Infect Immun 73(9): 5291-5300

Novel Sialic Acid Transporter of <em>Haemophilus influenzae</em><sup><a href="#fn1" rid="fn1" class=" fn">†</a></sup>

Buck Institute for Age Research, 8001 Redwood Blvd., Novato, California 94945,^{Department of Microbiology, University of Iowa, Iowa City, Iowa 52242,}^{Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446}³

^{Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7807. Fax: (319) 335-9006. E-mail:}ude.awoiu@allecipa-leahcim.

Received 2005 Feb 7; Revised 2005 Mar 30; Accepted 2005 May 24.

Abstract

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.

Abstract

Haemophilus influenzae is exclusively adapted to infect or colonize humans. Strains can be encapsulated or nonencapsulated (nontypeable). Nontypeable Haemophilus influenzae (NTHi) is a frequent colonizer of the nasopharynx. When the airway is compromised, NTHi can cause local infections such as otitis media in young children (24 million physician visits per year in the United States [25]) and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease.

Lipooligosaccharides (LOS) are a major component of the NTHi outer membrane and have been shown to play a role in microbial virulence and pathogenicity (22). LOS contains carbohydrate epitopes which mimic human glycosphingolipids, allowing the bacteria to avoid the host immune response (17). LOS present on the surface of NTHi is a heterogeneous mixture of glycoforms, the most abundant of which has been extensively studied and is known to consist of a lactose moiety (Galβ1-4Glc) attached to the first heptose (Hep^{) of a conserved core structure [Hep}^α1,2-Hep^α1,3-Hep^{α1,5-Kdo(P)-lipid A] (}19, 20, 24). Importantly, NTHi is also capable of incorporating the acidic sugar N-acetylneuraminic acid (Neu5Ac, or sialic acid) as terminal nonreducing units into its LOS, giving the bacterium protection from complement-mediated killing by normal human serum (10, 11). The acceptors for sialic acid are lactose (18), N-acetyllactosamine, and possibly N-acetylgalactosamine, although the precise structures of most of these sialylated LOS species have not been conclusively identified (12).

The sialic acid is incorporated into the LOS before it reaches the cell surface by one of three sialyltransferases, SiaA, Lic3a, or LsgB (10, 12). The donor for this transfer is CMP-sialic acid, which is synthesized from sialic acid and CTP by the CMP-sialic acid synthetase (SiaB) (11). The fate of sialic acid in NTHi is not solely incorporation into the LOS; sialic acid can also be utilized as a carbon and nitrogen source via its breakdown to N-acetylmannosamine and pyruvate by the neuraminyl lyase (NanA) (28).

NTHi is incapable of synthesizing sialic acid and thus requires an exogenous source of sialic acid for incorporation to occur. In Escherichia coli, sialic acid is imported via symport with a proton through a specific transporter (NanT) of the major facilitator superfamily (30). A gene (HI1104) was identified in the H. influenzae genome that has high homology to the E. coli sialic acid transporter. HI1104 was deleted in this study and shown to have no effect on sialic acid uptake in the H. influenzae strain studied here. Recent publications have suggested that sialic acid transport in H. influenzae is mediated via a novel class of transporter, a tripartite ATP-independent periplasmic (TRAP) transporter (15, 29). TRAP transporters consist of three components: an extracellular solute receptor (ESR) and two distinct integral membrane components of unequal size which are sometimes fused (14). These transporters differ from the better-characterized ABC-protein transporter family (4) in that they do not possess an ATP-binding cassette protein and are not driven by ATP hydrolysis but rather by an electrochemical ion gradient (14).

The gene HI0147 (siaT) was previously identified by Rabus et al. (21) as the fused transmembrane domains of a TRAP transporter and named Y147; it was recently suggested to be part of a sialic acid transporter (15, 29). In this paper we provide evidence that the siaT gene product is indeed a component of the sialic acid TRAP transporter in the NTHi strain 2019. Deletion of the gene encoding this protein has a marked effect on the incorporation of sialic acid into the LOS and on the survival of the organism when exposed to human serum.

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Acknowledgments

This work was supported by grants from the National Institutes of Health (AI24616 to M.A.A. and B.W.G.; AI30040 to M.A.A.).

Acknowledgments

Notes

Editor: J. N. Weiser

Notes

Editor: J. N. Weiser

Footnotes

^{Supplemental material for this article may be found at}http://iai.asm.org/.

Footnotes

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