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Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control.

ABSTRACT: The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with nonrandom (modal) chain length distribution. The number of the repeat O units in each individual O-antigen polymer depends on the Wzz chain length regulator, an inner membrane protein belonging to the polysaccharide copolymerase (PCP) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to >100 repeat units), despite having remarkably conserved structural folds. The molecular mechanism responsible for the selective preference for a certain number of O units is unknown. Guided by the three-dimensional structures of PCPs, we constructed a panel of chimeric molecules containing parts of two closely related Wzz proteins from Salmonella enterica and Shigella flexneri which confer different O-antigen chain length distributions. Analysis of the O-antigen length distribution imparted by each chimera revealed the region spanning amino acids 67 to 95 (region 67 to 95), region 200 to 255, and region 269 to 274 as primarily affecting the length distribution. We also showed that there is no synergy between these regions. In particular, region 269 to 274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269 to 274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by cross-linking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex.

SUBMITTER: Kalynych S

PROVIDER: S-EPMC3147518 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control.

Kalynych Sergei S Ruan Xiang X Valvano Miguel A MA Cygler Miroslaw M

Journal of bacteriology 20110603 15

The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with nonrandom (modal) chain length distribution. The number of the repeat O units in each individual O-antigen polymer depends on the Wzz chain length regulator, an inner membrane protein belonging to the polysaccharide copolymerase (PCP) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to >100 repeat units), despite having remarkably conserved structural ...[more]

PMID: 21642455

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Project description:The outer leaflet of the outer membrane of nearly all Gram-negative bacteria contains lipopolysaccharide (LPS). The distal end of LPS may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length. The chain length of the polysaccharide has many implications for bacterial survival and consequently is tightly controlled. In the Wzx/Wzy-dependent route of O antigen synthesis, one or more Wzz proteins determine the chain length via an unknown mechanism. To gain insight into this mechanism, we identified and characterized important regions of two Wzz proteins in Pseudomonas aeruginosa serotype O13, which confer the production of "long" (Wzz1) and "very long" (Wzz2) chain lengths, respectively. We found that compared to Wzz1, Wzz2 has distinct amino acid insertions in the central α-helices (insα6 and insα7) and in membrane-distal (insL4) and -proximal (insIL) loops. When these regions were deleted in Wzz2, the mutant proteins conferred drastically shortened chain lengths. Within these regions we identified several conserved amino acid residues that were then targeted for site-directed mutagenesis. Our results implicate an RTE motif in loop 4 and a "hot spot" of charged and polar residues in insα7 in the function of Wzz2 We present evidence that the functionally important residues of insα7 are likely involved in stabilizing Wzz through coiled-coil interactions.IMPORTANCE O antigen is an important virulence factor presented on the cell surface of Gram-negative bacteria that is critical for bacterial physiology and pathogenesis. However, some aspects of O antigen biosynthesis, such as the mechanisms for determining polysaccharide chain length, are poorly understood. In this study, we identified unique regions in the O antigen chain length regulators (termed Wzz) of the problematic opportunistic pathogen Pseudomonas aeruginosa We show that these regions are critical for determining O antigen chain length, which provides new insight into the model of the Wzz mechanism. Ultimately, our work adds knowledge toward understanding an important step in the biosynthesis of this virulence factor, which is applicable to a wide range of Gram-negative pathogens.

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Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control.

Publications

Structure-guided investigation of lipopolysaccharide O-antigen chain length regulators reveals regions critical for modal length control.

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