Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.
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ABSTRACT:
SUBMITTER: Zamze SE
PROVIDER: S-EPMC1148161 | biostudies-other | 1987 Jul
REPOSITORIES: biostudies-other
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Project description:In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.
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Project description:Nontypeable Haemophilus influenzae is an opportunistic pathogen and a common cause of otitis media in children and of chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The lipooligosaccharides, a major component of the outer membrane of H. influenzae, play an important role in microbial virulence and pathogenicity. N-Acetylneuraminic acid (sialic acid) can be incorporated into the lipooligosaccharides as a terminal nonreducing sugar. Although much of the pathway of sialic acid incorporation into lipooligosaccharides is understood, the transporter responsible for N-acetylneuraminic acid uptake in H. influenzae has yet to be characterized. In this paper we demonstrate that this transporter is a novel sugar transporter of the tripartite ATP-independent periplasmic transporter family. In the absence of this transporter, H. influenzae cannot incorporate sialic acid into its lipooligosaccharides, making the organism unable to survive when exposed to human serum and causing reduced viability in biofilm growth.
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Project description:Pasteurella multocida is the causative agent of a number of diseases in animals, including fowl cholera. P. multocida strains simultaneously express two lipopolysaccharide (LPS) glycoforms (glycoforms A and B) that differ only in their inner core structure. Glycoform A contains a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated by the Kdo kinase, KdkA, whereas glycoform B contains two unphosphorylated Kdo residues. We have previously shown that P. multocida mutants lacking the heptosyltransferase, HptA, produce full-length glycoform B LPS and a large amount of truncated glycoform A LPS, as they cannot add heptose to the glycoform A inner core. These hptA mutants were attenuated in chickens because the truncated LPS made them vulnerable to host defense mechanisms, including antimicrobial peptides. However, here we show that birds inoculated with high doses of the hptA mutant developed fowl cholera and the P. multocida isolates recovered from diseased birds no longer expressed truncated LPS. Sequencing analysis revealed that the in vivo-derived isolates had mutations in kdkA, thereby suppressing the production of glycoform A LPS. Interestingly, a number of the spontaneous KdkA mutant strains produced KdkA with a single amino acid substitution (A112V, R123P, H168Y, or D193N). LPS structural analysis showed that complementation of a P. multocida kdkA mutant with wild-type kdkA restored expression of glycoform A to wild-type levels, whereas complementation with any of the mutated kdkA genes did not. We conclude that in P. multocida KdkA, the amino acids A112, R123, H168, and D193 are critical for Kdo kinase function and therefore for glycoform A LPS assembly.
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Project description:The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.
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Project description:Otitis media, a common and often recurrent bacterial infection of childhood, is a major reason for physician visits and the prescription of antimicrobials. Haemophilus influenzae is the cause of approximately 20% of episodes of bacterial otitis media, but most strains lack the capsule, a factor known to play a critical role in the virulence of strains causing invasive H. influenzae disease. Here we show that in capsule-deficient (nontypeable) strains, sialic acid, a terminal residue of the core sugars of H. influenzae lipopolysaccharide (LPS), is a critical virulence factor in the pathogenesis of experimental otitis media in chinchillas. We used five epidemiologically distinct H. influenzae isolates, representative of the genetic diversity of strains causing otitis media, to inoculate the middle ear of chinchillas. All animals developed acute bacterial otitis media that persisted for up to 3 wk, whereas isogenic sialic acid-deficient mutants (disrupted sialyltransferase or CMP-acetylneuraminic acid synthetase genes) were profoundly attenuated. MS analysis indicated that WT bacteria used to inoculate animals lacked any sialylated LPS glycoforms. In contrast, LPS of ex vivo organisms recovered from chinchilla middle ear exudates was sialylated. We conclude that sialylated LPS glycoforms play a key role in pathogenicity of nontypeable H. influenzae and depend on scavenging the essential precursors from the host during the infection.
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Project description:High-molecular-weight proteins of Haemophilus influenzae mediate attachment to epithelial cells. Previous reports describe several allelic versions of hmwA genes that have different adherence properties. Here we report three new alleles of hmwA (hmwA from strain AAr96, hmwA from strain AAr105, and hmwA from strain G822), demonstrating the high degree of DNA variation of these genes among different strains.
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Project description:Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA. Approximately 20 percent of nontypeable H. influenzae strains contain copies of losA and losB in a genetic locus flanked by infA and ksgA. Of 30 strains containing losA at this site, 24 contained 2 tandem copies of the octanucleotide CGAGCATA, allowing full-length translation of losA (on), and 6 strains contained 3, 4, 6, or 10 tandem copies (losA off). For a serum-sensitive strain, R3063, with losA off (10 repeat units), selection for serum-resistant variants yielded a heterogeneous population in which colonies with increased serum resistance had losA on (2, 8, or 11 repeat units), and colonies with unchanged sensitivity to serum had 10 repeats. Inactivation of losA in strains R3063 and R2846 (strain 12) by insertion of the cat gene decreased the serum resistance of these strains compared to losA-on variants and altered the electrophoretic mobility of LPS. We conclude that expression of losA, a gene that contributes to LPS structure and affects serum resistance, is determined by octanucleotide repeat variation.
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Project description:Transcriptome analysis of NTHi 86-028NPrpsL, NTHi 86-028NPrpsL∆fur, and NTHi 86-028NPrpsL∆fur(pT-fur) strains Nontypeable Haemophilus influenzae (NTHi) is a commensal microorganism of the normal human nasopharyngeal flora, yet also an opportunistic pathogen of the upper and lower respiratory tracts. Changes in gene expression patterns in response to host microenvironments are likely critical for survival. One such system of gene regulation is the ability to carefully regulate iron uptake. A central regulatory system that controls iron uptake, mediated by the ferric uptake regulator Fur, is present in multiple bacteria, including NTHi. To understand the regulation of iron homeostasis in NTHi, fur was deleted in the NTHi strain 86-028NPrpsL. Using RNA-Seq, we identified both protein-encoding and small RNA genes whose expression was repressed or activated by Fur. Overall design: These data comprise transcriptional anaylses of an rpsL mutant of 86-028NP, an isogenic fur mutant of 86-028NPrpsL and a complemented fur mutant strain. All strains were grown in defined medium containing 10 µg/ml human hemoglobin to mid-log phase. Cells were then harvested and RNA extracted. A total of three biological replicates were generated for these analyses.
2015-07-01 | GSE58890 | GEO
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Project description:The type b capsule of pathogenic Haemophilus influenzae is a critical factor for H. influenzae survival in the blood and the establishment of invasive infections. Other pathogenic factors associated with type b strains may also play a role in invasion and sustained bacteremia, leading to the seeding of deep tissues. The gene encoding haemocin is the only noncapsular gene found to be specific to type b strains until now. Here we report the discovery of an approximately 16-kb genetic locus, HiGI1, that is present primarily in type b strains. Pulsed-field gel electrophoresis and Southern hybridization were used to map this new locus between secG (HI0445) and fruA (HI0446), which are contiguous in Rd, a nonpathogenic derivative of a serotype d strain. It is inserted at the 3' end of tRNA(4)(Leu) and has regions whose G+C content differs from the average genomic G+C content of H. influenzae. An integrase gene, which encodes a CP4-57 like integrase, is located downstream of tRNA(4)(Leu). Hybridization probes based on the sequences within the HiGI1 locus have been used to screen 61 H. influenzae strains (2 type a, 22 type b, 2 type c, 1 type d, 3 type e, 7 type f, and 21 nontypeable H. influenzae [NTHi]) from our collection. This HiGI1 locus exists in all 22 type b strains and two NTHi strains and is likely to have been acquired by an ancestral type b strain.
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