The NanA neuraminidase of Streptococcus pneumoniae is involved in biofilm formation

Abstract

Streptococcus pneumoniae remains a major cause of bacteremia, pneumonia, and otitis media despite vaccines and effective antibiotics. The neuraminidase of S. pneumoniae, which catalyzes the release of terminal sialic acid residues from glycoconjugates, is involved in host colonization in animal models of infection and may provide a novel target for preventing pneumococcal infection. We demonstrate that the S. pneumoniae neuraminidase (NanA) cleaves sialic acid and show that it is involved in biofilm formation, suggesting an additional role in pathogenesis, and that it shares this property with the neuraminidase of Pseudomonas aeruginosa even though we show that the two enzymes are phylogenetically divergent. Using an in vitro model of biofilm formation incorporating human airway epithelial cells, we demonstrate that small-molecule inhibitors of NanA block biofilm formation and may provide a novel target for preventative therapy. This work highlights the role played by the neuraminidase in pathogenesis and represents an important step in drug development for prevention of colonization of the respiratory tract by this important pathogen.

FIG. 1.

Activity of S. pneumoniae neuraminidase. (A) Titration of activity using different concentrations of purified NanA. (B) Effect of divalent cations on the activity of purified NanA compared to the activity of the wild-type enzyme (control). The assay was performed using 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MNN). *, P < 0.05.

FIG. 2.

Inhibition of NanA neuraminidase activity by the sialic acid compounds NANA and DANA. The activity is expressed as a percentage of the activity of NanA without an inhibitor (control). The assay was performed using 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. *, P < 0.05.

FIG. 3.

Phylogenetic analysis of neuraminidases: unrooted phylogenetic tree based on our broad survey of neuraminidase phylogeny. The tree is the strict consensus of three most parsimonious trees (45,885steps; consistency index, 0.326; retention index, 0.385; rescaled consistency, 0.125). Black circles indicate branches with bootstrap values greater than 80%. Gray circles indicate branches with bootstrap values between 50 and 80%. Open circles indicate nodes with bootstrap values less than 50% for agreement with the consensus bootstrap tree. Branches without an indication of the bootstrap value are found in the maximum parsimony tree but not in the bootstrap tree.

FIG. 4.

Release of sialic acid from the surface of airway epithelial cells by NanA. (A) Exposure of asialo-GM1 to concentrated supernatant from wild-type (WT) and nanA strains. (B) Exposure of asialo-GM1 to purified NanA. Cells were stained with antibody to asialo-GM1 and quantified by flow cytometry, and the results are expressed as the changes compared to a medium-only control. *, P < 0.05.

FIG. 5.

S. pneumoniae biofilm formation. (A) Encapsulated (D39 background) strains were grown in microtiter trays without (filled bars) or with (striped bars) previous exposure to epithelial cells. Unencapsulated R6 strains were grown in microtiter trays without exposure to epithelial cells. (B) Incubation with NANA results in reduced biofilm formation by wild-type strain D39 (WT). Biofilms were measured by using crystal violet staining. Biofilm formation was normalized to growth and expressed as a percentage compared to the R6 wild-type strain. *, P < 0.05.

FIG. 6.

Imaging of S. pneumoniae biofilms. (A) Images of crystal violet-stained biofilms in microtiter wells for wild-type (WT) and nanA strains with D39 (after exposure to epithelial cells) and R6 backgrounds. (B) Fluorescence microscopy of wild-type strain D39 and nanA biofilms grown in microtiter trays after exposure to epithelial cells and stained with BacLight live/dead stain. Magnification, ×200. (C) Three-dimensional reconstruction of the biofilm structure for the wild-type strain shown in panel B. (D) Three-dimensional reconstruction of cells of the nanA strain shown in panel B.

FIG. 7.

Inhibition of NanA by oseltamivir. The activity is expressed as a percentage of the activity without inhibitor. The assay was performed using 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. *, P < 0.05.

FIG. 8.

Inhibitory activity of potential NanA inhibitors. (A) Screening of potential inhibitors was performed with NanA and inhibitors at a concentration of 100 μM in the neuraminidase assay. Results are percentages of the level for the control without any inhibitor. (B) Dose-response curve for NanA with lead compound XX1. Data were fitted with a logarithmically based trend line. The data are the percentages of activity compared to the control with only the vehicle (dimethyl sulfoxide). (C) Biofilm formation by wild-type strain D39 (WT) grown in the presence of XX1 during exposure to epithelial cells and growth in microtiter trays. The nanA strain was used as a reference. Biofilm formation was normalized to growth and was expressed as a percentage compared to the results for the wild-type control. (D) Chemical structure of XX1. *, P < 0.05.