Deletion of the DNA Ligase IV Gene in Candida glabrata Significantly Increases Gene-Targeting Efficiency

Candida glabrata is reported as the second most prevalent human opportunistic fungal pathogen in the United States. Over the last decades, its incidence increased, whereas that of Candida albicans decreased slightly. One of the main reasons for this shift is attributed to the inherent tolerance of C. glabrata toward the commonly used azole antifungal drugs. Despite a close phylogenetic distance to Saccharomyces cerevisiae, homologous recombination works with poor efficiency in C. glabrata compared to baker''s yeast, in fact limiting targeted genetic alterations of the pathogen''s genome. It has been shown that nonhomologous DNA end joining is dominant over specific gene targeting in C. glabrata. To improve the homologous recombination efficiency, we have generated a strain in which the LIG4 gene has been deleted, which resulted in a significant increase in correct gene targeting. The very specific function of Lig4 in mediating nonhomologous end joining is the reason for the absence of clear side effects, some of which affect the ku80 mutant, another mutant with reduced nonhomologous end joining. We also generated a LIG4 reintegration cassette. Our results show that the lig4 mutant strain may be a valuable tool for the C. glabrata research community.     

盘基网柄菌——研究致病机制的宿主模型

盘基网柄菌作为致病菌宿主模型的研究主要有:筛选致病菌株及相应突变菌株毒性;鉴别对致病菌易感性和抗性的突变细胞宿主;宿主细胞的有效标记、已完成的基因组计划以及宿主细胞与致病菌间信号转导通路的相互作用;这些都表明盘基网柄菌是致病机制研究的理想宿主模型。     

Relationship Between the Quantity of Oral Candida and Systemic Condition/Diseases of the Host: Oral Candida Increases with Advancing Age and Anemia

Background

The impact of host systemic conditions/diseases on the prosperity of oral Candida colonies remains unclear. The aim of the present study was to investigate whether a relationship exists between the quantity of oral Candida and the systemic condition/diseases of the host.

Patients and Methods

The cross-sectional relationship between Candida mannan concentrations and health check-up results was analyzed in consideration of local conditions that influence the prevalence of oral Candida.

Results

Candida mannan concentrations correlated with age, the number of untreated decayed teeth, number of prosthetic teeth, salivary pH, HbA1c, and the red blood cell count in a univariate analysis. In a multivariate analysis, Candida mannan concentrations correlated with age, the number of untreated decayed teeth, number of prosthetic teeth, salivary pH, and the red blood cell count. Candida mannan concentrations were higher in subjects older than 80 years, with a higher number of either untreated or prosthetic teeth, with a lower salivary pH, and with a decreased red blood cell count. Mannan concentrations were slightly higher in subjects with elevated HbA1c.

Conclusions

The present results suggest a close relationship between the quantity of oral Candida and the systemic condition/diseases of the host. Oral Candida may increase in immunocompromised hosts.

     

Nutrient Enrichment Increases Mortality of Mangroves

Nutrient enrichment of the coastal zone places intense pressure on marine communities. Previous studies have shown that growth of intertidal mangrove forests is accelerated with enhanced nutrient availability. However, nutrient enrichment favours growth of shoots relative to roots, thus enhancing growth rates but increasing vulnerability to environmental stresses that adversely affect plant water relations. Two such stresses are high salinity and low humidity, both of which require greater investment in roots to meet the demands for water by the shoots. Here we present data from a global network of sites that documents enhanced mortality of mangroves with experimental nutrient enrichment at sites where high sediment salinity was coincident with low rainfall and low humidity. Thus the benefits of increased mangrove growth in response to coastal eutrophication is offset by the costs of decreased resilience due to mortality during drought, with mortality increasing with soil water salinity along climatic gradients.     

Benzyladenine Treatment Significantly Increases the Seed Yield of the Biofuel Plant Jatropha curcas

Jatropha curcas, a monoecious perennial biofuel shrub belonging to the family Euphorbiaceae, has few female flowers, which is one of the most important reasons for its poor seed yield. This study was undertaken to determine the effects of the plant growth regulator 6-benzyladenine (BA) on floral development and floral sex determination of J. curcas. Exogenous application of BA significantly increased the total number of flowers per inflorescence, reaching a 3.6-fold increase (from 215 to 784) at 160 mg/l of BA. Furthermore, BA treatments induced bisexual flowers, which were not found in control inflorescences, and a substantial increase in the female-to-male flower ratio. Consequently, a 4.5-fold increase in fruit number and a 3.3-fold increase in final seed yield were observed in inflorescences treated with 160 mg/L of BA, which resulted from the greater number of female flowers and the newly induced bisexual flowers in BA-treated inflorescences. This study indicates that the seed yield of J. curcas can be increased by manipulation of floral development and floral sex expression.     

Helicobacter pylori Infection Significantly Increases Insulin Resistance in the Asymptomatic Japanese Population

Background: Helicobacter pylori infection has been shown to contribute to atherosclerosis and cardiovascular diseases. Insulin resistance is the pathophysiologic background of the clinical features of atherosclerosis and cardiovascular diseases. We examined the association between H. pylori infection and insulin resistance in a large Japanese population. Materials and Methods: Fifteen hundred ninety‐eight consecutive asymptomatic subjects that underwent a complete medical survey in our institute between May 2007 and July 2008 were recruited. Cases under medication for hypertension, hyperlipidemia, diabetes mellitus, hyperuricemia, or cardiovascular diseases were excluded from the study. Cases suffering from chronic renal or liver failure were also excluded. The homeostasis model assessment of insulin resistance (HOMA‐IR) score was used to quantitatively estimate insulin resistance. Visceral and subcutaneous adipose tissues (SAT) were measured by computed tomography. The association between H. pylori serostatus and HOMA‐IR score was investigated by multivariate regression analysis. Results: A total of 988 men and 119 women were eventually eligible for this cross‐sectional survey. Helicobacter pylori seropositivity was significantly higher in 99 cases with insulin resistance (HOMA‐IR ≥2.5) compared with 1008 cases without insulin resistance (HOMA‐IR <2.5) (39.4 vs 28.7%, p = .027). There was a significant association between H. pylori serostatus and HOMA‐IR score by multiple linear regression analysis (coefficients = 0.152, 95% CI = 0.058–0.246, p = .001), after adjusting for sex, age, body mass index, waist girth, visceral and subcutaneous adipose tissues, smoking status, alcohol consumption, dietary habits, and physical activity. Conclusions: Helicobacter pylori infection significantly and independently contributed to promoting insulin resistance in a large asymptomatic population.     

Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections

Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.     

Characterization of Host Cell Mutants Significantly Resistant to Cryptosporidium parvum Infection

Cryptosporidium parvum is a parasitic protist and a causative agent of mild‐to‐severe diarrheal diseases in humans and animals. Despite its globally recognized importance, knowledge on the mechanism of parasite invasion and molecular interactions between host cells and the parasite is limited. Here, we report the establishment of 43 mutant cell lines derived from HCT‐8 cells by UV‐induced mutagenesis and the characterization of three mutants with significantly reduced susceptibility to cryptosporidial infection. Based on qRT‐PCR assay performed at 18 h postinfection time, the parasite loads could be reduced by ~45%, ~35%, and ~20% in mutants A05, B08, and B12, respectively (p < 0.001 in all three mutants vs. HCT‐8 cells). The mutagenesis mainly affected the attachment of parasite in A05 (i.e. ~30% reduction, p < 0.001 vs. HCT‐8), and intracellular development in B08 and B12. The three cell mutants may serve as valuable reagents to further investigate the mechanism of parasite invasion and intracellular development by identifying the gene mutations associated with the parasite attachment (A05) and intracellular development (B08 and B12).     

Parasite-induced Host Mortality: Indirect Evidence From a Long-term Study

A long-term field study of a perturbed host–helminth system provides indirect evidence that a long-lived swimbladder nematode, Cystidicola farionis, induces mortality of Arctic charr, Salvelinus alpinus. The prevalence and abundance of this parasite has changed little over the period from 1987 to 1999. The cumulative numbers of L₃-stage larvae steadily increased with increasing host age, indicating a continuous exposure to infection throughout the life of the target fish host. Indirect methods, which used data pooled over years and long-term cohort analyses, indicate that parasite-induced host mortality (PIHM) occurs in hosts older than 10 years. Furthermore, using a short-term cohort method adjusted for worm recruitment, we found indications of PIHM occurrence even in younger age groups. These patterns do not seem to be caused by high parasite mortality rates since dead worms are rarely observed inside the swimbladder. Age-related changes in infection rates or in resistance to infection seem to play only a minor role as there were only slight changes in the preference of charr for feeding on amphipods (which are intermediate hosts) and in the acquisition rate of L₃ larvae in older hosts. Mortality of the most heavily infected hosts is the most probable explanation for the observed patterns.     

Disease Severity and Mortality Can Be Independently Regulated in a Mouse Model of Experimental Graft versus Host Disease

Graft versus host disease (GVHD) is the major limitation of allogeneic hematopoietic stem cell transplantation (HSCT) presenting high mortality and morbidity rates. However, the exact cause of death is not completely understood and does not correlate with specific clinical and histological parameters of disease. Here we show, by using a semi-allogeneic mouse model of GVHD, that mortality and morbidity can be experimentally separated. We injected bone marrow-derived dendritic cells (BMDC) from NOD2/CARD15-deficient donors into semi-allogeneic irradiated chimaeras and observed that recipients were protected from death. However, no protection was observed regarding clinical or pathological scores up to 20 days after transplantation. Protection from death was associated with decreased bacterial translocation, faster hematologic recovery and epithelial integrity maintenance despite mononuclear infiltration at day 20 post-GVHD induction with no skew towards different T helper phenotypes. The protected mice recovered from aGVHD and progressively reached scores compatible with healthy animals. Altogether, our data indicate that severity and mortality can be separate events providing a model to study transplant-related mortality.     

The In Vitro Mass-Produced Model Mycorrhizal Fungus,Rhizophagus irregularis,Significantly Increases Yields of the Globally Important Food Security Crop Cassava

The arbuscular mycorrhizal symbiosis is formed between arbuscular mycorrhizal fungi (AMF) and plant roots. The fungi provide the plant with inorganic phosphate (P). The symbiosis can result in increased plant growth. Although most global food crops naturally form this symbiosis, very few studies have shown that their practical application can lead to large-scale increases in food production. Application of AMF to crops in the tropics is potentially effective for improving yields. However, a main problem of using AMF on a large-scale is producing cheap inoculum in a clean sterile carrier and sufficiently concentrated to cheaply transport. Recently, mass-produced in vitro inoculum of the model mycorrhizal fungus Rhizophagus irregularis became available, potentially making its use viable in tropical agriculture. One of the most globally important food plants in the tropics is cassava. We evaluated the effect of in vitro mass-produced R. irregularis inoculum on the yield of cassava crops at two locations in Colombia. A significant effect of R. irregularis inoculation on yield occurred at both sites. At one site, yield increases were observed irrespective of P fertilization. At the other site, inoculation with AMF and 50% of the normally applied P gave the highest yield. Despite that AMF inoculation resulted in greater food production, economic analyses revealed that AMF inoculation did not give greater return on investment than with conventional cultivation. However, the amount of AMF inoculum used was double the recommended dose and was calculated with European, not Colombian, inoculum prices. R. irregularis can also be manipulated genetically in vitro, leading to improved plant growth. We conclude that application of in vitro R. irregularis is currently a way of increasing cassava yields, that there is a strong potential for it to be economically profitable and that there is enormous potential to improve this efficiency further in the future.     

Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion

The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution.     

Absence of MyoD Increases Donor Myoblast Migration into Host Muscle

Donor myoblast migration is a major limiting factor in the success of myoblast transfer therapy, a potential treatment for Duchenne muscular dystrophy. A possible strategy to promote the migration of donor myoblasts into host muscle is to enhance their proliferation and delay their fusion, two properties that are major characteristics of myoblasts in regenerating skeletal muscle in MyoD null (-/-) mice. Here we investigate whether the migration of MyoD (-/-) donor myoblasts into host muscle is enhanced in vivo. Sliced muscle grafts from male MyoD (-/-) or normal control (Balb/c) mice were transplanted into the muscles of female normal (Balb/c) host mice. Muscles were sampled at 1, 3, and 12 weeks after grafting, and the fate of male donor myoblasts within female host muscles determined by in situ hybridization with the mouse Y-chromosome-specific Y-1 probe. MyoD (-/-) donor myoblasts migrated into host muscle continuously over 1, 3, and 12 weeks after grafting, in contrast with Balb/c donor myoblasts, whose overall numbers and migratory distances did not increase significantly after 1 week. These results strongly support a role for elevated donor myoblast proliferation and/or their delayed fusion in enhancing migration into host muscle in vivo, and endorse the use of either genetically engineered donor myoblasts, or the administration of exogenous myoblast mitogens to improve donor myoblast migration in myoblast transfer therapy.     

Heterologous Leaky Production of Transglutaminase in Lactococcus lactis Significantly Enhances the Growth Performance of the Host

This study describes a novel strategy to improve the growth performance of Lactococcus lactis by heterologous production of food-grade transglutaminase. The mtg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into L. lactis subsp. cremoris NZ9000. The leaky expression of the mtg gene from the nisA promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of L. lactis, which is an important step for the purposes of producing proteins of commercial interest using L. lactis as a host.