Advisory Committee Chair
Janet Yother
Advisory Committee Members
William Benjamin
Michael Niederweis
David Pritchard
Andries Steyn
Document Type
Dissertation
Date of Award
2007
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
The polysaccharide capsules of Streptococcus pneumoniae represent the most important virulence determinant produced by this organism. Ninety-one different serotypes have been identified, but only a small number of these serotypes are responsible for most of the infections caused by S. pneumoniae. The reasons why certain serotypes predominate in infections over others are not well understood. One common serotype found in invasive diseases is type 2, however, it is unclear what characteristics of a type 2 capsule makes strains producing it virulent. The type 2 capsule consists of repeat units made of a backbone of Glc-Rha-Rha-Rha, with a side chain Glc-GlcUA linked to the terminal Rha. In type 2 S. pneumoniae, neither the role the capsular side chain Glc-UA has in capsule assembly nor the role it has in virulence is known. UDP-GlcUA is synthesized from the oxidation of UDP-Glc by the activity of cps2K, a type-specific gene encoded within the type 2 capsule locus containing homology to known UDP-Glc dehydrogenses. Deletion of cps2K should eliminate the production of UDP-GlcUA, allowing us to assess the effects of alteration of the side chain on both capsule synthesis and virulence. Deletion of cps2K resulted in the production of only low levels of high molecular weight polymer that were retained on the cell membrane but not transferred to the cell wall, demonstrating a requirement for GlcUA in this process. Repair of this mutation only partially restored capsule production, suggestive of secondary mutations selected for during isolation of Δcps2K mutants. Linkage analyses ii were used to localize the suppressor mutations to a region containing cps2E, the glycosytransferase responsible for the addition of Glc-1-P to a C55 polyprenol acceptor (Und-PP) to initiate repeat unit synthesis. Sequence analyses revealed that the original Δcps2K mutants contained different point mutations in cps2E, all reducing Cps2E activity. Construction of multiple independent Δcps2K mutants resulted in the same phenotype as the original and a selection for suppressor mutations, the majority of which were located in cps2E. Mutants that were reduced in capsule production due to either mutations in cps2E or the capsule promoter region, resulting in 0.1% and 60% of the parental levels of capsule, respectively, were unable to efficiently colonize or cause disease in murine models of pnuemococcal infection, demonstrating a requirement for full capsule production by type 2 for these processes. Deletion of cps2H, the repeat-unit polymerase, and cps2J, the repeat-unit flippase, also selected for suppressor mutations in cps2E. Collectively, the data presented here suggests a selection for suppressor mutations may have resulted from the sequestration of undecaprenyl-pyrophosphate due to retention of a repeat-unit on the cytoplasmic side of the cell membrane or an inability to transfer a single repeat-unit or altered polymer to the cell wall. Suppressor mutations which reduced capsule synthesis, such as those in cps2E, would help alleviate stress from accumulation of lipid-linked subunits and preclusion of lipid turnover for use in other cellular processes. Thus, in S. pneumoniae serotype 2, alterations to the capsule structure or assembly process prevent proper capsule synthesis by interfering with transfer to the cell wall and possibly other steps in the pathway. These alterations also result in reduced virulence of the bacteria, demonstrating a requirement for at least 60% of parental levels of capsule in type 2 to cause disease.
Recommended Citation
Xayarath, Bobbi, "Effects Of Specific Alterations In Capsule Structure On Streptococcus Pneumoniae Capsule Assembly And Virulence" (2007). All ETDs from UAB. 3828.
https://digitalcommons.library.uab.edu/etd-collection/3828