Stabilisation of mixed peptide/lipid complexes in selective antifungal hexapeptides

AbstractThe design of antimicrobial peptides could have benefited from structural studies of known peptides having specific activity against target microbes, but not toward other microorganisms. We have previously reported the identification of a series of peptides (PAF-series) active against certain postharvest fungal phytopathogens, and devoid of toxicity towards E. coli and S. cerevisiae [López-Garcı́a et al. Appl. Environ. Microbiol. 68 (2002) 2453]. The peptides inhibited the conidia germination and hyphal growth. Here, we present a comparative structural characterisation of selected PAF peptides obtained by single-amino-acid replacement, which differ in biological activity. The peptides were characterised in solution using fluorescence and circular dichroism (CD) spectroscopies. Membrane and membrane mimetic–peptide interactions and the lipid-bound structures were studied using fluorescence with the aid of extrinsic fluorescent probes that allowed the identification of mixed peptide/lipid complexes. A direct correlation was found between the capability of complex formation and antifungal activity. These studies provide a putative structural basis for the mechanism of action of selective antifungal peptides

Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum

Antifungal proteins (AFPs) from Ascomycetes are small cysteine-rich proteins that are abundantly secreted and show antifungal activity against non-producer fungi. A gene coding for a class B AFP (AfpB) was previously identified in the genome of the plant pathogen Penicillium digitatum. However, previous attempts to detect the AfpB protein were not successful despite the high expression of the corresponding afpB gene. In this work, the structure of the putative AfpB was modeled. Based on this model, four synthetic cysteine-containing peptides, PAF109, PAF112, PAF118, and PAF119, were designed and their antimicrobial activity was tested and characterized. PAF109 that corresponds to the gamma-core motif present in defensin-like antimicrobial proteins did not show antimicrobial activity. On the contrary, PAF112 and PAF118, which are cationic peptides derived from two surface-exposed loops in AfpB, showed moderate antifungal activity against P. digitatum and other filamentous fungi. It was also confirmed that cyclization through a disulfide bridge prevented peptide degradation. PAF116, which is a peptide analogous to PAF112 but derived from the Penicillium chrysogenum antifungal protein PAF, showed activity against P. digitatum similar to PAF112, but was less active than the native PAF protein. The two AfpB-derived antifungal peptides PAF112 and PAF118 showed positive synergistic interaction when combined against P. digitatum. Furthermore, the synthetic hexapeptide PAF26 previously described in our laboratory also exhibited synergistic interaction with the peptides PAF112, PAF118, and PAF116, as well as with the PAF protein. This study is an important contribution to the mapping of antifungal motifs within the AfpB and other AFPs, and opens up new strategies for the rational design and application of antifungal peptides and proteins

A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection

<p>Abstract</p> <p>Background</p> <p>Postharvest losses of citrus fruit due to green mold decay, caused by the fungus <it>Penicillium digitaum</it>, have a considerable economic impact. However, little is known about the molecular processes underlying the response of citrus fruit to <it>P. digitatum</it>.</p> <p>Results</p> <p>Here we describe the construction of a subtracted cDNA library enriched in citrus genes preferentially expressed in response to pathogen infection followed by cDNA macroarray hybridization to investigate gene expression during the early stages of colonization of the fruit's peel by <it>P. digitatum</it>. Sequence annotation of clones from the subtracted cDNA library revealed that induction of secondary and amino acid metabolisms constitutes the major response of citrus fruits to <it>P. digitatum </it>infection. Macroarray hybridization analysis was conducted with RNA from either control, wounded, ethylene treated or <it>P. digitatum </it>infected fruit. Results indicate an extensive overlap in the response triggered by the three treatments, but also demonstrated specific patterns of gene expression in response to each stimulus. Collectively our data indicate a significant presence of isoprenoid, alkaloid and phenylpropanoid biosynthetic genes in the transcriptomic response of citrus fruits to <it>P. digitatum </it>infection. About half of the genes that are up-regulated in response to pathogen infection are also induced by ethylene, but many examples of ethylene-independent gene regulation were also found. Two notable examples of this regulation pattern are the genes showing homology to a caffeine synthase and a berberine bridge enzyme, two proteins involved in alkaloid biosynthesis, which are among the most induced genes upon <it>P. digitatum </it>infection but are not responsive to ethylene.</p> <p>Conclusions</p> <p>This study provided the first global picture of the gene expression changes in citrus fruit in response to <it>P. digitatum </it>infection, emphasizing differences and commonalities with those triggered by wounding or exogenous ethylene treatment. Interpretation of the differentially expressed genes revealed that metabolism is redirected to the synthesis of isoprenes, alkaloids and phenylpropanoids.</p

A genomic approach highlights common and diverse effects and determinants of susceptibility on the yeast Saccharomyces cerevisiae exposed to distinct antimicrobial peptides

<p>Abstract</p> <p>Background</p> <p>The mechanism of action of antimicrobial peptides (AMP) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMP is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast <it>Saccharomyces cerevisiae </it>to characterize the antifungal effect of two unrelated AMP.</p> <p>Results</p> <p>Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene Ontology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW-related genes including those of MAPK signaling pathways revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as Δ<it>ecm33 </it>or Δ<it>ssd1</it>. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, <it>ARG </it>genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking <it>ARG </it>genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (<it>ARG1</it>) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene <it>IPT1 </it>was extended to the peptides studied here.</p> <p>Conclusions</p> <p>Reinforcement of CW is a general response common after exposure to distinct AMP, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMP. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMP, was also independently confirmed.</p

Quantitative And Qualitative Analysis Of Microorganisms In Root-filled Teeth With Persistent Infection: Monitoring Of The Endodontic Retreatment.

The aim of this study was to investigate in vivo microorganisms detected in root-filled teeth with post-treatment apical periodontitis and quantify colony-forming units (CFU) during endodontic retreatment. Fifteen root-filled teeth had their previous gutta-percha removed and were randomly instrumented before being divided into three groups and medicated with either [Ca(OH)2 + 2% CHX gel], [Ca(OH)2 + 0.9% NaCl] or 2% CHX gel. Samples were taken after removal of gutta-percha (S1), after chemomechanical preparation using 2% CHX gel (S2), and after inter-appointment dressing (S3) for 7 or 14 days later. Cultivable bacteria recovered from infected root canals at the three stages were counted and identified by means of culture and PCR assay (16S rDNA). Quantitative data were statistically analyzed by using Mann-Whitney test in which pairs of groups were compared (P < 0.05). CFU counts decreased significantly from S1 to S2 (P < 0.05). No significant difference was found between S2 and S3 (P = 0.3093) for all three experimental groups. Chemomechanical preparation and intra-canal dressing promoted significant median reductions of 99.61% and 99.57%, respectively, in the number of bacteria compared to S1 samples. A total of 110 cultivable isolates were recovered by culture technique from 32 different species and 7 different genera. Out of the 13 target species-specific primer of bacteria analyzed, 11 were detected during endodontic retreatment. The great majority of taxa found in post-treatment samples were Gram-positive bacteria, although Gram-negative bacteria were found by molecular methods. Moreover, our results showed that gutta-percha removal and chemomechanical preparation are effective for root canal disinfection, whereas additional intra-canal dressing did not improve disinfection.7302-

Supplementary information files for An iron ore-based catalyst for producing hydrogen and metallurgical carbon via catalytic methane pyrolysis for decarbonisation of the steel industry

Supplementary files for article An iron ore-based catalyst for producing hydrogen and metallurgical carbon via catalytic methane pyrolysis for decarbonisation of the steel industry Experiments to investigate the catalytic pyrolysis of methane using an iron ore-based catalyst were carried out to optimize catalytic activity and examine the purity of the carbon produced from the process for the first time. Ball milling of the iron ore at 300 rpm for varying times – from 30 to 330 minutes – was studied to determine the effect of milling time on methane conversion. Optimal milling for 270 minutes led to a five-fold increase in methane conversion from ca. 1% to 5%. Further grinding resulted in a decline of methane conversion to 4% shown by SEM to correspond to an increase in particle size caused by agglomeration. Data from Raman and Mössbauer spectroscopy and H2 temperature programmed reduction indicated a change in phase from magnetite to maghemite and hematite (at the particle surface) as the grinding time increased. Analysis of the carbon produced as a byproduct of the reaction indicated a highly pure material with the potential to be used as an additive for steel production. </p

Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium digitatum

Filamentous fungi encode distinct antifungal proteins (AFPs) that offer great potential to develop new antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum, the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene (afpB), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P. digitatum strains. Biotechnological production and secretion of AfpB were achieved in P. digitatum with the use of a P. chrysogenum-based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed antifungal activity higher than the P. chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P. digitatum, which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi

Depressive symptoms and alcohol correlates among Brazilians aged 14 years and older: a cross-sectional study

Background: the associations between depressive symptoms and alcohol-related disorders, drinking patterns and other characteristics of alcohol use are important public health issues worldwide. This study aims to study these associations in an upper middle-income country, Brazil, and search for related socio-demographic correlations in men and women.Methods: A cross-sectional study was conducted between November 2005 and April 2006. the sample of 3,007 participants, selected using a multistage probabilistic sampling method, represents the Brazilian population aged 14 and older. Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression Scale and alcohol dependence was assessed using the Composite International Diagnostic Interview. Associations assessed using bi-variate analysis were tested using Rao-Scott measures. Gender specific multinomial logistic regression models were developed.Results: Among the participants with alcohol dependence, 46% had depressive symptoms (17.2% mild/moderate and 28.8% major/severe; p < 0.01); 35.8% (p = 0.08) of those with alcohol abuse and 23.9% (p < 0.01) of those with a binge-drinking pattern also had depressive symptoms. Alcohol abstainers and infrequent drinkers had the highest prevalence of major/severe depressive symptoms, whereas frequent heavy drinkers had the lowest prevalence of major/severe depressive symptoms. in women, alcohol dependence and the presence of one or more problems related to alcohol consumption were associated with higher risks of major/severe depressive symptoms. Among men, alcohol dependence and being = 45 years old were associated with higher risks of major/severe depressive symptoms.Conclusions: in Brazil, the prevalence of depressive symptoms is strongly related to alcohol dependence; the strongest association was between major/severe depressive symptoms and alcohol dependence in women. This survey supports the possible association of biopsychosocial distress, alcohol consumption and the prevalence of depressive symptoms in Brazil. Investing in education, social programs, and care for those with alcohol dependence and major/severe depressive symptoms, especially for such women, and the development of alcohol prevention policies may be components of a strategic plan to reduce the prevalence of depression and alcohol problems in Brazil. Such a plan may also promote the socio-economic development of Brazil and other middle-income countries.Universidade Federal de São Paulo UNIFESP, Department Psychiat, BR-15085420 Sao Jos Rio Preto, SP, BrazilUniv São Paulo FMRP, Ribeirao Preto Med Sch, Dept Social Med, BR-14049900 Ribeirao Preto, SP, BrazilUniv Fed Santa Catarina, Ctr Ciencias Saude, Dept Clin Med, BR-88040900 Florianopolis, SC, BrazilUniv Texas Dallas, Dallas Reg Campus, Sch Publ Hlth, Dallas, TX 75390 USAUniv São Paulo, FMRP USP, Ribeirao Preto Med Sch, Dept Neurosci & Behav, BR-14048900 Ribeirao Preto, SP, BrazilINCT Translat Med, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Department Psychiat, BR-15085420 Sao Jos Rio Preto, SP, BrazilWeb of Scienc

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi

[EN] Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, readyto-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciensmediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA,P7760,Pef1¿, and PafpB constitutive promoters, and PglaA,PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.This work was supported by PROMETEO/2018/066 from "Conselleria d'Educacio" (Generalitat Valenciana, Comunitat Valenciana, Spain), grant PID2021-125858OB-100, and the Severo Ochoa Excellence Program CEX 2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe." EM-G was the recipient of a predoctoral grant FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future." SG holds a Juan de la Cierva Incorporacion grant (IJC 2020-042749-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR.Moreno-Giménez, E.; Gandía, M.; Sáez, Z.; Manzanares, P.; Yenush, L.; Orzáez Calatayud, DV.; Marcos, JF.... (2023). FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi. Frontiers in Bioengineering and Biotechnology. 11:1-17. https://doi.org/10.3389/fbioe.2023.12228121171