Functional analysis of the large periplasmic loop of the <i>Escherichia coli</i> K‐12 WaaL O‐antigen ligase

Pérez, Jose; McGarry, Megan A.; Marolda, Cristina L.; Valvano, Miguel A.

doi:10.1111/j.1365-2958.2008.06490.x

Cited by 65 publications

(88 citation statements)

References 61 publications

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“…The other pathway involves the synthesis and assembly of the O-antigen polysaccharide, which also begins at the cytosolic side of the inner membrane resulting in the formation of a lipid-linked molecule that is further translocated across the inner membrane. The formation of a complete LPS molecule containing O antigen is catalyzed by the O-antigen ligase WaaL (41). LPS molecules are further translocated to the outer leaflet of the outer membrane by the Lpt transport system involving a number of inner membrane, periplasmic, and outer membrane proteins (44,45,48,49).…”

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confidence: 99%

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Marolda¹,

Li²,

Lung³

et al. 2010

J Bacteriol

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Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx EcO157 ) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletionfusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx EcO157 consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PPlinked saccharide substrate across the membrane.Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, plays critical roles in bacterial cell physiology (36) and in disease (53). The structure of LPS is complex and consists at a minimum of lipid A and core oligosaccharide (OS) (42). Many Gram-negative bacteria also have an O-specific antigen polysaccharide (or O antigen) attached to one of the terminal residues of the core OS (42). The O antigen is the most variable portion of the LPS molecule and arises from the polymerization of discrete oligosaccharide units (42, 54).The biosynthesis of LPS requires many enzymes and assembly proteins and generally involves two separate pathways. One pathway results in the synthesis of the lipid A-core OS (42), which is translocated across the inner membrane by the lipid A flippase MsbA, an ABC transporter (14, 15, 60). The other pathway involves the synthesis and assembly of the O-antigen polysaccharide, which also begins at the cytosolic side of the inner membrane resulting in the formation of a lipid-linked molecule that is further translocated across the inner membrane. The formation of a complete LPS molecule containing O antigen is catalyzed by ...

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Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Marolda¹,

Li²,

Lung³

et al. 2010

J Bacteriol

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“…1B). Additionally, the F. tularensis FTT1238c protein sequence was compared to the E. coli WaaL and Salmonella WaaL sequences, revealing a conserved histidine residue and a conserved arginine residue that have been shown to be important for function in other organisms (32)(33)(34) (Fig. 1B).…”

Section: Resultsmentioning

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Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

et al. 2014

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The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD 50 ) values of these strains are increased 100-to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge.

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“…Elimination of the enzymatic site (residues 255 to 311) and replacement of critical active-site residues with alanine were performed as described elsewhere (26). pYYB5 (pEVP3-rafX) was used as the template.…”

Section: Methodsmentioning

confidence: 99%

A Novel Protein, RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

Huang

Zhang

et al. 2014

J Bacteriol

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bTeichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ⌬rafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ⌬rafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ⌬rafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ⌬rafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ⌬rafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.

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Functional analysis of the large periplasmic loop of the Escherichia coli K‐12 WaaL O‐antigen ligase

Cited by 65 publications

References 61 publications

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

A Novel Protein, RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

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