Sialic acid metabolism's dual function in <i>Haemophilus influenzae</i>

Vimr, Eric R.; Lichtensteiger, Carol A.; Steenbergen, Susan M.

doi:10.1046/j.1365-2958.2000.01925.x

Cited by 98 publications

(144 citation statements)

References 45 publications

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“…All isolates of NTHi studied to date incorporate Neu5Ac as a common constituent of LPS (8), but it is also known that the patterns of sialylation of LPS in Hi are complex (6,7,20), and at least three distinct sialyltransferases have been identified (7,20,21). On the basis of the complete genome sequence of Hi strain Rd, the intrinsic pathway for synthesis of sialic acid is missing several key enzymes, a finding consistent with the observation that an exogenous source of Neu5Ac is required (7,25). However, all NTHi isolates examined to date have the siaB gene (6), and its inactivation provides a convenient strategy to construct sialic acid-deficient LPS in any NTHi strain.…”

Section: Discussionsupporting

confidence: 53%

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

Bouchet

Hood

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

191

192

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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 Ϸ20% 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.ex vivo isolate ͉ phylogeny ͉ mass spectrometry C arried by up to 80% of humans, Haemophilus influenzae (Hi) is a common nasopharyngeal commensal. Capsule-deficient or nontypeable (NT) Hi can cause upper and lower respiratory tract infections, the most common being episodes of otitis media (OM) in young children. On average, children experience two or more episodes of acute OM by age 2 yr (1), making OM a major cause of physician visits (24 million per year in the U.S.) and of the profligate use of antibiotics in general practice. OM causes sequelae, including impaired hearing (Ϸ20% cases) and cognitive development (2). NTHi is also the most frequent pathogen recovered from the middle ear in children with recurrent OM (3). Although immunity against infection due to NTHi appears to develop after acute OM (4, 5), protection is strain specific, therefore permitting recurrent episodes due to distinct isolates.We have reported that all NTHi OM isolates have the potential to incorporate sialic acid [N-acetylneuraminic acid (Neu5Ac)] into their lipopolysaccharide (LPS), and that strains expressing this sugar are more resistant to the bactericidal activity of normal human serum in vitro (6-8). Although sialylation of LPS has been implicated in bacterial virulence (9), the contribution of sialylated LPS glycoforms of Hi has not been investigated in vivo.We have investigated the role of sialic acid as a virulence factor of NTHi in a well described chinchilla model of OM (10, 11). By comparing isogenic sialic acid-proficient and sialic acid-deficient strains, we show that sialylation of LPS is a major factor in...

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Section: Discussionsupporting

confidence: 53%

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

Bouchet

Hood

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

191

192

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“…Sialylation of LPS is a critical virulence factor in vivo required for NTHi to cause otitis media in a chinchilla model of infection (3). H. influenzae lacks the genes required for de novo synthesis of Neu5Ac and must therefore access an external source of the sugar as either a substrate for growth (24) or for modification of LPS via a number of specific sialyltransferases. Sialyltransferases determine the frequency and pattern of LPS sialylation across NTHi strains.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Bifunctional Lipopolysaccharide Sialyltransferase in Haemophilus influenzae

Fox

Cox

Gilbert

et al. 2006

Journal of Biological Chemistry

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The lipopolysaccharide (LPS) of non-typeableHere we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both ␣-2,3-and ␣-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance.

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“…This operon includes N-acetylneuraminate lyase (nanA) and N-acetylmannosamine-6-phosphate epimerase (nanE) genes, and is responsible for the utilization of N-acetylneuraminic acid. In H.in¯uenzae this can be either catabolized or used for sialylation of the surface lipopolysaccharide in the outer cell membrane (45,46). It is conceivable that TRAP-type transporter in H.in¯uenzae, V.cholerae, Pasteurella multocida and several other organisms is a nonorthologous replacement of the MFS superfamily transporter NanT found in E.coli.…”

Section: Genome Context Analysismentioning

confidence: 99%

Identification and functional analysis of 'hypothetical' genes expressed in Haemophilus influenzae

Kolker¹,

Makarova

Shabalina

et al. 2004

Nucleic Acids Research

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The progress in genome sequencing has led to a rapid accumulation in GenBank submissions of uncharacterized`hypothetical' genes. These genes, which have not been experimentally characterized and whose functions cannot be deduced from simple sequence comparisons alone, now comprise a signi®cant fraction of the public databases. Expression analyses of Haemophilus in¯uenzae cells using a combination of transcriptomic and proteomic approaches resulted in con®dent identi®ca-tion of 54`hypothetical' genes that were expressed in cells under normal growth conditions. In an attempt to understand the functions of these proteins, we used a variety of publicly available analysis tools. Close homologs in other species were detected for each of the 54`hypothetical' genes. For 16 of them, exact functional assignments could be found in one or more public databases. Additionally, we were able to suggest general functional characterization for 27 more genes (comprising~80% total). Findings from this analysis include the identi®cation of a pyruvate-formate lyase-like operon, likely to be expressed not only in H.in¯uenzae but also in several other bacteria. Further, we also observed three genes that are likely to participate in the transport and/or metabolism of sialic acid, an important component of the H.in¯uenzae lipo-oligosaccharide. Accurate functional annotation of uncharacterized genes calls for an integrative approach, combining expression studies with extensive computational analysis and curation, followed by eventual experimental veri®cation of the computational predictions.

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Sialic acid metabolism's dual function in Haemophilus influenzae

Cited by 98 publications

References 45 publications

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

Identification of a Bifunctional Lipopolysaccharide Sialyltransferase in Haemophilus influenzae

Identification and functional analysis of 'hypothetical' genes expressed in Haemophilus influenzae

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