binding to the promoter, the first example of an EσE-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of EσA was sufficient to stimulate transcription from the phoB-PV promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for PV promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

scholarly journals Cloning and Inactivation of a Branched-Chain-Amino-Acid Aminotransferase Gene from Staphylococcus carnosus and Characterization of the Enzyme

2002 ◽  
Vol 68 (8) ◽  
pp. 4007-4014 ◽  
Author(s):  
Søren M. Madsen ◽  
Hans Christian Beck ◽  
Peter Ravn ◽  
Astrid Vrang ◽  
Anne Maria Hansen ◽  
...  
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Biomass Yield ◽  
Degradation Products ◽  
Wild Type ◽  
Staphylococcus Carnosus ◽  
Branched Chain

ABSTRACT Staphylococcus carnosus and Staphylococcus xylosus are widely used as aroma producers in the manufacture of dried fermented sausages. Catabolism of branched-chain amino acids (BCAAs) by these strains contributes to aroma formation by production of methyl-branched aldehydes and carboxy acids. The first step in the catabolism is most likely a transamination reaction catalyzed by BCAA aminotransferases (IlvE proteins). In this study, we cloned the ilvE gene from S. carnosus by using degenerate oligonucleotides and PCR. We found that the deduced amino acid sequence was 80% identical to that of the corresponding enzyme in Staphylococcus aureus and that the ilvE gene was constitutively expressed as a monocistronic transcript. To study the influence of ilvE on BCAA catabolism, we constructed an ilvE deletion mutant by gene replacement. The IlvE protein from S. carnosus was shown mainly to catalyze the transamination of isoleucine, valine, leucine, and, to some extent, methionine using pyridoxal 5′-phosphate as a coenzyme. The ilvE mutant degraded less than 5% of the BCAAs, while the wild-type strain degraded 75 to 95%. Furthermore, the mutant strain produced approximately 100-fold less of the methyl-branched carboxy acids, 2-methylpropanoic acid, 2-methylbutanoic acid, and 3-methylbutanoic acid, which derived from the BCAA catabolism, clearly emphasizing the role of IlvE in aroma formation. In contrast to previous reports, we found that IlvE was the only enzyme that catalyzed the deamination of BCAAs in S. carnosus. The ilvE mutant strain showed remarkably lower growth rate and biomass yield compared to those of the wild-type strain when grown in rich medium. Normal growth rate and biomass yield were restored by addition of the three BCAA-derived α-keto acids, showing that degradation products of BCAAs were essential for optimal cell growth.

scholarly journals Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

2013 ◽  
Vol 81 (4) ◽  
pp. 1334-1340 ◽  
Author(s):  
Nelly Leung ◽  
Antonella Gianfelice ◽  
Scott D. Gray-Owen ◽  
Keith Ireton
Keyword(s):  
Listeria Monocytogenes ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Pathogen ◽  
Adaptor Protein ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

scholarly journals Construction of an In Vivo Nonsense Readthrough Assay System and Functional Analysis of Ribosomal Proteins S12, S4, and S5 in Bacillus subtilis

2001 ◽  
Vol 183 (17) ◽  
pp. 4958-4963 ◽  
Author(s):  
Takashi Inaoka ◽  
Koji Kasai ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Type Strain ◽  
Wild Type Strain ◽  
Assay System ◽  
Wild Type ◽  
Translational Accuracy ◽  
Free Translation

ABSTRACT To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in aram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.

scholarly journals Critical Role of Multidrug Efflux Pump CmeABC in Bile Resistance and In Vivo Colonization of Campylobacter jejuni

2003 ◽  
Vol 71 (8) ◽  
pp. 4250-4259 ◽  
Author(s):  
Jun Lin ◽  
Orhan Sahin ◽  
Linda Overbye Michel ◽  
Qijing Zhang
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
In Vitro Growth ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Bile Resistance

ABSTRACT CmeABC functions as a multidrug efflux pump contributing to the resistance of Campylobacter to a broad range of antimicrobials. In this study, we examined the role of CmeABC in bile resistance and its contribution to the adaptation of Campylobacter jejuni in the intestinal tract of the chicken, a natural host and a major reservoir for Campylobacter. Inactivation of cmeABC drastically decreased the resistance of Campylobacter to various bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of the cmeABC mutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth of Campylobacter was well correlated with the total bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 107 CFU/g of feces. In contrast, the cmeABC mutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for the cmeABC mutant was at least 2.6 × 104-fold higher than that of the wild-type strain. Complementation of the cmeABC mutants with a wild-type cmeABC allele in trans fully restored the in vitro growth in bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected with C. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating resistance to bile salts in the intestinal tract, is required for successful colonization of C. jejuni in chickens. Inhibition of CmeABC function may not only control antibiotic resistance but also prevent the in vivo colonization of pathogenic Campylobacter.

scholarly journals Transient Down-Regulation of Nucleoside Transporter 3 Gene Expression as a Drug Target in Leishmania major Using Antisense RNA Technology

2019 ◽  
Author(s):  
Farideh TOHIDI ◽  
Bahram KAZEMI ◽  
Mojgan BANDEHPOUR ◽  
Iraj SHARIFI ◽  
Mohammad Reza RABIEI ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antisense Rna ◽  
Type Strain ◽  
Wild Type Strain ◽  
Leishmania Major ◽  
Leishmania Infection ◽  
Nucleoside Transporter ◽  
Wild Type

Background: This study was aimed to silencing the Nucleoside transporter 3 (NT3) permease nucleobases involved in the salvage pathway of Leishmania in order to disrupt purine nucleotide uptake in the parasite and consequently, destruction of the parasite. Methods: Overall, 502 bp fragment of the NT3 gene sequence was designed to produce an antisense transcript upon entry of the vector into the parasite. The NT3 construct was transfected into L. major promastigotes and NT3 gene expression was studied in vivo and in vitro conditions. Results: Relative expression NT3 gene in transgenic Leishmania was decreased in tenth day. The percentages and the number of amastigotes infected macrophages with transgenic L. major were less than infected macrophages with wild-type strain. Our results in two groups of BALB/c female mice (wild-type strain and mutant, n=4 each group) were showed that size and number of ulcers in BALB/c mice infected with transgenic Leishmania promastigotes were less than the BALB/c mice infected with wild-type parasites. Conclusion: The results indicate the use of antisense RNA reduces of NT3 gene expression in L. major. More studies are required to obtain a new approach for treating Leishmania infection.

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