Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato1

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia.

Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks.

Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait.

NMR studies of muscle glycogen synthesis in insulin-resistant offspring of parents with non-insulin-dependent diabetes mellitus immediately after glycogen-depleting exercise.

To examine the impact of insulin resistance on the insulin-dependent and insulin-independent portions of muscle glycogen synthesis during recovery from exercise, we studied eight young, lean, normoglycemic insulin-resistant (IR) offspring of individuals with non-insulin-dependent diabetes mellitus and eight age-weight matched control (CON) subjects after plantar flexion exercise that lowered muscle glycogen to approximately 25% of resting concentration. After approximately 20 min of exercise, intramuscular glucose 6-phosphate and glycogen were simultaneously monitored with 31P and 13C NMR spectroscopies. The postexercise rate of glycogen resynthesis was nonlinear. Glycogen synthesis rates during the initial insulin independent portion (0-1 hr of recovery) were similar in the two groups (IR, 15.5 +/- 1.3 mM/hr and CON, 15.8 +/- 1.7 mM/hr); however, over the next 4 hr, insulin-dependent glycogen synthesis was significantly reduced in the IR group [IR, 0.1 +/- 0.5 mM/hr and CON, 2.9 +/- 0.2 mM/hr; (P < or = 0.001)]. After exercise there was an initial rise in glucose 6-phosphate concentrations that returned to baseline after the first hour of recovery in both groups. In summary, we found that following muscle glycogen-depleting exercise, IR offspring of parents with non-insulin-dependent diabetes mellitus had (i) normal rates of muscle glycogen synthesis during the insulin-independent phase of recovery from exercise and (ii) severely diminished rates of muscle glycogen synthesis during the subsequent recovery period (2-5 hr), which has previously been shown to be insulin-dependent in normal CON subjects. These data provide evidence that exercise and insulin stimulate muscle glycogen synthesis in humans by different mechanisms and that in the IR subjects the early response to stimulation by exercise is normal.

Host-parasite dynamics and outgrowth of virus containing a single K70R amino acid change in reverse transcriptase are responsible for the loss of human immunodeficiency virus type 1 RNA load suppression by zidovudine.

The association between human immunodeficiency virus type I (HIV-1) RNA load changes and the emergence of resistant virus variants was investigated in 24 HIV-1-infected asymptomatic persons during 2 years of treatment with zidovudine by sequentially measuring serum HIV-1 RNA load and the relative amounts of HIV-1 RNA containing mutations at reverse transcriptase (RT) codons 70 (K-->R), 41 (M-->L), and 215 (T-->Y/F). A mean maximum decline in RNA load occurred during the first month, followed by a resurgence between 1 and 3 months, which appeared independent of drug-resistance. Mathematical modeling suggests that this resurgence is caused by host-parasite dynamics, and thus reflects infection of the transiently increased numbers of CD4+ lymphocytes. Between 3 and 6 months of treatment, the RNA load returned to baseline values, which was associated with the emergence of virus containing a single lysine to arginine amino acid change at RT codon 70, only conferring an 8-fold reduction in susceptibility. Despite the relative loss of RNA load suppression, selection toward mutations at RT codons 215 and 41 continued. Identical patterns were observed in the mathematical model. While host-parasite dynamics and outgrowth of low-level resistant virus thus appear responsible for the loss of HIV-1 RNA load suppression, zidovudine continues to select for alternative mutations, conferring increasing levels of resistance.

Detection of a major gene for resistance to fusiform rust disease in loblolly pine by genomic mapping.

Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analysis over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated, organisms, where conventional genetic analysis often is limited or not feasible.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells.

Benzodiazepine (BZA)-5B, a CAAX farnesyl-transferase inhibitor, was previously shown to block the farnesylation of H-Ras and to reverse the transformed morphology of Rat1 cells expressing oncogenic H-RasV12. Non-transformed Rat1 cells were not affected by BZA-5B, suggesting that they produce a form of Ras whose prenylation is not blocked by this compound. The likely candidate is K-RasB, which differs from H-Ras primarily in the terminal 24 amino acids. In the current study we examined the effect of BZA-5B on the prenylation of a chimeric oncogenic Ras protein designated H/K-RasBV12, consisting of the first 164 amino acids of H-RasV12 followed by the last 24 amino acids of K-RasB. BZA-5B failed to block the prenylation of this chimera and was thus unable to reverse the transformed morphology of Rat1 cells in which it was expressed. Another potent inhibitor of H-Ras farnesylation, L-739,749, also failed to block prenylation of H/K-RasBV12. Similar results were obtained in transfected cells expressing a widely used version of K-RasBV12 containing a 10-amino acid extension at its NH2 terminus. Neither BZA-5B nor L-739,749 reversed the transformed morphology of cells expressing H/K-RasBV12. The resistance of K-RasB to farnesyltransferase inhibition provides a likely explanation for the resistance of nontransformed cells to the growth inhibitory effects of BZA-5B and L-739,749.

Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage.

pS2 is a member of the trefoil peptide family, all of which are overexpressed at sites of gastrointestinal injury. We hypothesized that they are important in stimulating mucosal repair. To test this idea, we have produced a transgenic mice strain that expresses human pS2 (hpS2) specifically within the jejunum and examined the effect of this overexpression on proliferation and susceptibility to indomethacin-induced damage. A transgenic mouse was produced by microinjecting fertilized oocytes with a 1.7-kb construct consisting of rat intestinal fatty acid binding protein promoter (positions -1178 to +28) linked to full-length (490 bp) hpS2 cDNA. Screening for positive animals was by Southern blot analysis. Distribution of hpS2 expression was determined by using Northern and Western blot analyses and immunohistochemical staining. Proliferation of the intestinal mucosa was determined by assessing the crypt cell production rate. Differences in susceptibility to intestinal damage were analyzed in animals that had received indomethacin (85 mg/kg s.c.) 0-30 h previously. Expression of hpS2 was limited to the enterocytes of the villi within the jejunum. In the nondamaged intestine, villus height and crypt cell production rate were similar in transgenic and negative (control) litter mates. However, there was a marked difference in the amount of damage caused by indomethacin in control and transgenic animals in the jejunum (30% reduction in villus height in controls vs. 12% reduction in transgenic animals, P < 0.01) but the damage sustained in the non-hpS2-expressing ileal region was similar in control and transgenic animals. These studies support the hypothesis that trefoil peptides are important in stimulating gastrointestinal repair.

High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.

We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance. A. thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3. Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB). Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested. Interestingly, a hypersensitive-like response was still induced by several of the strains. The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica. Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III. Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.

Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway.

Plants can recognize and resist invading pathogens by signaling the induction of rapid defense responses. Often these responses are mediated by single dominant resistance genes (R genes). The products of R genes have been postulated to recognize the pathogen and trigger rapid host defense responses. Here we describe isolation of the classical resistance gene N of tobacco that mediates resistance to the well-characterized pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator (Ac) transposon. We confirmed isolation of the N gene by complementation of the TMV-sensitive phenotype with a genomic DNA fragment. Sequence analysis of the N gene shows that it encodes a protein with an amino-terminal domain similar to that of the cytoplasmic domains of the Drosophila Toll protein and the interleukin 1 receptor in mammals, a putative nucleotide-binding site and 14 imperfect leucine-rich repeats. The presence of these functional domains in the predicted N gene product is consistent with the hypothesis that the N resistance gene functions in a signal transduction pathway. Similarities of N to Toll and the interleukin 1 receptor suggest a similar signaling mechanism leading to rapid gene induction and TMV resistance.

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

Background: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.