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1.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
2.
Annu Rev Microbiol ; 74: 521-543, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32680453

RESUMO

Polysaccharides are dominant features of most bacterial surfaces and are displayed in different formats. Many bacteria produce abundant long-chain capsular polysaccharides, which can maintain a strong association and form a capsule structure enveloping the cell and/or take the form of exopolysaccharides that are mostly secreted into the immediate environment. These polymers afford the producing bacteria protection from a wide range of physical, chemical, and biological stresses, support biofilms, and play critical roles in interactions between bacteria and their immediate environments. Their biological and physical properties also drive a variety of industrial and biomedical applications. Despite the immense variation in capsular polysaccharide and exopolysaccharide structures, patterns are evident in strategies used for their assembly and export. This review describes recent advances in understanding those strategies, based on a wealth of biochemical investigations of select prototypes, supported by complementary insight from expanding structural biology initiatives. This provides a framework to identify and distinguish new systems emanating from genomic studies.

3.
J Biol Chem ; 295(31): 10593-10609, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32424042

RESUMO

Lipopolysaccharides are critical components of bacterial outer membranes. The more conserved lipid A part of the lipopolysaccharide molecule is a major element in the permeability barrier imposed by the outer membrane and offers a pathogen-associated molecular pattern recognized by innate immune systems. In contrast, the long-chain O-antigen polysaccharide (O-PS) shows remarkable structural diversity and fulfills a range of functions, depending on bacterial lifestyles. O-PS production is vital for the success of clinically important Gram-negative pathogens. The biological properties and functions of O-PSs are mostly independent of specific structures, but the size distribution of O-PS chains is particularly important in many contexts. Despite the vast O-PS chemical diversity, most are produced in bacterial cells by two assembly strategies, and the different mechanisms employed in these pathways to regulate chain-length distribution are emerging. Here, we review our current understanding of the mechanisms involved in regulating O-PS chain-length distribution and discuss their impact on microbial cell biology.


Assuntos
Bactérias Gram-Negativas/fisiologia , Antígenos O/biossíntese , Lipídeo A/biossíntese
4.
Nat Chem Biol ; 16(4): 450-457, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152541

RESUMO

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.


Assuntos
Glicosiltransferases/metabolismo , Antígenos O/metabolismo , Antígenos O/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Glicosiltransferases/fisiologia , Hexosiltransferases , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Polissacarídeos Bacterianos/química
5.
J Bacteriol ; 202(5)2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31792013

RESUMO

Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a single example: the O:54 antigen from Salmonella enterica serovar Borreze. Synthases are complex enzymes that have the capacity to both polymerize and export bacterial polysaccharides. Although synthases like cellulose synthase are widespread, they typically polymerize a glycan without employing a lipid-linked intermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenol diphosphate-linked product. This raises questions about the overall similarity between WbbF and conventional synthases. In this study, we examine the topology of WbbF, revealing four membrane-spanning helices, compared to the eight in cellulose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indicates a similar architecture, and site-directed mutagenesis confirmed that residues important for catalysis and processivity in cellulose synthase are conserved in WbbF and required for its activity. These findings indicate that the glycosyltransferase mechanism of WbbF and classic synthases are likely conserved despite the use of a lipid acceptor for chain extension by WbbF.IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide variety of bacterial polysaccharides. These include O-antigenic polysaccharides, which form the distal component of lipopolysaccharides and provide a protective barrier important for survival and host-pathogen interactions. Synthases are a subset of glycosyltransferases capable of coupled synthesis and export of glycans. Currently, the O:54 antigen of Salmonella enterica serovar Borreze involves the only example of an O-polysaccharide synthase, and its generation of a lipid-linked product differentiates it from classical synthases. Here, we explore features conserved in the O:54 enzyme and classical synthases to shed light on the structure and function of the unusual O:54 enzyme.


Assuntos
Domínio Catalítico , Glicosiltransferases/química , Modelos Moleculares , Salmonella enterica/enzimologia , Sequência de Aminoácidos , Catálise , Antígenos O/biossíntese , Proteínas Recombinantes de Fusão , Salmonella enterica/imunologia
6.
Glycobiology ; 29(12): 822-838, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31504498

RESUMO

The structures of bacterial cell surface glycans are remarkably diverse. In spite of this diversity, the general strategies used for their assembly are limited. In one of the major processes, found in both Gram-positive and Gram-negative bacteria, the glycan is polymerized in the cytoplasm on a polyprenol lipid carrier and exported from the cytoplasm by an ATP-binding cassette (ABC) transporter. The ABC transporter actively participates in determining the chain length of the glycan substrate, which impacts functional properties of the glycoconjugate products. A subset of these systems employs an additional elaborate glycan capping strategy that dictates the size distribution of the products. The hallmarks of prototypical capped glycan systems are a chain-terminating enzyme possessing a coiled-coil molecular ruler and an ABC transporter possessing a carbohydrate-binding module, which recognizes the glycan cap. To date, detailed investigations are limited to a small number of prototypes, and here, we used our current understanding of these processes for a bioinformatics census of other examples in available genome sequences. This study not only revealed additional instances of existing terminators but also predicted new chemistries as well as systems that diverge from the established prototypes. These analyses enable some new functional hypotheses and offer a roadmap for future research.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Biologia Computacional , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Sítios de Ligação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Modelos Moleculares
7.
J Biol Chem ; 294(41): 14978-14990, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31416837

RESUMO

Escherichia coli serotype O9a provides a model for export of lipopolysaccharide (LPS) O-antigen polysaccharide (O-PS) via ABC transporters. In O9a biosynthesis, a chain-terminator enzyme, WbdD, caps the nonreducing end of the glycan with a methylphosphate moiety and thereby establishes chain-length distribution. A carbohydrate-binding module (CBM) in the ABC transporter recognizes terminated glycans, ensuring that only mature O-PS is exported and incorporated into LPS. Here, we addressed two questions arising from this model. Are both residues in the binary terminator necessary for termination and export? And is a terminal methylphosphate moiety sufficient for export of heterologous glycans? To answer the first question, we uncoupled WbdD kinase and methyltransferase activities. WbdD mutants revealed that although the kinase activity is solely responsible for chain-length regulation, both activities are essential for CBM recognition and export. Consistent with this observation, a saturation transfer difference NMR experiment revealed a direct interaction between the CBM and the terminal methyl group. To determine whether methylphosphate is the sole determinant of substrate recognition by the CBM, we exploited Klebsiella pneumoniae O7, whose O-PS repeat-unit structure differs from O9a, but, as shown here, offers the second confirmed example of a terminal methylphosphate serving in substrate recognition. In vitro and in vivo experiments indicated that each CBM can bind the O-PS only with the native repeat unit, revealing that methylphosphate is essential but not sufficient for substrate recognition and export. Our findings provide important new insight into the structural determinants in a prototypical quality control system for glycan assembly and export.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Metabolismo dos Carboidratos , Escherichia coli/metabolismo , Antígenos O/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Antígenos O/química , Organofosfatos/metabolismo , Polimerização , Ligação Proteica , Conformação Proteica
8.
Nat Chem Biol ; 15(6): 632-640, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31036922

RESUMO

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2→4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.


Assuntos
Cápsulas Bacterianas/metabolismo , Glicolipídeos/biossíntese , Bactérias Gram-Negativas/metabolismo , Transferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Transferases/química
9.
J Biol Chem ; 294(28): 10863-10876, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31138653

RESUMO

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-ß-d-Galp-(1→] or O2c [→3)-ß-d-GlcpNAc-(1→5)-ß-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding ß-(1→3)-linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked d-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.


Assuntos
Diversidade de Anticorpos/imunologia , Klebsiella pneumoniae/enzimologia , Antígenos O/imunologia , Diversidade de Anticorpos/fisiologia , Proteínas de Bactérias/metabolismo , Galactanos/metabolismo , Galactosiltransferases/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Família Multigênica/genética , Antígenos O/química , Antígenos O/metabolismo , Sorotipagem/métodos
10.
J Bacteriol ; 201(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036729

RESUMO

Capsular polysaccharides (CPSs) are virulence factors for many important pathogens. In Escherichia coli, CPSs are synthesized via two distinct pathways, but both require proteins from the outer membrane polysaccharide export (OPX) family to complete CPS export from the periplasm to the cell surface. In this study, we compare the properties of the OPX proteins from the prototypical group 1 (Wzy-dependent) and group 2 (ABC transporter-dependent) pathways in E. coli K30 (Wza) and E. coli K2 (KpsD), respectively. In addition, we compare an OPX from Salmonella enterica serovar Typhi (VexA), which shares structural properties with Wza, while operating in an ABC transporter-dependent pathway. These proteins differ in distribution in the cell envelope and formation of stable multimers, but these properties do not align with acylation or the interfacing biosynthetic pathway. In E. coli K2, murein lipoprotein (Lpp) plays a role in peptidoglycan association of KpsD, and loss of this interaction correlates with impaired group 2 capsule production. VexA also depends on Lpp for peptidoglycan association, but CPS production is unaffected in an lpp mutant. In contrast, Wza and group 1 capsule production is unaffected by the absence of Lpp. These results point to complex structure-function relationships between different OPX proteins.IMPORTANCE Capsules are protective layers of polysaccharides that surround the cell surface of many bacteria, including that of Escherichia coli isolates and Salmonella enterica serovar Typhi. Capsular polysaccharides (CPSs) are often essential for virulence because they facilitate evasion of host immune responses. The attenuation of unencapsulated mutants in animal models and the involvement of protein families with conserved features make the CPS export pathway a novel candidate for therapeutic strategies. However, appropriate "antivirulence" strategies require a fundamental understanding of the underpinning cellular processes. Investigating export proteins that are conserved across different biosynthesis strategies will give important insight into how CPS is transported to the cell surface.


Assuntos
Cápsulas Bacterianas/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Polissacarídeos Bacterianos/química , Proteínas da Membrana Bacteriana Externa/genética , Vias Biossintéticas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Lipoproteínas/química , Lipoproteínas/genética , Peptidoglicano/química , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Polissacarídeos Bacterianos/genética , Transporte Proteico , Salmonella typhi/química , Salmonella typhi/genética
11.
Methods Mol Biol ; 1954: 151-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864130

RESUMO

The incorporation of fluorescent tags into synthetic acceptor molecules for in vitro biochemical assays allows quick and easy detection of enzyme activity. Reaction products can be separated via thin-layer chromatography and visualized under UV light for rapid detection of reaction progress. Subsequent structural analysis of these reaction products through the use of NMR spectroscopy and mass spectrometry allows for complete functional characterization of enzyme activity. Here we describe an application of this technique which was previously used to functionally characterize a dual-domain glycosyltransferase enzyme, KpsC, involved in capsular polysaccharide biosynthesis in Escherichia coli.


Assuntos
Ensaios Enzimáticos/métodos , Escherichia coli/enzimologia , Corantes Fluorescentes/metabolismo , Glicosiltransferases/metabolismo , Cromatografia em Camada Delgada/métodos , Escherichia coli/metabolismo , Corantes Fluorescentes/análise , Glicosiltransferases/análise , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Especificidade por Substrato
12.
Methods Mol Biol ; 1954: 245-253, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864137

RESUMO

In vitro assays using fluorescently tagged sugar residues can facilitate the characterization of glycosyltransferase function. Here we describe the use of in vitro assays to characterize the three glycosyltransferase modules of the protein designated WbbB from Klebsiella pneumoniae O12. This protein combines key activities necessary to synthesize the O antigenic polysaccharide portion of lipopolysaccharide. The specificities of the three glycosyltransferases were investigated in vitro, using purified proteins, the activated donor sugars (dTDP-Rha, UDP-GlcNAc and CMP-ß-Kdo) and synthetic acceptors terminating in either α1,3-linked Rha or ß1,4-linked GlcNAc. The reaction products were verified by mass spectrometry and nuclear magnetic resonance methods.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Klebsiella pneumoniae/enzimologia , Proteínas de Bactérias/química , Cromatografia em Camada Delgada/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Glicosiltransferases/química , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/química , Klebsiella pneumoniae/metabolismo , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Antígenos O/metabolismo , Domínios Proteicos
13.
J Am Chem Soc ; 141(6): 2201-2204, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30698425

RESUMO

Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-ß-d- manno-oct-2-ulosonic acid (ß-Kdo). The ß-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for ß-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.


Assuntos
Inibidores Enzimáticos/farmacologia , Glicosiltransferases/antagonistas & inibidores , Química Click , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Açúcares Ácidos/química , Açúcares Ácidos/farmacologia
14.
ACS Chem Biol ; 13(11): 3161-3172, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30346688

RESUMO

Nonribosomal peptide synthetases (NRPSs) increase the chemical diversity of their products by acquiring tailoring domains. Linear gramicidin synthetase starts with a tailoring formylation (F) domain, which likely originated from a sugar formyltransferase (FT) gene. Here, we present studies on an Anoxybacillus kamchatkensis sugar FT representative of the prehorizontal gene transfer FT. Gene cluster analysis reveals that this FT acts on a UDP-sugar in a novel pathway for synthesis of a 7-formamido derivative of CMP-pseudaminic acid. We recapitulate the pathway up to and including the formylation step in vitro, experimentally demonstrating the role of the FT. We also present X-ray crystal structures of the FT alone and with ligands, which unveil contrasts with other structurally characterized sugar FTs and show close structural similarity with the F domain. The structures reveal insights into the adaptations that were needed to co-opt and evolve a sugar FT into a functional and useful NRPS domain.


Assuntos
Hidroximetil e Formil Transferases/química , Peptídeo Sintases/química , Anoxybacillus/enzimologia , Cristalografia por Raios X , Transferência Genética Horizontal , Hidroliases/química , Hidroliases/genética , Hidroximetil e Formil Transferases/genética , Ligantes , Família Multigênica , Peptídeo Sintases/genética , Domínios Proteicos , Transaminases/química , Transaminases/genética
15.
Proc Natl Acad Sci U S A ; 115(21): E4870-E4879, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29735649

RESUMO

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogen Salmonella enterica serovar Typhi produces "Vi antigen" CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of the Burkholderiales, which are paradoxically distinguished from S. Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel ß-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced into S Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Liases/metabolismo , Periplasma/enzimologia , Polissacarídeos Bacterianos/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Transporte Biológico , Liases/química , Filogenia , Conformação Proteica
16.
J Biol Chem ; 293(13): 4666-4679, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29602878

RESUMO

Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-ß-d-Galf-(1→3)-ß-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.


Assuntos
Proteínas de Bactérias , Klebsiella pneumoniae , Antígenos O , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Configuração de Carboidratos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Antígenos O/biossíntese , Antígenos O/química , Antígenos O/genética , Coelhos
17.
Nature ; 553(7688): 361-365, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29320481

RESUMO

O-antigens are cell surface polysaccharides of many Gram-negative pathogens that aid in escaping innate immune responses. A widespread O-antigen biosynthesis mechanism involves the synthesis of the lipid-anchored polymer on the cytosolic face of the inner membrane, followed by transport to the periplasmic side where it is ligated to the lipid A core to complete a lipopolysaccharide molecule. In this pathway, transport to the periplasm is mediated by an ATP-binding cassette (ABC) transporter, called Wzm-Wzt. Here we present the crystal structure of the Wzm-Wzt homologue from Aquifex aeolicus in an open conformation. The transporter forms a transmembrane channel that is sufficiently wide to accommodate a linear polysaccharide. Its nucleotide-binding domain and a periplasmic extension form 'gate helices' at the cytosolic and periplasmic membrane interfaces that probably serve as substrate entry and exit points. Site-directed mutagenesis of the gates impairs in vivo O-antigen secretion in the Escherichia coli prototype. Combined with a closed structure of the isolated nucleotide-binding domains, our structural and functional analyses suggest a processive O-antigen translocation mechanism, which stands in contrast to the classical alternating access mechanism of ABC transporters.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Antígenos O/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Escherichia coli/química , Hidrólise , Modelos Moleculares , Polissacarídeos/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade
19.
mBio ; 8(3)2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28536290

RESUMO

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates.IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis.


Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Proteínas Periplásmicas/metabolismo , Soro/microbiologia , Escherichia coli Uropatogênica/fisiologia , Animais , Bacteriemia/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Atividade Bactericida do Sangue , Modelos Animais de Doenças , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Camundongos , Viabilidade Microbiana , Mutação , Peptidoglicano/genética , Escherichia coli Uropatogênica/genética
20.
Structure ; 25(5): 806-815.e3, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28434914

RESUMO

Wzz is an integral inner membrane protein involved in regulating the length of lipopolysaccharide O-antigen glycans and essential for the virulence of many Gram-negative pathogens. In all Wzz homologs, the large periplasmic domain is proposed to be anchored by two transmembrane helices, but no information is available for the transmembrane and cytosolic domains. Here we have studied purified oligomeric Wzz complexes using cryoelectron microscopy and resolved the transmembrane regions within a semi-continuous detergent micelle. The transmembrane helices of each monomer display a right-handed super-helical twist, and do not interact with the neighboring transmembrane domains. Modeling, flexible fitting and multiscale simulation approaches were used to study the full-length complex and to provide explanations for the influence of the lipid bilayer on its oligomeric status. Based on structural and in silico observations, we propose a new mechanism for O-antigen chain-length regulation that invokes synergy of Wzz and its polymerase partner, Wzy.


Assuntos
Proteínas de Bactérias/química , Membrana Celular/ultraestrutura , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica
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