Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Mechanisms and diversity of resistance to sorghum midge, Stenodiplosis sorghicola in Sorghum bicolor

Sorghum midge, Stenodiplosis sorghicola (Coquillett) is the most important pest of grain sorghum worldwide, and plant resistance is an important component for the control of this pest. To identify sorghum genotypes with diverse mechanisms of resistance to sorghum midge, we studied oviposition, larval survival, and midge damage in 27 sorghum midge-resistant genotypes, and a susceptible check under greenhouse conditions. Observations were also recorded on floral characteristics and compensation in grain mass. Of the 28 sorghum genotypes tested, 19 showed high levels of antixenosis to oviposition as a component of resistance, and had

Mechanisms of inducible resistance against Bacillus thuringiensis endotoxins in invertebrates

Abstract Resistance in insect pests against the endotoxin of Bacillus thuringiensis (Berliner) (Bt) is a major threat to the usefulness of this biopesticide, both used as traditional formulations and in transgenic crops. A crucial requirement for the development of successful resistance management strategies is a molecular understanding of the nature and inheritance of resistance mechanisms. This information can be used to design management strategies that will delay or counteract Bt resistance. The best known Bt resistance mechanism is inactivation of brush border membrane receptors. This type of resistance has a largely recessive mode of inheritance, which has enabled the design of resistance management approaches involving high dose and refuge strategies. Recent observations suggest that other resistance mechanisms are possible, including a mechanism that sequesters the toxin in the gut lumen through inducible immune reactions. The elevated immune status associated with tolerance to the toxin can be transmitted to subsequent generations by a maternal effect, which has implications for resistance management in the field. The high dose/refuge strategy may not be appropriate for the management of these alternative resistance mechanisms and other strategies have to be developed if inducible dominant resistance or tolerance mechanisms occur frequently in the field.

Structure of thermolysin cleaved microcin J25: Extreme stability of a two-chain antimicrobial peptide devoid of covalent links

sThe structure of a two-chain peptide formed by the treatment of the potent antimicrobial peptide microcin J25 (MccJ25) with thermolysin has been characterized by NMR spectroscopy and mass spectrometry. The native peptide is 21 amino acids in size and has the remarkable structural feature of a ring formed by linkage of the side chain of Glu8 to the N-terminus that is threaded by the C-terminal tail of the peptide. Thermolysin cleaves the peptide at the Phe10-Val11 amide bond, but the threading of the C-terminus through the N-terminal ring is so tight that the resultant two chains remain associated both in the solution and in the gas phases. The three-dimensional structure of the thermolysin-cleaved peptide derived using NMR spectroscopy and simulated annealing calculations has a well-defined core that comprises the N-terminal ring and the threading C-terminal tail. In contrast to the well-defined core, the newly formed termini at residues Phe10 and Val11 are disordered in solution. The C-terminal tail is associated to the ring both by hydrogen bonds stabilizing a short beta-sheet and by hydrophobic interactions. Moreover, unthreading of the tail through the ring is prevented by the bulky side chains of Phe19 and Tyr20, which flank the octapeptide ring. This noncovalent two-peptide complex that has a remarkable stability in solution and in highly denaturing conditions and that survives in the gas phase is the first example of such a two-chain peptide lacking disulfide or interchain covalent bonds.

Pathogenic variation of Fusarium isolates associated with head blight of wheat in Australia

This paper examines the level of pathogenic diversity in Australian Fusarium pseudograminearum and Fusarium graminearum isolates for head blight from the assessment of 51 wheat germplasm lines, barley, triticale, rye, maize and sorghum plants. A set of nine putative wheat differentials were selected and assessed with 10 F. graminearum and 12 F. pseudograminearum isolates. Isolates of both species were pathogenic on all the wheat germplasm lines, barley triticale and rye. The isolates differed largely in a quantitative way with only small differential effects and were statistically demarcated into three pathogenicity groups: low, intermediate and high. Such distribution patterns suggest that wheat germplasm lines employ different resistance mechanisms to each group of isolates and the three pathogenicity groups may have different mechanisms controlling pathogenicity. The aggressiveness of F. graminearum and F. pseudograminearum isolates on the wheat germplasm lines were marginally correlated (r = 0.40). Durum wheats were ranked as the most susceptible while Sumai 3, Ituo Komugi, Sotome A, Sotome and Nobeokabouzu komugi were consistently grouped as resistant by both species. These findings reiterate the need to consider pathogen variability in the screening, selection and improvement of resistance to head blight in wheat.

Neutrophil depletion increases susceptibility to systemic and vaginal candidiasis in mice, and reveals differences between brain and kidney in mechanisms of host resistance

Infections caused by the yeast Candida albicans represent an increasing threat to debilitated and immunosuppressed patients, and neutropenia is an important risk factor. Monoclonal antibody depletion of neutrophils in mice was used to study the role of these cells in host resistance. Ablation of neutrophils increased susceptibility to both systemic and vaginal challenge. The fungal burden in the kidney increased threefold on day 1, and 100-fold on day 4, and infection was associated with extensive tissue destruction. However, a striking feature of the disseminated disease in neutrophil-depleted animals was the altered pattern of organ involvement. The brain, which is one of the primary target organs in normal mice, was little affected. There was a threefold increase in the number of organisms recovered from the brains of neutrophil-depleted mice on day 4 after infection, but detectable abscesses were rare. In contrast, the heart, which in normal mice shows only minor lesions, developed severe tissue damage following neutrophil depletion. Mice deficient in C5 demonstrated both qualitative and quantitative increases in the severity of infection after neutrophil depletion when compared with C5-sufficient strains. The results are interpreted as reflecting organ-specific differences in the mechanisms of host resistance.

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen- dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

Insect peptides with improved protease-resistance protect mice against bacterial infection

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia call infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Compensation for obesity-induced insulin resistance in dogs: Causal web analysis of the associations of leptin and GLP-1.

Obesity affects aspects of glucose homeostasis such as insulin secretion and insulin sensitivity. Hormones secreted by adipocytes like leptin mediate the metabolic consequences of obesity. Incretin hormones like glucagon-like peptide-1 (GLP-1) increase insulin secretion in response to changes in blood glucose concentration and have been proposed to regulate insulin secretion in fasting, overweight dogs. The aim of this study was to examine hormonal mechanisms by which adiposity alters glucose homeostasis, plasma insulin concentration, and insulin sensitivity in spontaneously overweight dogs.

Mechanisms of biocontrol by Pantoea dispersa of sugar cane leaf scald disease caused by Xanthomonas albilineans

Albicidins, a family of phytotoxins and antibiotics produced by Xanthomonas albilineans, are important in sugar cane leaf scald disease development. The albicidin detoxifying bacterium Pantoea dispersa (syn. Erwinia herbicola) SB1403 provides very effective biocontrol against leaf scald disease in highly susceptible sugar cane cultivars. The P. dispersa gene (albD) for enzymatic detoxification of albicidin has recently been cloned and sequenced. To test the role of albicidin detoxification in biocontrol of leaf scald disease, albD was inactivated in P. dispersa by site-directed mutagenesis. The mutants, which were unable to detoxify albicidin, were less resistant to the toxin and less effective in biocontrol of leaf scald disease than their parent strain. This indicates that albicidin detoxification contributes to the biocontrol capacity of P. dispersa against X. albilineans. Rapid growth and ability to acidify media are other characteristics likely to contribute to the competitiveness of P. dispersa against X. albilineans at wound sites used to invade sugar cane.

Exercise-induced alterations in neutrophil degranulation and respiratory burst activity: possible mechanisms of action

Neutrophils constitute 50-60% of all circulating leukocytes; they present the first line of microbicidal defense and are involved in inflammatory responses. To examine immunocompetence in athletes, numerous studies have investigated the effects of exercise on the number of circulating neutrophils and their response to stimulation by chemotactic stimuli and activating factors. Exercise causes a biphasic increase in the number of neutrophils in the blood, arising from increases in catecholamine and cortisol concentrations. Moderate intensity exercise may enhance neutrophil respiratory burst activity, possibly through increases in the concentrations of growth hormone and the inflammatory cytokine IL-6. In contrast, intense or long duration exercise may suppress neutrophil degranulation and the production of reactive oxidants via elevated circulating concentrations of epinephrine (adrenaline) and cortisol. There is evidence of neutrophil degranulation and activation of the respiratory burst following exercise-induced muscle damage. In principle, improved responsiveness of neutrophils to stimulation following exercise of moderate intensity could mean that individuals participating in moderate exercise may have improved resistance to infection. Conversely, competitive athletes undertaking regular intense exercise may be at greater risk of contracting illness. However there are limited data to support this concept. To elucidate the cellular mechanisms involved in the neutrophil responses to exercise, researchers have examined changes in the expression of cell membrane receptors, the production and release of reactive oxidants and more recently, calcium signaling. The investigation of possible modifications of other signal transduction events following exercise has not been possible because of current methodological limitations. At present, variation in exercise-induced alterations in neutrophil function appears to be due to differences in exercise protocols, training status, sampling points and laboratory assay techniques.

Yield response of rice (Oryza sativa L.) genotypes to different types of drought under rainfed lowlands - Part 3. Plant factors contributing to drought resistance

A series of experiments were conducted in drought-prone northeast Thailand to examine the magnitude of yield responses of diverse genotypes to drought stress environments and to identify traits that may confer drought resistance to rainfed lowland rice. One hundred and twenty eight genotypes were grown under non-stress and four different types of drought stress conditions. Under severe drought conditions, the maintenance of PWP of genotypes played a significant role in determining final grain yield. Because of their smaller plant size (lower total dry matter at anthesis) genotypes that extracted less soil water during the early stages of the drought period, tended to maintain higher PWP and had a higher fertile panicle percentage, filled grain percentage and final grain yield than other genotypes. PWP was correlated with delay in flowering (r = -0.387) indicating that the latter could be used as a measure of water potential under stress. Genotypes with well-developed root systems extracted water too rapidly and experienced severe water stress at flowering. RPR which showed smaller coefficient of variation was more useful than root mass density in identifying genotypes with large root system. Under less severe and prolonged drought conditions, genotypes that could achieve higher plant dry matter at anthesis were desirable. They had less delay in flowering, higher grain yield and higher drought response index, indicating the importance of ability to grow during the prolonged stress period. Other shoot characters (osmotic potential, leaf temperature, leaf rolling, leaf death) had little effect on grain yield under different drought conditions. This was associated with a lack of genetic variation and difficulty in estimating trait values precisely. Under mild stress conditions (yield loss less than 50%), there was no significant relationship between the measured drought characters and grain yield. Under these mild drought conditions, yield is determined more by yield potential and phenotype than by drought resistant mechanisms per se. (C) 2002 Elsevier Science B.V. All rights reserved.

Immune correlate study on human Schistosoma japonicum in a well-defined population in Leyte, Philippines: I. Assessment of 'resistance' versus 'susceptibility' to S-japonicum infection

This study describes the categorical classification of 155 individuals living in an endemic village in Macanip, Leyte, Philippines as 'resistant' or 'susceptible' to Schistosoma japonicum infection using available exposure, infection and reinfection data collected from a 3-year water contact (WC) study. Epidemiological parameters including age, sex, and infection intensities in relation to observed reinfection patterns are also described. This classification was used in subsequent immunological studies described in two accompanying papers to identify protective immune mechanisms among resistant individuals induced by defined candidate vaccine molecules for S. japonicum. The study suggests that individuals who were most vulnerable to rapid reinfection were children belonging to the 5-14 age group. A drop in incidence at age group 15-19 and decreased intensity of infection starting at this age group and older (15+) suggests development of immunity. Controlling for the effect of the other variables, a multivariate analysis showed significant association for sex, in that females were more likely to be resistant. This implies that other than acquired immunity to infection, some age-dependent host factors may also play an important role in the overall changes of reinfection patterns seen in schistosomiasis japonica in this population. Crown Copyright (C) 2002 Published by Elsevier Science B.V. All rights reserved.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.