Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Key Amino Acid Residues Involved in the Transitions of L- to R-Type Protofilaments of the Salmonella Flagellar Filament

The flagellar filament enables bacteria to swim by functioning as a helical propeller. The filament is a supercoiled assembly of a single protein, flagellin, and is formed by 11 protofilaments arranged in a circle. Bacterial swimming and tumbling correlate with changes of the various helical structures, called polymorphic transformation, that are determined by the ratios of two distinct forms of protofilaments, the L and R types. The polymorphic transformation is caused by transition of the protofilament between L and R types. Elucidation of this transition mechanism has been addressed by comparing the atomic structures of L- and R-type straight filaments or using massive molecular dynamic simulation. Here, we found amino acid residues required for the transition of the protofilament using fliC-intragenic suppressor analysis. We isolated a number of revertants producing supercoiled filaments from mutants with straight filaments and identified the second-site mutations in all of the revertants. The results suggest that Asp107, Gly426, and Ser448 and Ser106, Ala416, Ala427, and Arg431 are the key residues involved in inducing supercoiled filaments from the R- and the L-type straight filaments, respectively. Considering the structures of the R- and L-type protofilaments and the relationship between the rotation of the flagellar motor and the polymorphic transformation, we propose that Gly426, Ala427, and Arg431 contribute to the first stage of the transition and that Ser106, Asp107, and Ala416 play a role in propagating the transitions along the flagellar filament.     

Bacteriolytic Activity of Enzymes Derived from Streptomyces Species

Streptomyces species H–402 and 1829 possessing high lytic activities against cariogenic streptococci which induce dental plaque and caries, were isolated by the screening from soils and sewers. They were identified as Streptomyces griseus and Streptomyces globisporus respectively. The former strain produced lytic enzyme accompanying spore formation during the surface culture, while the latter strain revealed a high activity in the submerged culture. These enzymes had wide substrate specificity against all groups of cariogenic streptococci. The lytic enzymes may be expected as an useful medicament for the prevention of dental caries.     

Genetic linkages between circadian clock-associated components and phytochrome-dependent red light signal transduction in Arabidopsis thaliana

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.     

GA2 and GA20-oxidase expressions are associated with the meristem position in Streptocarpus rexii (Gesneriaceae)

We examined genes involved in the regulatory pathway of gibberellin (GA) in meristems of Streptocarpus rexii. The plants do not possess a typical shoot apical meristem (SAM) and form unique meristems: the basal meristem extends the lamina area of one cotyledon to produce anisocotylous seedlings; the groove meristem forms new leaves at the base of the macrocotyledon. Exogenous application of GA significantly suppresses the basal meristem activity in developing cotyledons and the seedlings remain isocotyl. To examine the role of endogenous GA on these meristems in vivo, we isolated homologs of GA2-oxidase responsible for degrading active GAs (SrGA2ox), and GA20-oxidase regulating the rate limiting step of active GA synthesis (SrGA20ox). During embryogenesis, while first partly overlapping, the expression of SrGA2ox and SrGA20ox became more differentiated and mutually exclusive, ending with SrGA2ox being expressed solely in the adaxial–proximal domain of the embryo in regions with meristem activity, whereas SrGA20ox was restricted to the fork between the two cotyledons. The latter may be responsible for suppressing the formation of an embryonic SAM in S. rexii. In developing seedlings, SrGA2ox expression also followed the centers of meristem activity, where SrGA20ox expression was excluded. Our results suggest that low levels of GA are required in S. rexii meristems for their establishment and maintenance. Thus, the meristems in S. rexii share similar regulatory pathways suggested for the SAM in model plants, but that in S. rexii evolutionary modifications involving a lateral transfer of function, from shoot to leaves, is implicated in attaining the unusual morphology of the plants.     

A study on the relationship between Atlantic sea surface temperature and Amazonian greenness

The growth of tropical rainforest in Amazon is critically vulnerable to the change in rainfall and radiation than in temperature, and that amount of rainfall and cloudiness in the northeast region of South American is strongly affected by the Atlantic sea surface temperature (SST). Results from recent model experiments for future climate projection have indicated a reduction of Amazonian greenness by a weakening of tropical vapor circulation system related with the change in SST. Therefore, the observational investigation of the relations between the Amazon greenness and Atlantic SST is fundamental to understand the response of Amazonian tropical forest to climate change. In this study, the effect of Atlantic SST on the spatial and temporal change of the Normalized Difference Vegetation Index (NDVI) in the Amazonian region is examined by using satellite remote sensing data for the period of 1981–2001. A strong correlation between NDVI and SST is found for certain regions in Amazon during the periods of 1980s and 1990s, respectively. In addition, strong correlations with NDVI lagging behind SST for two months and one year, respectively, are also identified from the interannual December-to-February (rain season) variations during 1981–2001. Despite these findings, the mechanisms behind the identified correlation remain unclear. Further analyses using observed precipitation and radiation data are required to understand the potential changes of Amazonian rainforest in the context of global warming.     

A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay

In hybridoma screening, quantitative kinetic evaluation is difficult since the concentration of each antibody in the hybridoma supernatant is unknown. From modeling calculations, we hypothesized that the ratio of two different antigen-antibody concentrations might allow discrimination of high-affinity monoclonal antibodies irrespective of the antibody concentration. Using anti-alpha-fetoprotein monoclonal antibodies of known affinity, we set the signal ratio of a time-resolved assay at >0.1, in which the antigen concentrations were 10 and 100 ng/mL. From anti-alpha-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1x10(-8) M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of alpha-fetoprotein has been established using selected high-affinity monoclonal antibodies. This screening method is useful for selection of high-affinity monoclonal antibodies of potential diagnostic value.     

Comparison of DNA Microarray,Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of <Emphasis Type="Italic">Fusarium</Emphasis> spp. Obtained from Patients with Hematologic Malignancies

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.     

Glycogen metabolism of the anammox bacterium “Candidatus Brocadia sinica”

Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)⁻¹ while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.Subject terms: Applied microbiology, Water microbiology     

Insulin mediates the linkage acceleration of muscle protein synthesis, thermogenesis, and heat storage by amino acids

Amino acid (AA) administration can stimulate heat accumulation in the body, as especially found under anesthetic conditions. To test our hypothesis that marked rise in plasma insulin concentrations following AA administration plays an important role in the heat storage, we intravenously administered either a balanced AA mixture or saline over 3 h, both with and without a primed-constant infusion of somatostatin in propofol-anesthetized rats. Rats on AA but lacking marked rise in plasma insulin by somatostatin treatment failed to show: attenuation of fall in core body temperature; partial increases in oxygen consumption; and stimulated muscle protein synthesis. Furthermore, the AA’s stimulatory effects on phosphorylation of mTOR, 4E-BP1, and S6K1 were partially blocked by somatostatin. Our findings strongly suggest that the marked rise in insulin following AA administration promote translation initiation activities and stimulate muscle protein synthesis, which facilitates heat accumulation in the body.