RESUMO
The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.
Assuntos
Infecções por Klebsiella/metabolismo , Klebsiella pneumoniae/metabolismo , Lipídeo A/biossíntese , Lipídeo A/química , Animais , Humanos , Infecções por Klebsiella/genética , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/genética , Lipídeo A/genética , Pulmão/microbiologia , Camundongos , Estrutura Molecular , Especificidade de ÓrgãosRESUMO
The NF-κB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identified 17 kinases that when targeted by siRNA restored IL-1ß-dependent NF-κB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)-phosphatidylinositol 3-OH kinase (PI3K)-AKT-PAK4-ERK-GSK3ß signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-κB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR-PI3K-AKT-PAK4-ERK-GSK3ß signalling pathway. Our efforts to identify the bacterial factor(s)responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence suggesting that CPS could mediate the activation of EGFR. Supporting this notion, purified CPS did activate EGFR as well as the EGFR-dependent PI3K-AKT-PAK4-ERK-GSK3ß signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4-MyD88-c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.
Assuntos
Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/patogenicidade , Proteínas Quinases/metabolismo , Transdução de Sinais , Cápsulas Bacterianas/imunologia , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , HumanosRESUMO
Protein N-glycosylation requires flipping of the glycolipid Man(5)GlcNAc(2)-diphosphate dolichol (Man(5)GlcNAc(2)-PP-Dol) across the endoplasmic reticulum (ER). Helenius et al. report genetic evidence suggesting that Rft1, an essential ER membrane protein in yeast, is required directly to translocate Man(5)GlcNAc(2)-PP-Dol. We now show that a specific ER protein(s), but not Rft1, is required to flip Man(5)GlcNAc(2)-PP-Dol in reconstituted vesicles. Rft1 may have a critical accessory role in translocating Man(5)GlcNAc(2)-PP-Dol in vivo, but the Man(5)GlcNAc(2)-PP-Dol flippase itself remains to be identified.
Assuntos
Dolicóis/análogos & derivados , Mananas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dolicóis/metabolismo , Proteínas de Membrana Transportadoras , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/citologiaRESUMO
Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-κB and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1ß-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1ß-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.
Assuntos
Evasão da Resposta Imune , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/patogenicidade , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Animais , Linhagem Celular , Enzima Desubiquitinante CYLD , Fosfatase 1 de Especificidade Dupla/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Inflamação/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD1/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Understanding the virulence mechanisms of K. pneumoniae is a priority and timely to design new therapeutics. Here, we demonstrate that K. pneumoniae limits the SUMOylation of host proteins in epithelial cells and macrophages (mouse and human) to subvert cell innate immunity. Mechanistically, in lung epithelial cells, Klebsiella increases the levels of the deSUMOylase SENP2 in the cytosol by affecting its K48 ubiquitylation and its subsequent degradation by the ubiquitin proteasome. This is dependent on Klebsiella preventing the NEDDylation of the Cullin-1 subunit of the ubiquitin ligase complex E3-SCF-ßTrCP by exploiting the CSN5 deNEDDylase. Klebsiella induces the expression of CSN5 in an epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-extracellular signal-regulated kinase (ERK)-glycogen synthase kinase 3 beta (GSK3ß) signaling pathway-dependent manner. In macrophages, Toll-like receptor 4 (TLR4)-TRAM-TRIF-induced type I interferon (IFN) via IFN receptor 1 (IFNAR1)-controlled signaling mediates Klebsiella-triggered decrease in the levels of SUMOylation via let-7 microRNAs (miRNAs). Our results revealed the crucial role played by Klebsiella polysaccharides, the capsule, and the lipopolysaccharide (LPS) O-polysaccharide, to decrease the levels of SUMO-conjugated proteins in epithelial cells and macrophages. A Klebsiella-induced decrease in SUMOylation promotes infection by limiting the activation of inflammatory responses and increasing intracellular survival in macrophages.IMPORTANCEKlebsiella pneumoniae has been singled out as an urgent threat to human health due to the increasing isolation of strains resistant to "last-line" antimicrobials, narrowing the treatment options against Klebsiella infections. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of multidrug-resistant Klebsiella infections; this pathogen is exemplary of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline. Furthermore, there is still limited evidence on K. pneumoniae pathogenesis at the molecular and cellular levels in the context of the interactions between bacterial pathogens and their hosts. In this research, we have uncovered a sophisticated strategy employed by Klebsiella to subvert the activation of immune defenses by controlling the modification of proteins. Our research may open opportunities to develop new therapeutics based on counteracting this Klebsiella-controlled immune evasion strategy.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Evasão da Resposta Imune , Imunidade Inata , Klebsiella pneumoniae/imunologia , Klebsiella pneumoniae/metabolismo , Sumoilação , Células A549 , Animais , Feminino , Humanos , Interferon Tipo I/imunologia , Infecções por Klebsiella/microbiologia , Pulmão/microbiologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaRESUMO
Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at approximately 4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25 degrees C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity approximately 5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase.
Assuntos
Retículo Endoplasmático/metabolismo , Glicerofosfolipídeos/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Transporte Biológico , Lipossomos/metabolismo , Microssomos/metabolismo , Modelos Biológicos , Oligossacarídeos/química , Proteolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Membrane transporter proteins are essential for the maintenance of cellular ion homeostasis. In the secretory pathway, the P-type ATPase family of transporters is found in every compartment and the plasma membrane. Here, we report the identification of COD1/SPF1 (control of HMG-CoA reductase degradation/SPF1) through genetic strategies intended to uncover genes involved in protein maturation and endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control pathway that rids misfolded proteins. Cod1p is a putative ER P-type ATPase whose expression is regulated by the unfolded protein response, a stress-inducible pathway used to monitor and maintain ER homeostasis. COD1 mutants activate the unfolded protein response and are defective in a variety of functions apart from ERAD, which further support a homeostatic role. COD1 mutants display phenotypes similar to strains lacking Pmr1p, a Ca(2+)/Mn(2+) pump that resides in the medial-Golgi. Because of its localization, the previously reported role of PMR1 in ERAD was somewhat enigmatic. A clue to their respective roles came from observations that the two genes are not generally required for ERAD. We show that the specificity is rooted in a requirement for both genes in protein-linked oligosaccharide trimming, a requisite ER modification in the degradation of some misfolded glycoproteins. Furthermore, Cod1p, like Pmr1p, is also needed for the outer chain modification of carbohydrates in the Golgi apparatus despite its ER localization. In strains deleted of both genes, these activities are nearly abolished. The presence of either protein alone, however, can support partial function for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they reside.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Adenosina Trifosfatases/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , ATPases Transportadoras de Cálcio/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Peroxinas , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia , Especificidade por SubstratoRESUMO
Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Recent studies highlight the emergence of multidrug-resistant K. pneumoniae strains which show resistance to colistin, a last-line antibiotic, arising from mutational inactivation of the mgrB regulatory gene. However, the precise molecular resistance mechanisms of mgrB-associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an mgrB gene K. pneumoniae mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mgrB mutation induces PhoPQ-governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances K. pneumoniae virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence.
Assuntos
Farmacorresistência Bacteriana , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colistina/farmacologia , Modelos Animais de Doenças , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Lepidópteros , Lipídeo A/química , Pulmão/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Polimixinas/farmacologia , Análise de Sobrevida , VirulênciaRESUMO
Infectious diseases are a leading cause of global human mortality. The use of antimicrobials remains the most common strategy for treatment. However, the isolation of pathogens resistant to virtually all antimicrobials makes it urgent to develop effective therapeutics based on new targets. Here we review a new drug discovery paradigm focusing on identifying and targeting host factors important for infection as well as pathogen determinants involved in disease progression. We summarize innovative strategies which by combining bioinformatics with transcriptomics and chemical genetics have already identified host factors essential for pathogen entry, survival and replication. We describe how the discovery of RNA interference which allows loss-of-function studies has facilitated functional genomic studies in human cells. It is expected that these studies will identify targets to be used as host-directed drug therapy which, together with antimicrobials targeting microbial virulence factors, will efficiently eliminate the invading pathogen.
RESUMO
N-linked protein glycosylation follows a conserved pathway in eukaryotic cells. The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for the oligosaccharyltransferase (OST), is catalyzed by different glycosyltransferases located at the membrane of the endoplasmic reticulum (ER). The substrate specificity of the different glycosyltransferase guarantees the ordered assembly of the branched oligosaccharide and ensures that only completely assembled oligosaccharide is transferred to protein. The glycosyltransferases involved in this pathway are highly specific, catalyzing the addition of one single hexose unit to the lipid-linked oligosaccharide (LLO). Here, we show that the dolichylphosphomannose-dependent ALG9 mannosyltransferase is the exception from this rule and is required for the addition of two different alpha-1,2-linked mannose residues to the LLO. This report completes the list of lumen-oriented glycosyltransferases required for the assembly of the LLO.
Assuntos
Lipídeos/fisiologia , Manosiltransferases/metabolismo , Oligossacarídeos/biossíntese , Dolicol Monofosfato Manose/metabolismo , Retículo Endoplasmático/metabolismo , Manose/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/enzimologiaRESUMO
We describe the second case of congenital disorder of glycosylation type IL (CDG-IL) caused by deficiency of the ALG9 a1,2 mannosyltransferase enzyme. The female infant's features included psychomotor retardation, seizures, hypotonia, diffuse brain atrophy with delayed myelination, failure to thrive, pericardial effusion, cystic renal disease, hepatosplenomegaly, esotropia, and inverted nipples. Lipodystrophy and dysmorphic facial features were absent. Magnetic resonance imaging of the brain showed volume loss in the cerebral hemispheres and cerebellum and delayed myelination. Laboratory investigations revealed low levels of multiple serum proteins including antithrombin III, factor XI, and cholesterol. Hypoglycosylation was confirmed by the typical CDG type 1 pattern of serum transferrin analyzed by isoelectric focusing. A defect in the ALG9 enzyme was suggested by the accumulation of the DolPP-GlcNAc2Man6 and DolPP-GlcNAc2Man8 in the patient's fibroblasts and confirmed by mutation analysis: the patient is homozygous for the ALG9 mutation p.Y286C. The causal effect of the mutation was shown by complementation assays in alg9 deficient yeast cells. The child described here further delineates the clinical spectrum of CDG-IL and confirms the significant clinical overlap amongst CDG subtypes.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Manosiltransferases/genética , Mutação , Catepsina A/metabolismo , Defeitos Congênitos da Glicosilação/enzimologia , Defeitos Congênitos da Glicosilação/patologia , Feminino , Teste de Complementação Genética , Glicosilação , Humanos , Lactente , Manosiltransferases/deficiência , Manosiltransferases/metabolismo , Hipotonia Muscular/patologia , Fenótipo , Transtornos Psicomotores/patologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Convulsões/patologiaRESUMO
Defects of lipid-linked oligosaccharide assembly lead to alterations of N-linked glycosylation known as "type I congenital disorders of glycosylation" (CDG). Dysfunctions along this stepwise assembly pathway are characterized by intracellular accumulation of intermediate lipid-linked oligosaccharides, the detection of which contributes to the identification of underlying enzymatic defects. Using this approach, we have found, in a patient with CDG, a deficiency of the ALG9 alpha 1,2 mannosyltransferase enzyme, which causes an accumulation of lipid-linked-GlcNAc(2)Man(6) and -GlcNAc(2)Man(8) structures, which was paralleled by the transfer of incomplete oligosaccharides precursors to protein. A homozygous point-mutation 1567G-->A (amino acid substitution E523K) was detected in the ALG9 gene. The functional homology between the human ALG9 and Saccharomyces cerevisiae ALG9, as well as the deleterious effect of the E523K mutation detected in the patient with CDG, were confirmed by a yeast complementation assay lacking the ALG9 gene. The ALG9 defect found in the patient with CDG--who presented with developmental delay, hypotonia, seizures, and hepatomegaly--shows that efficient lipid-linked oligosaccharide synthesis is required for proper human development and physiology. The ALG9 defect presented here defines a novel form of CDG named "CDG-IL."
Assuntos
Defeitos Congênitos da Glicosilação/enzimologia , Defeitos Congênitos da Glicosilação/genética , Manosiltransferases/genética , Mutação Puntual , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Defeitos Congênitos da Glicosilação/diagnóstico , Feminino , Teste de Complementação Genética , Glicosilação , Hepatomegalia/genética , Homozigoto , Humanos , Recém-Nascido , Lipopolissacarídeos/metabolismo , Manosiltransferases/deficiência , Dados de Sequência Molecular , Hipotonia Muscular/genética , Saccharomyces cerevisiae/genética , Convulsões/genética , Homologia de Sequência de AminoácidosRESUMO
In the endoplasmic reticulum (ER) of eukaryotes, N-linked glycans are first assembled on the lipid carrier dolichyl pyrophosphate. The GlcNAc(2)Man(9)Glc(3) oligosaccharide is transferred to selected asparagine residues of nascent polypeptides. Defects along the biosynthetic pathway of N-glycans are associated with severe multisystemic syndromes called congenital disorders of glycosylation. Here, we describe a deficiency in the ALG12 ER alpha1,6-mannosyltransferase resulting in a novel type of glycosylation disorder. The severe disease was identified in a child presenting with psychomotor retardation, hypotonia, growth retardation, dysmorphic features and anorexia. In the patient's fibroblasts, the biosynthetic intermediate GlcNAc(2)Man(7) oligosaccharide was detected both on the lipid carrier dolichyl pyrophosphate and on newly synthesized glycoproteins, thus pointing to a defect in the dolichyl pyrophosphate-GlcNAc(2)Man(7)-dependent ALG12 alpha1,6 mannosyltransferase. Analysis of the ALG12 cDNA in the CDG patient revealed compound heterozygosity for two point mutations that resulted in the amino acid substitutions T67M and R146Q, respectively. The impact of these mutations on ALG12 protein function was investigated in the Saccharomyces cerevisiae alg12 glycosylation mutant by showing that the yeast ALG12 gene bearing the homologous mutations T61M and R161Q and the human mutant ALG12 cDNA alleles failed to normalize the growth defect phenotype of the alg12 yeast model, whereas expression of the normal ALG12 cDNA complemented the yeast mutation. The ALG12 mannosyltransferase defect defines a new type of congenital disorder of glycosylation, designated CDG-Ig.
Assuntos
Manosiltransferases/genética , Erros Inatos do Metabolismo/genética , Transtornos Psicomotores/genética , Sequência de Aminoácidos , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Retículo Endoplasmático/metabolismo , Glicosilação , Humanos , Lactente , Masculino , Manosiltransferases/deficiência , Manosiltransferases/metabolismo , Dados de Sequência Molecular , Hipertonia Muscular/genética , Fenótipo , Alinhamento de SequênciaRESUMO
Defects of N-linked glycosylation represent diseases with multiple organ involvements that are classified as congenital disorders of glycosylation (CDG). In recent years, several CDG types have been attributed to defects of dolichol-linked oligosaccharide assembly in the endoplasmic reticulum. The profiling of [3H]mannose-labeled lipid-linked oligosaccharides was instrumental in identifying most of these glycosylation disorders. However, this method is poorly suited for the identification of short lipid-linked oligosaccharide biosynthesis defects. To adequately resolve deficiencies affecting the first steps of lipid-linked oligosaccharide formation, we have used a non-radioactive procedure employing the fluorescence detection of 2-aminobenzamide-coupled oligosaccharides after HPLC separation. By applying this method, we have detected the accumulation of dolichylpyrophosphate-GlcNAc2 in a previously untyped CDG patient. The accumulation pattern suggested a deficiency of the ALG1 beta1,4 mannosyltransferase, which adds the first mannose residue to lipid-linked oligosaccharides. This was supported by the finding that this CDG patient was compound heterozygous for three mutations in the ALG1 gene, leading to the amino acid substitutions S150R and D429E on one allele and S258L on the other. The detrimental effect of these mutations on ALG1 protein function was demonstrated in a complementation assay using alg1 Saccharomyces cerevisiae yeast mutants. The ALG1 mannosyltransferase defect described here represents a novel type of CDG, which should be referred to as CDG-Ik.