The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall - PubMed (original) (raw)
Comparative Study
The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall
Lynn E Hancock et al. Proc Natl Acad Sci U S A. 2002.
Abstract
With the goal of identifying and characterizing traits of Enterococcus faecalis that play key roles in human disease, we identified an operon specifying synthesis of a capsular carbohydrate of the type most commonly expressed by clinical isolates. This surface-exposed carbohydrate consists of glycerol phosphate, glucose, and galactose residues, and its biosynthesis is encoded by a determinant that includes 11 ORFs. Insertional inactivation of genes in this pathway yielded mutants with enhanced susceptibility to phagocytic killing in vitro and compromised in the ability to persist in regional lymph nodes in vivo.
Figures
Figure 1
(A) Genetic organization of the cps locus in E. faecalis strain V583. Restriction sites for _Eco_RI (E) and _Cla_I (C) are shown above the map. Solid bars mark regions used for complementing _cps_I in strain HG101. The direction of transcription and relative size of each gene are designated by bold arrows. (B) Hybridization analysis of MMH594, V583, and FA2–2 total DNA, digested with _Hin_dIII and probed with radiolabeled _cps_I. Lane 1, MMH594; lane 2, V583; lane 3, FA2–2. The relative position of λ _Hin_dIII size standard (kb) is shown to the left of lane 1, and the size (bp) of the _Hin_dIII fragment detected by the _cps_I probe is shown to the right of lane 3. (C) Hybridization analysis of V583 and FA2–2 total DNA, digested with _Eco_RI (lanes 1 and 2) or _Cla_I (lanes 3 and 4), and probed with the radiolabeled _cps_A-K fragment. Lanes 1 and 3, V583; lanes 2 and 4, FA2–2. The relative position of λ _Hin_dIII size standard (kb) is shown to the left of lanes 1 and 3 and the size (bp) of fragments hybridizing to the probe is shown to the right of lanes 2 and 4.
Figure 2
Detection of variation in cell wall carbohydrate polymer species among cps mutants by electrophoresis through a 10% polyacrylamide gel with Stains-All detection. Lane 1, FA2–2; lane 2, HG101 (_cps_I−); lane 3, HG103 (HG101 complemented with pEUIK); lane 4, HG102 (HG101 complemented with pEUI); lane 5, HG303 (_cps_C−); lane 6, HG202 (_cps_K−); lane 7, HG404 (insertional inactivation of an ORF 3′ to _cps_K). The mean molecular size (kDa) of each carbohydrate, as assessed by gel filtration, is shown at the left.
Figure 3
(A) Persistence in infection for FA2–2 and HG101. Persistence was monitored at 2 and 4 days. The mean from 13 animals is shown, with error bars representing SEM. * represent statistically significant differences (P < 0.01). (B) Opsonophagocytic killing of E. faecalis strains FA2–2, HG101, and HG103. Percent survival was calculated by dividing the cfu recovered after 90 min (t = 90) by the cfu at time 0 (t = 0), multiplied by 100. Experiments were performed in triplicate on two separate occasions, and the data from both experiments were pooled. Error bars for six independent measures show SEM. * represent statistically significant (P < 0.01) differences relative to cfu recovered from strains FA2–2 and HG103.
Figure 4
Schematic representation of a model for the organization of cell wall polymers in the cell wall of E. faecalis.
References
- Hancock L E, Gilmore M S. In: Gram-Positive Pathogens. Fischetti V, Novick R, Ferretti J, Portnoy D, Rood J, editors. Washington, DC: Am. Soc. Microbiol.; 2000. pp. 251–258.
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