Inactivation of the srtA gene affects localization of surface proteins and decreases adhesion of Streptococcus pneumoniae to human pharyngeal cells in vitro

Abstract

Inactivation of sortase gene srtA in Streptococcus pneumoniae strain R6 caused the release of beta-galactosidase and neuraminidase A (NanA) from the cell wall into the surrounding medium. Both of these surface proteins contain the LPXTG motif in the C-terminal domain. Complementation with plasmid-borne srtA reversed protein release. Deletion of murM, a gene involved in the branching of pneumococcal peptidoglycan, also caused partial release of beta-galactosidase, suggesting preferential attachment of the protein to branched muropeptides in the cell wall. Inactivation of srtA caused decreased adherence to human pharyngeal cells in vitro but had no effect on the virulence of a capsular type III strain of S. pneumoniae in the mouse intraperitoneal model. The observations suggest that--as in other gram-positive bacteria--sortase-dependent display of proteins occurs in S. pneumoniae and that some of these proteins may be involved in colonization of the human host.

FIG. 1.

Clustal V alignment of the SrtA sequences of S. aureus strain Mu50 (S.au.) and S. pneumoniae strain R6 and the CHP of S. pneumoniae strain TIGR4. Nonidentical amino acid residues are indicated in red. Broken lines indicate gaps.

FIG. 2.

Phylogenetic tree constructed from the sortase protein sequences identified in various streptococcal species and in S. aureus strain Mu50. The SrtA protein sequences of S. suis, S. gordonii, S. pyogenes, S. pneumoniae strain R6, ORF SP1218 of S. pneumoniae strain TIGR4, and S. aureus strain Mu50 as well as the SrtB, SrtC, and SrtD protein sequences of S. pneumoniae strain TIGR4 were aligned for homology by using Clustal V and megaline DNASTAR software.

FIG. 3.

Effect of srtA mutation on the localization of β-galactosidase at the surface of S. pneumoniae. Filled bars indicate cell pellet-associated enzyme activity; empty bars indicate activity in the culture medium. S, presence of plasmid pAKSRTAR64 carrying the srtA gene for complementation; P, presence of plasmid vector pLS578 without the srtA insert (control for complementation). Enzyme activity is expressed as a percentage of the total recovered in association with the bacteria and in the culture medium. Error bars indicate standard deviations.

FIG. 4.

Effect of srtA mutation on the localization of NanA in S. pneumoniae. Bacterial cultures were centrifuged, and the pelleted cells (lanes 1 and 3) and their cell-free culture media (lanes 2 and 4) were assayed for NanA by Western blotting as described in Materials and Methods.

FIG. 5.

Effect of srtA mutation and deletion of murM on the localization of β-galactosidase in strains of S. pneumoniae carrying different types of branched peptides in their cell walls. The distributions of enzyme activities associated with the bacteria and released into the culture medium are reported as described in the legend to Fig. 3. Error bars indicate standard deviations.

FIG. 6.

Effect of srtA mutation on the adherence of S. pneumoniae to human pharyngeal cells. S. pneumoniae bacteria were mixed with human pharyngeal cells of the Detroit 562 cell line, and the fraction of bacteria that remained associated with the pharyngeal cells was estimated as described in Materials and Methods. Filled bars indicate adherence by the wild-type strains; empty bars indicate adherence by the corresponding srtA mutant strains. Data were normalized so that the adherence of the wild-type cells was expressed as 100%. Error bars indicate standard deviations.

FIG. 7.

Effect of srtA mutation on the ability of S. pneumoniae to invade human pharyngeal cells. In a follow-up of the experiment described in the legend to Fig. 6, bacteria that remained associated with the pharyngeal cells after three consecutive washes were treated with antimicrobial agents that can kill extracellular but not intracellular pneumococci as described in Materials and Methods. After removal of the antibiotics by several washes, the pharyngeal cells were lysed and the number of internalized bacteria was determined as described in Materials and Methods. Data were normalized so that invasion by the wild-type cells was expressed as 100%. Error bars indicate standard deviations.

FIG. 8.

Effect of srtA mutation on the virulence of capsular type III S. pneumoniae in the mouse intraperitoneal model. The Kaplan-Meier plot shows the percentages of survival of mice which received 10³ CFU of R36ASIII (closed symbols) and R36ASIIIsrtA (open symbols).

FIG. 9.

Distributions of β-galactosidase activities in S. pneumoniae strain TIGR4 and its srtA, srtB, and srtD mutants. Filled bars indicate cell pellet-associated enzyme activity; empty bars indicate activity in the culture medium. Enzyme activity is expressed as a percentage of the total recovered in association with the bacteria and in the culture medium. Error bars indicate standard deviations.