Nucleus-Independent Chemical Shift (NICS) as a Criterion for the Design of New Antifungal Benzofuranones.

The assertion made by Wu et al. that aromaticity may have considerable implications for molecular design motivated us to use nucleus-independent chemical shifts (NICS) as an aromaticity criterion to evaluate the antifungal activity of two series of indol-4-ones. A linear regression analysis of NICS and antifungal activity showed that both tested variables were significantly related (p < 0.05); when aromaticity increased, the antifungal activity decreased for series I and increased for series II. To verify the validity of the obtained equations, a new set of 44 benzofuran-4-ones was designed by replacing the nitrogen atom of the five-membered ring with oxygen in indol-4-ones. The NICS(0) and NICS(1) of benzofuran-4-ones were calculated and used to predict their biological activities using the previous equations. A set of 10 benzofuran-4-ones was synthesized and tested in eight human pathogenic fungi, showing the validity of the equations. The minimum inhibitory concentration (MIC) in yeasts was 31.25 µg·mL-1 for Candida glabrata, Candida krusei and Candida guilliermondii with compounds 15-32, 15-15 and 15-1. The MIC for filamentous fungi was 1.95 µg·mL-1 for Aspergillus niger for compounds 15-1, 15-33 and 15-34. The results obtained support the use of NICS in the molecular design of compounds with antifungal activity.

Study of Titanium-Silver Monolayer and Multilayer Films for Protective Applications in Biomedical Devices.

The search for coatings that extend the useful life of biomedical devices has been of great interest, and titanium has been of great relevance due to its innocuousness and low reactivity. This study contributes to the investigation of Ti/Ag films in different configurations (monolayer and multilayer) deposited by magnetron sputtering. The sessile droplet technique was applied to study wettability; greater film penetrability was obtained when Ag is the external layer, conferring high efficiency in cell adhesion. The morphological properties were characterized by SEM, which showed porous nuclei on the surface in the Ag coating and crystals embedded in the Ti film. The structural properties were studied by XRD, revealing the presence of TiO2 in the anatase crystalline phase in a proportion of 49.9% and the formation of a silver cubic network centered on the faces. Tafel polarization curves demonstrated improvements in the corrosion current densities of Ag/Ti/Ag/Ti/Ag/Ti/Ag/Ti and Ti/Ag compared to the Ag coating, with values of 0.1749, 0.4802, and 2.044 nA.m-2, respectively. Antimicrobial activity was evaluated against the bacteria Pseudomonas aeruginosa and Bacillus subtilis and the yeasts Candida krusei and Candida albicans, revealing that the Ti/Ag and Ag/Ti/Ag/Ti/Ag/Ti/Ag/Ti coatings exhibit promise in biomedical material applications.

Epidemiological Trends of Fungemia in Greece with a Focus on Candidemia during the Recent Financial Crisis: a 10-Year Survey in a Tertiary Care Academic Hospital and Review of Literature.

Updated information on the epidemiology of candidemia, particularly during severe socioeconomic events, is important for proper management of these infections. A systematic literature review on candidemia in Greece and a retrospective surveillance study were conducted in a tertiary university hospital during the years of the recent financial crisis (2009 to 2018) in order to assess changes in incidence rates, patient characteristics, species distribution, antifungal susceptibilities, and drug consumption. The average annual incidence of 429 candidemic episodes was 2.03/10,000 bed days, with 9.88 in adult intensive care units (ICUs), 1.74 in surgical wards, and 1.81 in internal medicine wards, where a significant increase was observed (1.15, 1.85, and 2.23/10,000 bed days in 2009 to 2011, 2012 to 2014, and 2015 to 2018, respectively; P = 0.004). Candida albicans was the most common species (41%), followed by Candida parapsilosis species complex [SC] (37%), Candida glabrata SC (11%), Candida tropicalis (7%), Candida krusei (1%), and other rare Candida spp. (3%). Mixed infections were found in 20/429 (4.7%) cases, while 33 (7%) cases were due to non-Candida spp. Overall, 44/311 (14%) isolates were resistant/non-wild type (WT) to the nine antifungals tested, with 23/113 (20%) C. parapsilosis SC and 2/34 (6%) C. glabrata SC isolates being resistant to fluconazole (1 panechinocandin and 2 panazole resistant). All isolates were susceptible/WT to amphotericin B and flucytosine. While the overall consumption of antifungals diminished (P = 0.02), with a mean of 17.93 defined daily doses (DDD)/100 bed days, increased micafungin use was correlated with the rise in C. parapsilosis SC (P = 0.04). A significant increase of candidemia in internal medicine wards and of C. parapsilosis SC infections was found during the years of financial crisis. Although resistance rates remain low (<14%), fluconazole-resistant C. parapsilosis SC and multidrug-resistant C. glabrata SC isolates are of major concern.

Prolonged Outbreak of Candida krusei Candidemia in Paediatric Ward of Tertiary Care Hospital.

BACKGROUND: A sudden rise of Candida krusei candidemia cases was noticed in our hospital within 1 year with maximum cases from paediatric unit. The present study reports the results of epidemiological investigation of possible outbreak of candidemia by C. krusei in paediatric unit at our tertiary care centre. METHODS: Clinical characteristics and risk factors associated with C. krusei candidemia were evaluated. Yeast identification and antifungal susceptibility testing was performed according to standard protocol. To find the potential source of C. krusei in hospital environment and hand colonization, swabs were collected from different fomites (n = 40) and hand washings from 24 health care workers (HCW), respectively. Infection control and prevention practices were intensified following the recognition of outbreak. Genetic typing was done by fluorescent amplified fragment length polymorphism (FAFLP) technique. Case-control comparison was performed with C. tropicalis and C. pelliculosa cases. RESULTS: Candida krusei fungaemia significantly affected paediatric group (82/186, 44%) as compared to adults (14/130, 10.8%; p < 0.001). Among paediatric group, maximum isolation was reported from neonatal unit of paediatric emergency (NUPE). C. krusei was isolated from hands of one HCW and washbasin in NUPE. FAFLP revealed clonality between blood and environmental isolates indicating cross-transmission of C. krusei. Gastrointestinal disease (p = 0.018), previous antibiotics (p = 0.021) especially to carbapenems (p = 0.039), was significant among C. krusei candidemia cases compared to C. pelliculosa cases. CONCLUSION: We report the largest outbreak of C. krusei candidemia in paediatric unit within 1 year with isolation of related strains from environment and hands of HCW. Routine screening of hand hygiene practices revealed non-compliance to standard practices leading to the increase in C. krusei candidemia cases.

Elucidating redox balance shift in Scheffersomyces stipitis' fermentative metabolism using a modified genome-scale metabolic model.

BACKGROUND: Scheffersomyces stipitis is an important yeast species in the field of biorenewables due to its desired capacity for xylose utilization. It has been recognized that redox balance plays a critical role in S. stipitis due to the different cofactor preferences in xylose assimilation pathway. However, there has not been any systems level understanding on how the shift in redox balance contributes to the overall metabolic shift in S. stipitis to cope with reduced oxygen uptake. Genome-scale metabolic network models (GEMs) offer the opportunity to gain such systems level understanding; however, currently the two published GEMs for S. stipitis cannot be used for this purpose, as neither of them is able to capture the strain's fermentative metabolism reasonably well due to their poor prediction of xylitol production, a key by-product under oxygen limited conditions. RESULTS: A system identification-based (SID-based) framework that we previously developed for GEM validation is expanded and applied to refine a published GEM for S. stipitis, iBB814. After the modified GEM, named iDH814, was validated using literature data, it is used to obtain genome-scale understanding on how redox cofactor shifts when cells respond to reduced oxygen supply. The SID-based framework for GEM analysis was applied to examine how the environmental perturbation (i.e., reduced oxygen supply) propagates through the metabolic network, and key reactions that contribute to the shifts of redox and metabolic state were identified. Finally, the findings obtained through GEM analysis were validated using transcriptomic data. CONCLUSIONS: iDH814, the modified model, was shown to offer significantly improved performance in terms of matching available experimental results and better capturing available knowledge on the organism. More importantly, our analysis based on iDH814 provides the first genome-scale understanding on how redox balance in S. stipitis was shifted as a result of reduced oxygen supply. The systems level analysis identified the key contributors to the overall metabolic state shift, which were validated using transcriptomic data. The analysis confirmed that S. stipitis uses a concerted approach to cope with the stress associated with reduced oxygen supply, and the shift of reducing power from NADPH to NADH seems to be the center theme that directs the overall shift in metabolic states.

A putative phospholipase C is involved in Pichia fermentans dimorphic transition.

BACKGROUND: Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi. METHODS: Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition. RESULTS: Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18. CONCLUSION: Phospholipase C is part of a putative "methionine sensing machinery" that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition. GENERAL SIGNIFICANCE: Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.

Alternative methods for the control of postharvest citrus diseases.

The postharvest diseases of citrus fruit cause considerable losses during storage and transportation. These diseases are managed principally by the application of synthetic fungicides. However, the increasing concern for health hazards and environmental pollution due to chemical use has required the development of alternative strategies for the control of postharvest citrus diseases. Management of postharvest diseases using microbial antagonists, natural plant-derived products and Generally Recognized As Safe compounds has been demonstrated to be most suitable to replace the synthetic fungicides, which are either being banned or recommended for limited use. However, application of these alternatives by themselves may not always provide a commercially acceptable level of control of postharvest citrus diseases comparable to that obtained with synthetic fungicides. To provide more effective disease control, a multifaceted approach based on the combination of different postharvest treatments has been adopted. Actually, despite the distinctive features of these alternative methods, several reasons hinder the commercial use of such treatments. Consequently, research should emphasize the development of appropriate tools to effectively implement these alternative methods to commercial citrus production.

Identification of differentially expressed genes associated with changes in the morphology of Pichia fermentans on apple and peach fruit.

Pichia fermentans (strain DISAABA 726) is an effective biocontrol agent against Monilinia fructicola and Botrytis cinerea when inoculated in artificially wounded apple fruit but is an aggressive pathogen when inoculated on wounded peach fruit, causing severe fruit decay. Pichia fermentans grows as budding yeast on apple tissue and exhibits pseudohyphal growth on peach tissue, suggesting that dimorphism may be associated with pathogenicity. Two complementary suppressive subtractive hybridization (SSH) strategies, that is, rapid subtraction hybridization (RaSH) and PCR-based subtraction, were performed to identify genes differentially expressed by P. fermentans after 24-h growth on apple vs. peach fruit. Gene products that were more highly expressed on peach than on apple tissue, or vice versa, were sequenced and compared with available yeast genome sequence databases. Several of the genes more highly expressed, when P. fermentans was grown on peach, were related to stress response, glycolysis, amino acid metabolism, and alcoholic fermentation but surprisingly not to cell wall degrading enzymes such as pectinases or cellulases. The dual activity of P. fermentans as both a biocontrol agent and a pathogen emphasizes the need for a thorough risk analysis of potential antagonists to avoid unpredictable results that could negatively impact the safe use of postharvest biocontrol strategies.

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain.

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce ('biopreservation') has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers' yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.

Past, present and future research directions with Pichia anomala.

The first International Pichia anomala Symposium provided a survey of past, recent and ongoing research on this yeast. The research community working with this yeast has focussed on several areas. Based on molecular data, a revision of the taxonomy is required: the name P. anomala is no longer applicable, as the genus Pichia is polyphyletic. The current debate centres on whether the yeast should be designated as Wickerhamomyces anomalus or if the previous name, Hansenula anomala, should be re-instated. The anti-microbial activities of this yeast received considerable attention during the symposium. H. anomala has been extensively studied as a biopreservation agent in many different post-harvest systems. Several mechanisms account for its anti-microbial activities, including the production of killer proteins and toxic volatile metabolites. Anti-idiotypic antibodies generating an "internal image" of a killer protein have been found to possess therapeutic activity against a broad range of microorganisms. A great diversity of H. anomala strains was reported at the symposium. Strains have been isolated from several food and feed systems and even from the intestine and reproductive organs of a malaria vector (Anopheles stephensi). Feed and food supplemented with certain H. anomala strains show an improved quality due, for example, to the addition of advantageous proteins and phytase activity. However, a number of apparent opportunistic pathogenic strains have also been isolated. Strain differentiation, especially the recognition of potentially pathogenic isolates, is an important challenge for the future commercialisation of this yeast. Future industrial and agricultural application of this yeast also raises questions of the economics of large-scale production, its survival during storage (formulation) and of safety regulations, all of which require further investigation.

In situ microscopy as online tool for detecting microbial contaminations in cell culture.

Microbial contamination in mammalian cell cultures causing rejected batches is costly and highly unwanted. Most methods for detecting a contamination are time-consuming and require extensive off-line sampling. To circumvent these efforts and provide a more convenient alternative, we used an online in situ microscope to estimate the cell diameter of the cellular species in the culture to distinguish mammalian cells from microbial cells depending on their size. A warning system was set up to alert the operator if microbial cells were present in the culture. Hybridoma cells were cultured and infected with either Candida utilis or Pichia stipitis as contaminant. The warning system could successfully detect the introduced contamination and alert the operator. The results suggest that in situ microscopy could be used as an efficient online tool for early detection of contaminations in cell cultures.

Identification of a Killer Toxin from Wickerhamomyces anomalus with ß-Glucanase Activity.

The yeast Wickerhamomyces anomalus has several applications in the food industry due to its antimicrobial potential and wide range of biotechnological properties. In particular, a specific strain of Wickerhamomyces anomalus isolated from the malaria mosquito Anopheles stephensi, namely WaF17.12, was reported to secrete a killer toxin with strong anti-plasmodial effect on different developmental stages of Plasmodium berghei; therefore, we propose its use in the symbiotic control of malaria. In this study, we focused on the identification/characterization of the protein toxin responsible for the observed antimicrobial activity of the yeast. For this purpose, the culture medium of the killer yeast strain WaF17.12 was processed by means of lateral flow filtration, anion exchange and gel filtration chromatography, immunometric methods, and eventually analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on this concerted approach, we identified a protein with a molecular weight of approximately 140 kDa and limited electrophoretic mobility, corresponding to a high molecular weight ß-glucosidase, as confirmed by activity tests in the presence of specific inhibitors.

An outbreak of Pichia anomala fungemia in a Brazilian pediatric intensive care unit.

OBJECTIVE: To report an outbreak of Pichia anomala fungemia that occurred in a Brazilian pediatric intensive care unit (ICU) from October 2002 to January 2004. DESIGN: Unmatched case-control study. METHODS: We randomly selected four control-patients for each case-patient from a list of all patients admitted to the ICU for at least 48 hours during the outbreak. A second control group was composed of all consecutive patients with nosocomial candidemia in the ICU during the outbreak. An environmental study was performed, and genetic relatedness among the clinical isolates was characterized by randomly amplified polymorphic DNA assay. RESULTS: During the study period, 1,046 children were admitted to the pediatric ICU, 17 of whom developed P. anomala fungemia (attack rate, 1.6%). The median age was 1.1 years, and the main underlying conditions were congenital malformations (35.3%) and neoplastic diseases (11.8%). The overall mortality rate was 41.2%. Two patients received no antifungal treatment; all of the others were treated with amphotericin B. On multivariate analysis, only the presence of a central venous catheter was significantly associated with P. anomala fungemia. The yeast was not found on healthcare workers' hands or in the environment. Molecular studies showed that the outbreak was caused by a single strain. The distribution of risk factors was similar between patients with P. anomala fungemia and control-patients with candidemia. CONCLUSIONS: This study highlights the importance of P. anomala as an emerging nosocomial fungal pathogen. Patients with P. anomala fungemia seem to have risk factors in common with those who have candidemia.

A tobacco S-like RNase inhibits hyphal elongation of plant pathogens.

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

Hansenula anomala infection after bone marrow transplantation.

Hansenula anomala is a yeast which has seldom been reported as a human pathogen. A case of fungaemia with this organism is described in a 22-year-old patient with chronic myeloid leukaemia undergoing a second HLA-matched sibling transplant. A Hickman catheter was in situ and hyperalimentation commenced on day -1. Fever developed on day +10 and H. anomala was isolated from blood cultures. The patient was receiving cyclosporin and methotrexate as prophylaxis against graft-versus-host disease and was severely neutropenic. Treatment with amphotericin B was commenced and the patient's Hickman catheter was removed. Fever resolved and subsequent blood cultures were negative. Amphotericin was continued to a cumulative dose of 680 mg and oral fluconazole 400 mg/day was given for a further week. H. anomala infection has been reported in premature babies and in immunosuppressed individuals but has not been previously observed in patients undergoing bone marrow transplantation. Clinical features of previously reported cases of infection with H. anomala are reviewed.

Fungemia caused by Hansenula anomala: successful treatment with fluconazole.

Fungemia, due to Hansenula anomala, developed in an adult patient with small cell lung cancer who received anti-cancer chemotherapy and plasmapheresis for a sensori-motor neuropathy complication. Treatment with intravenous infusion of fluconazole in addition to the removal of the central venous catheter was successful in treating the fungemia. Pathogenic Hansenula anomala infections are rare, but reports of this infection have been increasing. The use of fluconazole treatment for this infection has not been reported in the literature, and this is the first case of an adult infection of Hansenula anomala in Japan.

Pichia fermentans dimorphic changes depend on the nitrogen source.

Pichia fermentans DiSAABA 726 is a biofilm-forming yeast that undergoes dimorphic transition. Under yeast-like morphology it controls brown rot caused by Monilia spp. on apple fruit, while under pseudohyphal form, it shows pathogenic behaviour itself on peach fruit. The present study investigates the nutritional factors that induce and separate yeast-like and pseudohyphal morphologies under laboratory conditions. We show that P. fermentans DiSAABA 726 produces mainly yeast-like cells on media containing millimolar concentrations of urea and diammonium phosphate, and forms pseudohyphae at micromolar concentrations of these two salts. With ammonium sulphate, yeast-like or pseudohyphal morphology depends on the N concentration and the pH of the culture media. Amino acids such as methionine, valine, and phenylalanine invariably induce pseudohyphal morphology irrespective of the N concentration and the pH of the culture media. Methionol, 1-butanol, isobutanol, and isopropanol induce pseudohyphal growth, while phenylethanol and isoamyl alcohol fail to induce the formation of filaments. Thus, the morphogenesis of P. fermentans DiSAABA 726 depends more on the nitrogen source than on the N concentration, and is regulated by the quorum-sensing molecules that are generally produced from amino-acid assimilation under nitrogen starvation.

A double WAP domain-containing protein from Chinese mitten crab Eriocheir sinensis with antimicrobial activities against Gram-negative bacteria and yeast.

The whey acidic protein (WAP) domain is characterized by a 'four-disulfide-core' (4-DSC) motif comprising of approximately 50 amino acids with eight highly conserved cysteine residues. Previous research indicated that WAP domain-containing proteins played an important role in the innate immunity of crustaceans. In the present study, a novel double WAP domain (DWD)-containing protein gene was identified from Chinese mitten crab Eriocheir sinensis (designated EsDWD) by expressed sequence tag (EST) analysis and PCR techniques. The full-length cDNA of EsDWD was of 593 bp, consisting of a 5'-terminal untranslated region (UTR) of 71 bp, a 3' UTR of 120 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 402 bp. The ORF encoded a polypeptide of 133 amino acids with the predicted molecular weight of 14.4 kDa and the theoretical isoelectric point of 8.14, including a signal peptide of 22 amino acids and two WAP domains. The EsDWD mRNA transcripts were ubiquitously expressed in all the tested tissues, and its expression level in gill was significantly higher than that in other tissues. The mRNA expression of EsDWD in haemocytes was up-regulated after challenge of Vibrio anguillarum and Pichia pastoris GS115, as well as injury treatment. The cDNA encoding the mature EsDWD protein was cloned and expressed in Escherichia coli BL21 (DE3) pLysS, and the purified recombinant EsDWD (rEsDWD) protein exhibited antimicrobial activities against Gram-negative bacteria V. anguillarum, yeast P. pastoris GS115 and Candida parapsilosis. The results collectively suggested that EsDWD was a novel member of double WAP domain (DWD)-containing proteins, and involved in the immune defense against microorganism and wound healing in E. sinensis.

The second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis.

The anti-lipopolysaccharide factor (ALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724bp, consisting of an open reading frame (ORF) of 363bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonella anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris, indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity.

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of C1qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by d-mannose and PGN but not by LPS, glucan or d-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway.