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Purpose Pathogenic variants in GNPTAB and GNPTG, encoding different subunits of GlcNAc-1-phosphotransferase, cause mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma. This study aimed to investigate the cellular and molecular bases underlying skeletal abnormalities in patients with MLII and MLIII. Methods We analyzed bone biopsies from patients with MLIII alpha/beta or MLIII gamma by undecalcified histology and histomorphometry. The skeletal status of Gnptgko and Gnptab-deficient mice was determined and complemented by biochemical analysis of primary Gnptgko bone cells. The clinical relevance of the mouse data was underscored by systematic urinary collagen crosslinks quantification in patients with MLII, MLIII alpha/beta, and MLIII gamma. Results The analysis of iliac crest biopsies revealed that bone remodeling is impaired in patients with GNPTAB-associated MLIII alpha/beta but not with GNPTG-associated MLIII gamma. Opposed to Gnptab-deficient mice, skeletal remodeling is not affected in Gnptgko mice. Most importantly, patients with variants in GNPTAB but not in GNPTG exhibited increased bone resorption. Conclusion The gene-specific impact on bone remodeling in human individuals and in mice proposes distinct molecular functions of the GlcNAc-1-phosphotransferase subunits in bone cells. We therefore appeal for the necessity to classify MLIII based on genetic in addition to clinical criteria to ensure appropriate therapy.

Mutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/β-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients. Brazilian National Council for Scientific and Technological Development/International; 125440785-SFB877/Deutsche Forschungsgemeinschaft/International; PO 1539/1-1/Deutsche Forschungsgemeinschaft/International; 395238399-PO 1539/1-1/Deutsche Forschungsgemeinschaft/International; KU 1240/10-1/Deutsche Forschungsgemeinschaft/International; Cinque per mille e Ricerca Corrente, Ministero della Salute/International. This study was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 125440785‐ SFB877, 395238399‐PO 1539/1‐1 to S. P. and 1240/10‐1 to K. K.), the Brazilian National Council for Scientific and Technological Development (CNPq) to N. F. L. and by unrestricted grants from Cinque per mille e Ricerca Corrente, Ministero della Salute to M. F. and B. T. info:eu-repo/semantics/publishedVersion

Research question What is the potential role of immune cells and their inflammatory cytokines in the pathogenesis, development and establishment of endometriosis? Design Peritoneal fluid from 59 women (43 with endometriosis and 16 controls) who had undergone laparoscopic surgery was analysed. Changes in the population of innate and adaptive immune cells, cytokines, chemokines and growth factor expression were measured by flow cytometry, Luminex Technology and enzyme-linked immunosorbent assay. Results No differences were found in the frequencies of the innate and adaptive immune cells between women with and without endometriosis. In the peritoneal fluid of women with endometriosis, IL-1β, IL-1RN, IL-2, IL-4, IL-8, IL-10, IL-12 (p70), IL-17α, FGF2, G-CSF, MCP-1, MIP-1α and TNF-α were significantly increased compared with controls. A correlation between IL-2, MCP-1, MIP-1α, TNF-α and the severity of endometriosis was observed. The concentration of neopterin, a possible biomarker for this disease, was increased in women with endometriosis compared with controls. Conclusions The functional activity of immune cells seemed to be reduced despite their numbers remaining unchanged. The data indicate that a shift of TH cytokine profile occurs, which increases the TH1–TH2 ratio. This is driven by the increased levels of the cytokines (TNF-α and IL-2) in women with severe endometriosis.

Skeletal pathologies are frequently observed in lysosomal storage disorders, yet the relevance of specific lysosomal enzymes in bone remodeling cell types is poorly defined. Two lysosomal enzymes, ie, cathepsin K (Ctsk) and Acp5 (also known as tartrate-resistant acid phosphatase), have long been known as molecular marker proteins of differentiated osteoclasts. However, whereas the cysteine protease Ctsk is directly involved in the degradation of bone matrix proteins, the molecular function of Acp5 in osteoclasts is still unknown. Here we show that Acp5, in concert with Acp2 (lysosomal acid phosphatase), is required for dephosphorylation of the lysosomal mannose 6-phosphate targeting signal to promote the activity of specific lysosomal enzymes. Using an unbiased approach we identified the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), mutated in mucopolysaccharidosis type VI (MPS-VI), as an osteoclast marker, whose activity depends on dephosphorylation by Acp2 and Acp5. Similar to Acp2/Acp5-/- mice, Arsb-deficient mice display lysosomal storage accumulation in osteoclasts, impaired osteoclast activity, and high trabecular bone mass. Of note, the most prominent lysosomal storage accumulation was observed in osteocytes from Arsb-deficient mice, yet this pathology did not impair production of sclerostin (Sost) and Fgf23. Because the influence of enzyme replacement therapy (ERT) on bone remodeling in MPS-VI is still unknown, we additionally treated Arsb-deficient mice by weekly injection of recombinant human ARSB from 12 to 24 weeks of age. We found that the high bone mass phenotype of Arsb-deficient mice and the underlying bone cell deficits were fully corrected by ERT in the trabecular compartment. Taken together, our results do not only show that the function of Acp5 in osteoclasts is linked to dephosphorylation and activation of lysosomal enzymes, they also provide an important proof-of-principle for the feasibility of ERT to correct bone cell pathologies in lysosomal storage disorders. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α(2)β(2)γ(2)). Upon proteolytic cleavage by site-1 protease, the α/β-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptg(ko) mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/β-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptg(ko) fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptg(ko) lysosomes. Incubation of Gnptg(ko) fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

Mucolipidosis II and III (ML II and III) alpha/beta and ML III gamma are lysosomal diseases caused by GlcNAc-1-phosphotransferase deficiency. Previous data indicate that MLII patients have functionally impaired immune system that contributes to predisposition to infections.We evaluated the immunological phenotype of three Brazilian patients with ML III gamma. Our data suggest that the residual activity of GlcNAc-1-phosphotransferase in patients with ML III gamma is enough to allow the targeting of the lysosomal enzymes required for B-cell functions maintenance.

Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α⁄β-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α⁄β-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity.

The Golgi-resident site-1 protease (S1P) is a key regulator of cholesterol homeostasis and ER stress responses by converting latent transcription factors sterol regulatory element binding proteins (SREPBs) and activating transcription factor 6 (ATF6), as well as viral glycoproteins to their active forms. S1P is also essential for lysosome biogenesis via proteolytic activation of the hexameric GlcNAc-1-phosphotransferase complex required for modification of newly synthesized lysosomal enzymes with the lysosomal targeting signal, mannose 6-phosphate. In the absence of S1P, the catalytically inactive α/β-subunit precursor of GlcNAc-1-phosphotransferase fails to be activated and results in missorting of newly synthesized lysosomal enzymes, and lysosomal accumulation of non-degraded material, which are biochemical features of defective GlcNAc-1-phosphotransferase subunits and the associated pediatric lysosomal diseases mucolipidosis type II and III. The early embryonic death of S1P-deficient mice and the importance of various S1P-regulated biological processes, including lysosomal homeostasis, cautioned for clinical inhibition of S1P. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Background The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms. Methods MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis. Results Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis. Conclusions Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.

Mucolipidosis II and III (ML II and III) alpha/beta and ML III gamma are lysosomal diseases caused by GlcNAc-1-phosphotransferase deficiency. Previous data indicate that MLII patients have a functionally impaired immune system that contributes to the high predisposition to infections. We evaluated the immunological phenotype of three Brazilian patients with ML III gamma. Our data suggest that the residual activity of the enzyme in patients with ML III gamma is enough to allow the targeting lysosomal enzymes required for B-cell functions maintenance. Herein, we evaluate the immunological phenotype of Brazilian patients with ML III gamma.

Schwartz, Ida/0000-0002-7933-6687; Tuysuz, Beyhan/0000-0002-9620-5021; Pohl, Sandra/0000-0001-7409-7042 WOS:000490781000001 PubMed ID: 31579991 Mucolipidosis (ML) II and III alpha/beta are inherited lysosomal storage disorders caused by mutations in GNPTAB encoding the alpha/beta-precursor of GlcNAc-1-phosphotransferase. This enzyme catalyzes the initial step in the modification of more than 70 lysosomal enzymes with mannose 6-phosphate residues to ensure their intracellular targeting to lysosomes. The so-called stealth domains in the alpha- and beta-subunit of GlcNAc-1-phosphotransferase were thought to be involved in substrate recognition and/or catalysis. Here, we performed in silico alignment analysis of stealth domain-containing phosphotransferases and showed that the amino acid residues Glu(389), Asp(408), His(956), and Arg(986) are highly conserved between different phosphotransferases. Interestingly, mutations in these residues were identified in patients with MLII and MLIII alpha/beta. To further support the in silico findings, we also provide experimental data demonstrating that these four amino acid residues are strictly required for GlcNAc-1-phosphotransferase activity and thus may be directly involved in the enzymatic catalysis. Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [KU 1240/10-1, PO 1539/1-1, SFB877-B3]; Brazilian National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq) Deutsche Forschungsgemeinschaft, Grant/Award Numbers: KU 1240/10-1, PO 1539/1-1, SFB877-B3; Brazilian National Council for Scientific and Technological Development (CNPq)

The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2 β2 γ2 ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

Mucolipidosis (ML) III gamma is a rare autosomal-recessive disorder caused by pathogenic mutations in the GNPTG gene. GNPTG encodes the γ-subunit of GlcNAc-1-phosphotransferase that catalyzes mannose 6-phosphate targeting signal synthesis on soluble lysosomal enzymes. ML III gamma patients are characterized by missorting of lysosomal enzymes. In this report, we describe the probable occurrence of mRNA editing in two ML III gamma patients. Patients A and B (siblings) presented at the adult age with a typical clinical picture of ML III gamma, mainly compromising bone and joints, and high levels of lysosomal enzymes in plasma and low levels in fibroblasts. Both were found to be homozygous for c.-112C>G and c.328G>T (p.Glu110Ter) mutations in genomic DNA (gDNA) analysis of GNPTG. Analysis of complementary DNA (cDNA), however, showed normal genotypes for both patients. Low GNPTG mRNA expression was observed in both patients. The mRNA editing can explain the differences found in patients A and B regarding gDNA and cDNA analysis, and the mild clinical phenotype associated with homozygosity for a nonsense mutation. Our results suggest that mRNA editing can be more frequent than expected in monogenic disorders and that GNPTG analysis should be performed on gDNA.

The multimeric GlcNAc-1-phosphotransferase complex catalyzes the formation of mannose 6-phosphate recognition marker on lysosomal enzymes required for receptor-mediated targeting to lysosomes. GNPTAB and GNPTG encode the α/β-subunit precursor membrane proteins and the soluble γ-subunits, respectively. Performing extensive mutational analysis, we identified the binding regions of γ-subunits in a previously uncharacterized domain of α-subunits comprising residues 535-698, named GNPTG binding (GB) domain. Both the deletion of GB preventing γ-subunit binding and targeted deletion of GNPTG led to significant reduction in GlcNAc-1-phosphotransferase activity. We also identified cysteine 70 in α-subunits to be involved in covalent homodimerization of α-subunits which is, however, required neither for interaction with γ-subunits nor for catalytic activity of the enzyme complex. Finally, binding assays using various γ-subunit mutants revealed that residues 130-238 interact with glycosylated α-subunits suggesting a role for the mannose 6-phosphate receptor homology domain in α-subunit binding. These studies provide new insight into the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

With the advance and popularization of molecular techniques, the identification of genetic mutations that cause diseases has increased dramatically. Thus, the number of laboratories available to investigate a given disorder and the number of subsequent diagnosis have increased over time. Although it is necessary to identify mutations and provide diagnosis, it is also critical to develop specific therapeutic approaches based on this information. This review aims to highlight recent advances in mutation-targeted therapies with chemicals that mitigate mutational pathology at the molecular level, for disorders that, for the most part, have no effective treatment. Currently, there are several strategies being used to correct different types of mutations, including the following: the identification and characterization of translational readthrough compounds; antisense oligonucleotide-mediated splicing redirection; mismatch repair; and exon skipping. These therapies and other approaches are reviewed in this paper. Com o avanço e a popularização de técnicas moleculares, a identificação de mutações genéticas que causam doenças aumentou drasticamente. Deste modo, o número de laboratórios disponíveis para investigar uma determinada doença e, consequentemente, o número de diagnósticos, aumentou ao longo do tempo. Embora seja necessário identificar mutações e fornecer diagnósticos, é crucial também desenvolver abordagens terapêuticas específicas baseadas nestas informações. Esta revisão tem como objetivo destacar os avanços recentes em terapias mutação-alvo com produtos químicos que mitiguem a patologia mutacional ao nível molecular, para doenças que, para a maior parte, não têm tratamento eficaz. Atualmente, existem várias estratégias sendo utilizadas para corrigir diferentes tipos de mutações, incluindo as seguintes: a identificação e caracterização de compostos readthroughtraducionais; redirecionamento de splicingmediado por oligonucleotídeos de sentido contrário; reparo por mismatch; e salto de exon. Estas terapias e outras abordagens são revistas neste artigo.

Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/β-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/β-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/β-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive β-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype–phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity.

Mucolipidosis II and III alpha/beta (ML II/III alpha/beta) are rare autosomal recessive lysosomal storage diseases that are caused by a deficiency of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the enzyme responsible for the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. A Brazilian patient suspected of having a very mild ML III was investigated using whole next-generation sequencing (NGS). Two mutations in the GNPTAB gene were detected and confirmed to be in trans status by parental analysis: c.1208T>C (p.Ile403Thr), previously reported as being pathogenic, and the novel mutation c.1723G>A (p.Gly575Arg). This study demonstrates the effectiveness of using whole NGS for the molecular diagnosis of very mild ML III alpha/beta patients.

Mucolipidosis III (ML III) gamma is a very rare autosomal-recessive disorder characterized by the abnormal trafficking and subcellular localization of lysosomal enzymes due to mutations in the GNPTG gene. The present study consists of a report of a Brazilian compound heterozygote patient with ML III gamma resulting from one mutant paternal allele and one allele that had most likely undergone a de novo or maternal germline mutation. This is the first report of a de novo mutation in ML III gamma. This finding has significant implications for genetic counseling.

Texto completo: acesso restrito. p. 59–64 Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2014-05-15T15:07:00Z No. of bitstreams: 1 1-s2.0-S0378111913003740-main.pdf: 742037 bytes, checksum: 2ed9c976f8da0546f888090180ab5ded (MD5) Approved for entry into archive by Patricia Barroso (pbarroso@ufba.br) on 2014-08-11T19:07:38Z (GMT) No. of bitstreams: 1 1-s2.0-S0378111913003740-main.pdf: 742037 bytes, checksum: 2ed9c976f8da0546f888090180ab5ded (MD5) Made available in DSpace on 2014-08-11T19:07:38Z (GMT). No. of bitstreams: 1 1-s2.0-S0378111913003740-main.pdf: 742037 bytes, checksum: 2ed9c976f8da0546f888090180ab5ded (MD5) Previous issue date: 2013 Mucolipidosis II and III (MLII and MLIII) alpha/beta are rare autosomal recessive lysosomal storage diseases (LSDs) caused by pathogenic variations in the GNPTAB gene. GNPTAB gene codes for the α and β subunits of phosphotransferase, the enzyme responsible for synthesis of the mannose-6-phosphate (M6P) marker that directs lysosomal enzymes to the lysosome. Objectives: The objective of this study is to identify sequence variations of the GNPTAB gene in Brazilian patients with MLII and MLIII alpha/beta. Method: Sequencing of the GNPTAB gene was performed in samples of gDNA extracted from the peripheral blood of patients with MLII/III diagnosed at a national reference center for LSDs. Results: Twelve unrelated patients, from several regions of Brazil, were included in this study. Only one was born of consanguineous parents. All patients were found to carry at least one nonpathogenic variation. Nine causal sequence variations were found: c.242G>T (p.W81L); c.1123C>T (p.R375X); c.1196C>T (p.S399F); c.1208T>C (p.I403T); c.1514G>A (p.C505Y); c.1759C>T (p.R587X); c.2808A>G (p.Y937_M972del, novel mutation); c. 2269_2273delGAAAC (p.E757KfsX2, novel mutation); and c.3503_3504delTC (p.L1168QfsX5). Both pathogenic variations were identified in 8 of 12 patients; in four patients, only one pathogenic variation was identified. Mutation c.3503_3504delTC, located in exon 19, was the most frequent pathogenic variation found (n = 11/24 alleles). The deleterious effect of the c.2808A>C mutation on splicing was confirmed by cDNA analysis. Discussion/conclusions: Our findings confirm that the GNPTAB gene presents broad allelic heterogeneity and suggests that, in Brazilian ML II and III patients, screening for mutations should begin at exon 19 of the GNPTAB gene. Further analyses will be conducted on patients in whom both pathogenic mutations have not been found in this study.

Temperature and pH are key factors influencing the production of antimicrobial peptides. In this work, qRT-PCR methodology was used to demonstrate the effect of these two variables on sboA (subtilosin A) and ituD (iturin A) expression in Bacillus sp. P11, an isolate from aquatic environment of the Amazon. Bacillus sp. P11 was incubated in BHI broth for 36 h at 30, 37 and 42 °C, and the pH values were 6.0, 7.4 and 8.0. The production of subtilosin A and iturin A was confirmed by mass spectrometry. The sboA expression increased 200-fold when the initial pH was 8.0. In contrast, ituD expression was maximum at pH 6.0. Increased temperature (42 °C) was adverse for both genes, but ituD expression increased at 37 °C. Expression of sboA and ituD was strongly affected by pH and temperature and qRT-PCR proved to be a powerful tool to investigate the potential of Bacillus strains to produce subtilosin A and iturin A.

TwelveLactobacillusisolates from Brazilian starter-free ovine cheeses were evaluated for their probiotic potential. The strains were identified by 16S rDNA sequencing asLactobacillus plantarum(7),Lb. brevis(2),Lb. casei(2) andLb. parabuchneri(1). All strains showed variable resistance to gastric juices and relative tolerance to pancreatin and bile salts. Only five strains ofLb. plantarumcould not deconjugate the sodium salt of taurodeoxycholic acid. Autoaggregation ability after 24 h was above 50% and hydrophobicity was higher than 60% for most strains. All lactobacilli could inhibit linolenic acid oxidation, exceptLb. parabuchneristrain, whereas none of them could scavenge DPPH radical. β-Galactosidase activity ranged from 47·7 to 2503 Miller units. Inhibition of food pathogensListeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coliandSalmonella typhimuriumwas demonstrated and the production of organic acids could be associated with this effect. TheLactobacillusstrains from Brazilian regional ovine cheese showed interesting functional characteristics, mainly the strainsLb. brevisSM-B andLb. plantarumSM-I. Both presented high acid tolerance. In addition,Lb. brevisSM-B also displayed remarkable antioxidant activity andLb. plantarumSM-I was the highest β-galactosidase producer, exhibited high autoaggregation and hydrophobicity properties.

The biological activity and the presence of genes sfp and ituD (surfactin and iturin A) among Bacillus strains isolated from the Amazon basin were determined. Bacillus spp. were tested for hemolytic activity and inhibition of fungal growth by agar plate assays in parallel with PCR for identification of sfp and ituD genes. All strains tested produced surface-active compounds, giving evidence by lysis of erythrocytes and emulsifying activity on mineral oil and soybean oil. These strains of Bacillus caused growth inhibition of several phytopathogenic fungi, including Fusarium spp., Aspergillus spp., and Bipolaris sorokiniana. The presence of genes ituD and sfp was confirmed by PCR and sequence analysis. The only exception was Bacillus sp. P34 that lacks sfp gene. Lipopeptides were isolated from culture supernatants and analyzed by mass spectrometry. Characteristic m/z peaks for surfactin and iturin were observed, and some strains also produced fengycin and bacillomycin. The remarkable antifungal activity showed by the strains could be associated with the co-production of three or more lipopeptide antibiotics. Screening for novel bacteria producing useful biosurfactants or biocontrol agents for agriculture is a topic of greatest importance to eliminate chemical pollutants.

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42°C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

Bacillus amyloliquefaciens LBM 5006 produces antagonistic activity against pathogenic bacteria and phytopathogenic fungi, including Aspergillus spp., Fusarium spp., and Bipolaris sorokiniana. PCR analysis revealed the presence of ituD, but not sfp genes, coding for iturin and surfactin, respectively. The antimicrobial substance produced by this strain was isolated by ammonium sulfate precipitation, gel filtration chromatography and 1-butanol extraction. The ultraviolet spectrum was typical of a polypeptide and the infrared spectrum indicates the presence of peptide bonds and acyl group(s). The antimicrobial substance was resistant to proteolytic enzymes and heat treatment, and was reactive with ninhydrin. Mass spectroscopy analysis indicated that B. amyloliquefaciens LBM 5006 produces two antimicrobial peptides, with main peaks at m/z 1,058 Da and 1,464 Da, corresponding to iturin-like and fengycin-like peptides, respectively. B. amyloliquefaciens LBM 5006 showed significant activity against phytopatogenic fungi, showing potential for use as a biocontrol agent or production of antifungal preparations.

Dentre as inúmeras espécies da flora nativa brasileira, encontra-se a Ilex paraguariensis, conhecida popularmente como erva-mate. Estudos atribuem diversas atividades biológicas para essa espécie. O trabalho teve por objetivo avaliar a atividade antifúngica e antibacteriana do óleo volátil, extrato das folhas e dos ramos da I. paraguariensis. O material vegetal foi coletado na URI (28°16’41.4”S 54°16’13.0”W) em Santo Ângelo – RS/Brasil, sendo moído e filtrado e o óleo volátil obtido por hidrodestilação. A atividade antimicrobiana foi avaliada empregando a técnica de difusão em meio sólido Brain heart infusion (BHI). As bactérias utilizadas foram Corynebacterium fimi, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Salmonella enteritidis e Escherichia coli. Para a atividade antifúngica, foi empregado o método de difusão em meio sólido ágar Patato dextrose (PDA), e os fungos utilizados foram Aspergillus phoenicis isolado, Aspergillus niger, Fusarium sp. e Fusarium sp. isolado clínico. As alíquotas empregadas foram as mesmas, tanto para as bactérias quanto para os fungos, de 25 e 50 µL nas concentrações de 50 e 100 mg/mL do óleo e extratos. Os resultados obtidos, quando comparados aos resultados de inibição do antifúngico comercial Fox®, usados como padrão comparativo, comprovaram a acentuada atividade antimicrobiana apresentada pelo óleo de I. paraguariensis. Entre os diferentes extratos testados, o de folhas apresentou melhor resultado para atividade antimicrobiana, com a concentração de 100 mg/mL do óleo. O óleo volátil de I. paraguariensis, por apresentar halos de inibição superiores a 10 mm, indica uma boa atividade antimicrobiana.

Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms.

This report demonstrates the usefulness of PCR for the genes spaS and sboA as a means of identifying Bacillus strains with a potential to produce subtilin and subtilosin A. One collection strain and five Bacillus spp. isolated from aquatic environments in the Amazon basin were screened by PCR using primers for sboA and spaS designed specifically for this study. The sequences of the PCR products showed elevated homology with previously described spaS and sboA genes. Antimicrobial peptides were isolated from culture supernatants and analyzed by mass spectrometry. For all samples, the mass spectra revealed clusters with peaks at m/z 3300-3500 Da, corresponding to subtilosin A, subtilin and isoforms of these peptides. These results suggest that the antimicrobial activity of these strains may be associated with the production of subtilosin A and/or subtilin. The PCR used here was efficient in identifying novel Bacillus strains with the essential genes for producing subtilosin A and subtilin.

Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.

A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.