First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil.

Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. All isolates were completely resistant to ß-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: ß-lactams (blaNDM-1, blaKPC-2, blaTEM-1, blaCTX-M-1 group, blaOXA-1 group and blaSHVvariants in 20-80% of the strains), aminoglycoside resistance genes [aac(6')-Ib and aph(3')-VI, 70 and 80%], a fluoroquinolone resistance gene (qnrS, 80%), a sulfonamide resistance gene (sul-2, 80%) and a multidrug efflux system transporter (mdtK, 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC. Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including ß-lactamases (blaOXA-9, blaTEM-1, blaNDM-1, blaCTX-M-15, blaAmpC-1, blaAmpC-2), the bleomycin resistance gene (bleMBL), an erythromycin resistance methylase (ermC), aminoglycoside-modifying enzymes [aac(6')-Ib, aadA/ant(3")-Ia, aph(3')-VI], a sulfonamide resistance enzyme (sul-2), a chloramphenicol acetyltransferase (catA-like), a plasmid-mediated quinolone resistance protein (qnrS1), a glutathione transferase (fosA), PEtN transferases (eptA, eptB) and a glycosyltransferase (arnT). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes, co-harboring blaKPC-2 and blaNDM-1, is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms.

Peroxiredoxins in erythrocytes: far beyond the antioxidant role.

The red blood cells (RBCs) are essential to transport oxygen (O2) and nutrients throughout the human body. Changes in the structure or functioning of the erythrocytes can lead to several deficiencies, such as hemolytic anemias, in which an increase in reactive oxidative species generation is involved in the pathophysiological process, playing a significant role in the severity of several clinical manifestations. There are important lines of defense against the damage caused by oxidizing molecules. Among the antioxidant molecules, the enzyme peroxiredoxin (Prx) has the higher decomposition power of hydrogen peroxide, especially in RBCs, standing out because of its abundance. This review aimed to present the recent findings that broke some paradigms regarding the three isoforms of Prxs found in RBC (Prx1, Prx2, and Prx6), showing that in addition to their antioxidant activity, these enzymes may have supplementary roles in transducing peroxide signals, as molecular chaperones, protecting from membrane damage, and maintenance of iron homeostasis, thus contributing to the overall survival of human RBCs, roles that seen to be disrupted in hemolytic anemia conditions.

Xylose Isomerase Depletion Enhances Virulence of Xanthomonas citri subsp. citri in Citrus aurantifolia.

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence.

Assessment of cecal microbiota modulation from piglet dietary supplementation with copper.

BACKGROUND: Swine production expanded in the last decades. Efforts have been made to improve meat production and to understand its relationship to pig gut microbiota. Copper (Cu) is a usual supplement to growth performance in animal production. Here, two performance studies were conducted to investigate the effects of three different sources of Cu on the microbiota of piglets. A total of 256 weaned piglets were randomly allocated into 4 treatments (10 replicates per treatment of 4 piglets per pen in Trial 1 and 8 replicates of 3 piglets per pen in Trial 2). Treatments included a control group (fed 10 mg/kg of Cu from CuSO4), a group fed at 160 mg/kg of Copper (II) sulfate (CuSO4) or tri-basic copper chloride (TBCC), and a group fed with Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1 (24-35 d), 2 (36-49 d), and 3 (50-70 d), respectively. At 70 d, the cecum luminal contents from one pig per pen were collected and polled for 16 S rRNA sequencing (V3/V4 regions). Parameters were analyzed in a completely randomized block design, in which each experiment was considered as a block. RESULTS: A total of 1337 Operational Taxonomic Units (OTUs) were identified. Dominance and Simpson ecological metrics were statistically different between control and treated groups (P < 0.10) showing that different Cu sources altered the gut microbiota composition with the proliferation of some bacteria that improve gut health. A high abundance of Prevotella was observed in all treatments while other genera were enriched and differentially modulated, according to the Cu source and dosage. The supplementation with Cu-MHAC can modify a group of bacteria involved in feed efficiency (FE) and short chain fatty acids (SCFA) production (Clostridium XIVa, Desulfovibrio, and Megasphera). These bacteria are also important players in the activation of ghrelin and growth hormones that were previously reported to correlate with Cu-MHAC supplementation. CONCLUSIONS: These results indicated that some genera seem to be directly affected by the Cu source offered to the animals. TBCC and Cu-MHAC (even in low doses) can promote healthy modifications in the gut bacterial composition, being a promising source of supplementation for piglets.

Influence of Melatonin Treatment on Cellular Mechanisms of Redox Adaptation in K562 Erythroleukemic Cells.

Melatonin (MEL) presents well-documented pleiotropic actions against oxidative stress (OS), acting indirectly through activation of transcription factors, e.g., FoxO3 and Nrf2. Thus, this study aimed to investigate the possible modulating effects of MEL on the redox signaling pathways PI3K/AKT/FoxO3 and Keap1/Nrf2/ARE in K562 erythroleukemic cells subjected to OS induction. For this, the viability, and transcript levels of genes involved in redox adaptation were evaluated in K562 cells in different periods of erythroid differentiation: under OS induction by hydrogen peroxide (100 µM H2O2); treated with 1 nM (C1) and 1 mM (C2) MEL; and associated or not with stress induction. We observed a restoration of physiological levels of Nrf2 in both MEL concentrations under OS. The C1 was related to enhanced expression of antioxidant and proteasome genes through the Nrf2-ARE pathway, while C2 to the induction of FOXO3 expression, suggesting an involvement with apoptotic pathway, according to BIM transcript levels. The effects of MEL administration in these cells showed a period and dose-dependent pattern against induced-OS, with direct and indirect actions through different pathways of cellular adaptation, reinforcing the importance of this indolamine in the regulation of cellular homeostasis, being a promising therapeutic alternative for diseases that present an exacerbated OS.

Potential Cytoprotective and Regulatory Effects of Ergothioneine on Gene Expression of Proteins Involved in Erythroid Adaptation Mechanisms and Redox Pathways in K562 Cells.

This study aimed to establish the importance of ergothioneine (ERT) in the erythroid adaptation mechanisms by appraising the expression levels of redox-related genes associated with the PI3K/AKT/FoxO3 and Nrf2-ARE pathways using K562 cells induced to erythroid differentiation and H2O2-oxidative stress. Cell viability and gene expression were evaluated. Two concentrations of ERT were assessed, 1 nM (C1) and 100 µM (C2), with and without stress induction (100 µM H2O2). Assessments were made in three periods of the cellular differentiation process (D0, D2, and D4). The C1 treatment promoted the induction of FOXO3 (D0 and 2), PSMB5, and 6 expressions (D4); C1 + H2O2 treatment showed the highest levels of NRF2 transcripts, KEAP1 (D0), YWHAQ (D2 and 4), PSMB5 (D2) and PSMB6 (D4); and C2 + H2O2 (D2) an increase in FOXO3 and MST1 expression, with a decrease of YWHAQ and NRF2 was observed. in C2 + H2O2 (D2) an increase in FOXO3 and MST1, with a decrease in YWHAQ and NRF2 was observed All ERT treatments increased gamma-globin expression. Statistical multivariate analyzes highlighted that the Nrf2-ARE pathway presented a greater contribution in the production of PRDX1, SOD1, CAT, and PSBM5 mRNAs, whereas the PI3K/AKT/FoxO3 pathway was associated with the PRDX2 and TRX transcripts. In conclusion, ERT presented a cytoprotective action through Nrf2 and FoxO3, with the latter seeming to contribute to erythroid proliferation/differentiation.

Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil.

Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including ß-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to ß-lactamases (bla IND-13, bla CIA-3, bla TEM-116, bla OXA-209, bla VEB-15), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.

Vitamin D deficiency predicts 30-day hospital mortality of adults with COVID-19.

BACKGROUND & AIMS: Several studies have shown conflicting results for the relationship between vitamin D deficiency and COVID-19 outcomes. Here, we aimed to evaluate whether plasma 25(OH)D levels predict mortality in adults admitted with COVID-19, considering potential confounders. METHODS: We conducted a retrospective cohort study that included 115 adults (age 62.1 ± 17.6 years, 65 males) admitted to a Brazilian public hospital for severely symptomatic COVID-19. Subjects were classified into two groups according to their plasma levels of 25(OH)D: sufficiency (≥50 nmol/L) and the deficiency (<50 nmol/L). The diagnosis of COVID-19 was performed using real-time polymerase chain reaction (qPCR). In addition, direct competitive chemiluminescence immunoassay assessed serum 25(OH)D levels. RESULTS: The all-cause 30-day mortality was 13.8% (95% CI: 6.5%-21%) in the group of patients with sufficient plasma 25(OH)D levels and 32.1% (95% CI: 14.8%-49.4%) among those with deficient plasma 25(OH)D levels. Cox regression showed that plasma 25(OH)D levels remained a significant predictor of mortality even after adjusting for the covariates sex, age, length of the delay between symptom onset and hospitalization, and disease severity (HR = 0.98, 95% CI: 0.96-1.00; p = 0.02). CONCLUSION: Vitamin D deficiency predicts higher mortality risk in adults with COVID-19.

Retraction notice to "Use of ivermectin in the treatment of Covid-19: A pilot trial" [Toxicol. Rep. 8 (2021) 505-510].

[This retracts the article DOI: 10.1016/j.toxrep.2021.03.003.].

A Systematic Immuno-Informatic Approach to Design a Multiepitope-Based Vaccine Against Emerging Multiple Drug Resistant Serratia marcescens.

Serratia marcescens is now an important opportunistic pathogen that can cause serious infections in hospitalized or immunocompromised patients. Here, we used extensive bioinformatic analyses based on reverse vaccinology and subtractive proteomics-based approach to predict potential vaccine candidates against S. marcescens. We analyzed the complete proteome sequence of 49 isolate of Serratia marcescens and identified 5 that were conserved proteins, non-homologous from human and gut flora, extracellular or exported to the outer membrane, and antigenic. The identified proteins were used to select 5 CTL, 12 HTL, and 12 BCL epitopes antigenic, non-allergenic, conserved, hydrophilic, and non-toxic. In addition, HTL epitopes were able to induce interferon-gamma immune response. The selected peptides were used to design 4 multi-epitope vaccines constructs (SMV1, SMV2, SMV3 and SMV4) with immune-modulating adjuvants, PADRE sequence, and linkers. Peptide cleavage analysis showed that antigen vaccines are processed and presented via of MHC class molecule. Several physiochemical and immunological analyses revealed that all multiepitope vaccines were non-allergenic, stable, hydrophilic, and soluble and induced the immunity with high antigenicity. The secondary structure analysis revealed the designed vaccines contain mainly coil structure and alpha helix structures. 3D analyses showed high-quality structure. Molecular docking analyses revealed SMV4 as the best vaccine construct among the four constructed vaccines, demonstrating high affinity with the immune receptor. Molecular dynamics simulation confirmed the low deformability and stability of the vaccine candidate. Discontinuous epitope residues analyses of SMV4 revealed that they are flexible and can interact with antibodies. In silico immune simulation indicated that the designed SMV4 vaccine triggers an effective immune response. In silico codon optimization and cloning in expression vector indicate that SMV4 vaccine can be efficiently expressed in E. coli system. Overall, we showed that SMV4 multi-epitope vaccine successfully elicited antigen-specific humoral and cellular immune responses and may be a potential vaccine candidate against S. marcescens. Further experimental validations could confirm its exact efficacy, the safety and immunogenicity profile. Our findings bring a valuable addition to the development of new strategies to prevent and control the spread of multidrug-resistant Gram-negative bacteria with high clinical relevance.

The Heat Shock Transcription Factor HsfA Is Essential for Thermotolerance and Regulates Cell Wall Integrity in Aspergillus fumigatus.

The deleterious effects of human-induced climate change have long been predicted. However, the imminent emergence and spread of new diseases, including fungal infections through the rise of thermotolerant strains, is still neglected, despite being a potential consequence of global warming. Thermotolerance is a remarkable virulence attribute of the mold Aspergillus fumigatus. Under high-temperature stress, opportunistic fungal pathogens deploy an adaptive mechanism known as heat shock (HS) response controlled by heat shock transcription factors (HSFs). In eukaryotes, HSFs regulate the expression of several heat shock proteins (HSPs), such as the chaperone Hsp90, which is part of the cellular program for heat adaptation and a direct target of HSFs. We recently observed that the perturbation in cell wall integrity (CWI) causes concomitant susceptibility to elevated temperatures in A. fumigatus, although the mechanisms underpinning the HS response and CWI cross talking are not elucidated. Here, we aim at further deciphering the interplay between HS and CWI. Our results show that cell wall ultrastructure is severely modified when A. fumigatus is exposed to HS. We identify the transcription factor HsfA as essential for A. fumigatus viability, thermotolerance, and CWI. Indeed, HS and cell wall stress trigger the coordinated expression of both hsfA and hsp90. Furthermore, the CWI signaling pathway components PkcA and MpkA were shown to be important for HsfA and Hsp90 expression in the A. fumigatus biofilms. Lastly, RNA-sequencing confirmed that hsfA regulates the expression of genes related to the HS response, cell wall biosynthesis and remodeling, and lipid homeostasis. Our studies collectively demonstrate the connection between the HS and the CWI pathway, with HsfA playing a crucial role in this cross-pathway regulation, reinforcing the importance of the cell wall in A. fumigatus thermophily.

Transcriptional Control of the Production of Aspergillus fumigatus Conidia-Borne Secondary Metabolite Fumiquinazoline C Important for Phagocytosis Protection.

Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.

Use of ivermectin in the treatment of Covid-19: A pilot trial.

OBJECTIVES: In this randomized open-label trial pilot study we assessed the antiviral effects and safety of various doses of ivermectin in patients with mild clinical symptoms of COVID-19. METHODS: Patients were randomly assigned to receive standard of care (SOC) treatment at hospital admission; SOC plus ivermectin 100 mcg/kg; SOC plus ivermectin 200 mcg/kg; or SOC plus ivermectin 400 mcg/kg. The primary assessed endpoint was the proportion of patients who achieved two consecutive negative SARS-CoV-2 RT PCR tests within 7 days of the start of the dosing period. This study was registered at ClinicalTrials.gov (NCT04431466). RESULTS: A total of 32 patients were enrolled and randomized to treatment. SOC treatment together with ivermectin did not result in any serious adverse events. All patients exhibited a reduction in SARS-CoV-2 viral load within 7 days; however, those who received ivermectin had a more consistent decrease as compared to the SOC alone group, characterized by a shorter time for obtaining two consecutive negative SARS-CoV-2 RT PCR tests. CONCLUSIONS: Ivermectin is safe in patients with SARS-CoV-2, reducing symptomatology and the SARS-CoV-2 viral load. This antiviral effect appears to depend on the dose used, and if confirmed in future studies, it suggests that ivermectin may be a useful adjuvant to the SOC treatment in patients with mild COVID-19 symptoms.

Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation.

The initiation of Aspergillus fumigatus infection occurs via dormant conidia deposition into the airways. Therefore, conidial germination and subsequent hyphal extension and growth occur in a sustained heat shock (HS) environment promoted by the host. The cell wall integrity pathway (CWIP) and the essential eukaryotic chaperone Hsp90 are critical for fungi to survive HS. Although A. fumigatus is a thermophilic fungus, the mechanisms underpinning the HS response are not thoroughly described and important to define its role in pathogenesis, virulence and antifungal drug responses. Here, we investigate the contribution of the CWIP in A. fumigatus thermotolerance. We observed that the CWIP components PkcA, MpkA and RlmA are Hsp90 clients and that a PkcAG579R mutation abolishes this interaction. PkcAG579R also abolishes MpkA activation in the short-term response to HS. Biochemical and biophysical analyses indicated that Hsp90 is a dimeric functional ATPase, which has a higher affinity for ADP than ATP and prevents MpkA aggregation in vitro. Our data suggest that the CWIP is constitutively required for A. fumigatus to cope with the temperature increase found in the mammalian lung environment, emphasising the importance of this pathway in supporting thermotolerance and cell wall integrity.

The Lung Microbiome of Three Young Brazilian Patients With Cystic Fibrosis Colonized by Fungi.

Microbial communities infiltrate the respiratory tract of cystic fibrosis patients, where chronic colonization and infection lead to clinical decline. This report aims to provide an overview of the diversity of bacterial and fungal species from the airway secretion of three young CF patients with severe pulmonary disease. The bacterial and fungal microbiomes were investigated by culture isolation, metataxonomics, and metagenomics shotgun. Virulence factors and antibiotic resistance genes were also explored. A. fumigatus was isolated from cultures and identified in high incidence from patient sputum samples. Candida albicans, Penicillium sp., Hanseniaspora sp., Torulaspora delbrueckii, and Talaromyces amestolkiae were isolated sporadically. Metataxonomics and metagenomics detected fungal reads (Saccharomyces cerevisiae, A. fumigatus, and Schizophyllum sp.) in one sputum sample. The main pathogenic bacteria identified were Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex, and Achromobacter xylosoxidans. The canonical core CF microbiome is composed of species from the genera Streptococcus, Neisseria, Rothia, Prevotella, and Haemophilus. Thus, the airways of the three young CF patients presented dominant bacterial genera and interindividual variability in microbial community composition and diversity. Additionally, a wide diversity of virulence factors and antibiotic resistance genes were identified in the CF lung microbiomes, which may be linked to the clinical condition of the CF patients. Understanding the microbial community is crucial to improve therapy because it may have the opposite effect, restructuring the pathogenic microbiota. Future studies focusing on the influence of fungi on bacterial diversity and microbial interactions in CF microbiomes will be welcome to fulfill this huge gap of fungal influence on CF physiopathology.

Digestion of Intact Gluten Proteins by Bifidobacterium Species: Reduction of Cytotoxicity and Proinflammatory Responses.

Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the Bifidobacterium species Bifidobacterium bifidum, Bifidobacterium longum, Bembidion breve, Bifidobacterium animalis alone, and also a Bifidobacterium consortium on the digestion of intact gluten proteins (gliadins and glutenins) and the associated immunomodulatory responses elicited by the resulting peptides. The cytotoxicity and proinflammatory responses were evaluated through the activation of NF-kB p65 and the expression of cytokines TNF-α and IL-1ß in Caco-2 cell cultures exposed to gluten-derived peptides. The peptides induced a clear reduction in cytotoxic responses and proinflammatory marker levels compared to the gluten fragments generated during noninoculated gastrointestinal digestion. These results highlight the possible use of probiotics based on bifidobacteria as a prospective treatment for CD.

Recombinant expression, characterization and phylogenetic studies of novels cystatins-like proteins of sweet orange (Citrus sinensis) and clementine (Citrus clementina).

Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes.

The Cell Wall Integrity Pathway Contributes to the Early Stages of Aspergillus fumigatus Asexual Development.

Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcAG579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi.IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.

Analyses of the three 1-Cys Peroxiredoxins from Aspergillus fumigatus reveal that cytosolic Prx1 is central to H2O2 metabolism and virulence.

Standing among the front defense strategies against pathogens, host phagocytic cells release various oxidants. Therefore, pathogens have to cope with stressful conditions at the site of infection. Peroxiredoxins (Prx) are highly reactive and abundant peroxidases that can support virulence and persistence of pathogens in distinct hosts. Here, we revealed that the opportunistic human pathogen A. fumigatus presents three 1-Cys Prx (Prx6 subfamily), which is unprecedented. We showed that PrxB and PrxC were in mitochondria, while Prx1 was in cytosol. As observed for other Prxs, recombinant Prx1 and PrxC decomposed H2O2 at elevated velocities (rate constants in the 107 M-1s-1 range). Deletion mutants for each Prx displayed higher sensitivity to oxidative challenge in comparison with the wild-type strain. Additionally, cytosolic Prx1 was important for A. fumigatus survival upon electron transport dysfunction. Expression of Prxs was dependent on the SakAHOG1 MAP kinase and the Yap1YAP1 transcription factor, a global regulator of the oxidative stress response in fungi. Finally, cytosolic Prx1 played a major role in pathogenicity, since it is required for full virulence, using a neutropenic mouse infection model. Our data indicate that the three 1-Cys Prxs act together to maintain the redox balance of A. fumigatus.