Capsule Enhances Pneumococcal Colonization by Limiting Mucus-Mediated Clearance<sup><a href="#fn3" rid="fn3" class=" fn">▿</a></sup> <sup><a href="#fn2" rid="fn2" class=" fn">†</a></sup>

Aaron Nelson

Aoife Roche

Jane Gould

Kannie Chim

Adam Ratner

Jeffrey Weiser

Departments of Microbiology and Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

^{Corresponding author. Mailing address: 402A Johnson Pavilion, Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104-6076. Phone: (215) 573-3511. Fax: (215) 898-9557. E-mail:}ude.nnepu.dem.liam@resiew.

^{A.L.N. and A.M.R. contributed equally to this study.}

Received 2006 Sep 14; Revised 2006 Oct 14; Accepted 2006 Oct 29.

Abstract

Expression of a polysaccharide capsule is required for the full pathogenicity of many mucosal pathogens such as Streptococcus pneumoniae. Although capsule allows for evasion of opsonization and subsequent phagocytosis during invasive infection, its role during mucosal colonization, the organism's commensal state, remains unknown. Using a mouse model, we demonstrate that unencapsulated mutants remain capable of nasal colonization but at a reduced density and duration compared to those of their encapsulated parent strains. This deficit in colonization was not due to increased susceptibility to opsonophagocytic clearance involving complement, antibody, or the influx of Ly-6G-positive cells, including neutrophils seen during carriage. Rather, unencapsulated mutants remain agglutinated within lumenal mucus and, thus, are less likely to transit to the epithelial surface where stable colonization occurs. Studies of in vitro binding to immobilized human airway mucus confirmed the inhibitory effect of encapsulation. Likewise, pneumococcal variants expressing larger amounts of negatively charged capsule per cell were less likely to adhere to surfaces coated with human mucus and more likely to evade initial clearance in vivo. Removal of negatively charged sialic acid residues by pretreatment of mucus with neuraminidase diminished the antiadhesive effect of encapsulation. This suggests that the inhibitory effect of encapsulation on mucus binding may be mediated by electrostatic repulsion and offers an explanation for the predominance of anionic polysaccharides among the diverse array of unique capsule types. In conclusion, our findings demonstrate that capsule confers an advantage to mucosal pathogens distinct from its role in inhibition of opsonophagocytosis—escape from entrapment in lumenal mucus.

Abstract

Capsule, a surface coating generally comprised of polysaccharide, is a prominent feature of many pathogens, particularly those causing invasive infection. For example, the most common etiologic agents of bacterial meningitis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Neisseria meningitidis, are encapsulated—a requirement for the sustained bacteremia needed to breach the blood-brain barrier. The importance of capsule to their pathogenicity results from its inhibition of opsonophagocytosis, a process involving the recognition of underlying structures by complement components and/or antibody and leading to engulfment by professional phagocytes (1, 3, 40, 41). Unencapsulated mutants rarely cause invasive infection and are highly attenuated in models of infection due to more efficient opsonophagocytotic clearance (3, 36).

Many encapsulated pathogens, including each of these species noted above, exist primarily in a commensal relationship with their human host, where they reside on the mucosal surface of the nasopharynx. This suggests that the main selective pressures for adaptation to the host occur during colonization, an organism's carrier state. Any advantage capsule confers to bacteria residing on mucosal surfaces, where complement and phagocytes may be less abundant, has not been established. It is thought that the ability of a microbe to colonize for extended periods (persistence) in this niche requires binding to host cells and tissues. However, in vitro studies consistently show an antiadhesive effect of capsule, suggesting a potential biological disadvantage to capsule expression that must be balanced by its contribution to survival during mucosal colonization (9, 24, 27, 28).

In this study, we focus on the role of the capsule during colonization by S. pneumoniae (the pneumococcus), a species capable of expressing a repertoire of at least 90 unique capsular polysaccharide “types” (PnPSs) (10). Each of these PnPSs differs in composition and linkage of its component sugars as well as other substitutes (12). A further source of heterogeneity among pneumococcal isolates of most types is due to phase variation between two forms (opaque [O] and transparent [T] colony forms) differing by up to 5.6-fold in amounts of PnPS/cell as well as other characteristics (13, 39). Similar to the capsules of many other species, the only common structural feature among this large array of polysaccharides is that none has a net positive charge. In fact, structures of more than half of the known PnPS types have been determined and all but four types are negatively charged due to the presence of acidic sugars, pyruvate, or phosphate, with the remainder being neutral (12). Despite the diversity of PnPS structures, shared physical characteristics are thought to contribute to a conserved function in protecting the underlying bacterial surface structures from the deposition of antibody and complement.

Here we show that unencapsulated mutants successfully colonize nasal spaces but display altered colonization dynamics due to agglutination by mucus rather than enhanced susceptibility to opsonophagocytosis. These findings are consistent with capsule protecting underlying structures from host clearance mechanisms but suggest distinct functional roles during infection and colonization.

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Acknowledgments

This work was supported by grants from the U.S. Public Health Service to J.N.W. (AI44231 and AI38446) and to the Bacterial Respiratory Pathogen Research Unit (NO1 AI30040) and The Morphology Core of the Center for the Molecular Studies of Liver and Digestive Disease (P30 DK50306) and from the Howard Hughes Medical Institute (K.C.).

Acknowledgments

Notes

Editor: A. Casadevall

Notes

Editor: A. Casadevall

Footnotes

^{Published ahead of print on 6 November 2006.}

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

Footnotes

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