Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

Abstract

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role.

FIG 1

Pneumococcal metabolism and exoglycosidase activity. (A) Differential routes of S. pneumoniae pyruvate metabolism, with active pathways in the presence and absence of glucose-mediated CCR denoted with bold arrows. (B) Schematic representation of the exoglycosidase activity of the dominant pneumococcal neuraminidase A (NanA) and β-galactosidase A (BgaA), which cleave terminal sialic acid residues and β-1,4-linked galactose residues, respectively, off host glycoconjugates.

FIG 2

Galactose is present in the nasopharynx, and its exposure is increased following colonization with S. pneumoniae. (A) Relative levels of sugars present in serum and NALF of patients experimentally colonized with S. pneumoniae strain BHN418 (white squares) versus carriage-negative controls (black squares). Each square represents an individual clinical sample. Statistical analyses were done using the Mann-Whitney test. P values of ≤0.05 were considered statistically significant. Asterisks indicate specific P values. (B) Representative fluorescence microscopy images of sections obtained from septa of mice colonized with 6A-10, 6A-10 ΔnanA, or a mock-treated control (n = 3 per cohort). Labeling was performed using FITC-labeled E. cristagalli lectin to indicate the presence of exposed galactose (green) and DAPI (blue) to visualize cells. Exposed galactose present on the apical mucosal epithelial cell layer is denoted by white arrows.

FIG 3

Neuraminidase A and β-galactosidase A contribute to robust biofilm formation within the nasopharynx. Shown are representative scanning electron microscopy images (at low and high magnification) of nasal septal epithelia isolated from mice colonized with wild-type S. pneumoniae strain 6A-10, 6A-10ΔnanA, or 6A-10 ΔbgaA. A minimum of 5 septa were examined per cohort. Nasal septa of mice challenged with saline were used as negative controls. Septa were collected at 7 dpi.

FIG 4

Contribution of neuraminidase A and β-galactosidase A to biofilm formation and adhesion to host cells in vitro. (A) Biofilm-forming ability of strains 6A-10, 6A-10 ΔnanA, and 6A-10 ΔbgaA in a 48-h 6-well polystyrene plate model. Biofilm biomass was measured using the amount of crystal violet trapped within the biofilm at 540 nm. (B) Abilities of strains 6A-10, 6A-10 ΔnanA, and 6A-10 ΔbgaA to adhere to Detroit 562 cells as measured by recoverable CFU following washing and plating of bacteria. Values are expressed as fold increase in adhesion relative to wild-type strain 6A-10. Statistical analysis was performed using the Mann-Whitney test. **, P ≤ 0.01. Experiments were repeated three times. Mean results are shown, and error bars represent the standard error of the mean.

FIG 5

Non-CCR-acting carbohydrates are permissive for biofilm formation. (A) Pneumococcal biofilm formation was assessed in diluted medium (50% THB) supplemented with increasing concentrations of glucose, sucrose, fructose, galactose, lactose, and sialic acid in a 48-h 6-well polystyrene plate model. Statistical analysis was performed using one-way ANOVA. Statistical significance was calculated by comparing the biofilm biomass of supplemented media with that of unsupplemented sugar-free media. (B) Biofilm biomass at 48 h by the wild-type TIGR4 and 6A-10 strains and their respective spxB mutants. Statistical analyses compared the mutant to its wild-type counterpart using Student's t test. (C) Biofilm biomass by strain 6A-10 in the presence of different concentrations of the end metabolites acetate (black bars) and lactate (white bars). Statistical analyses were performed by comparing the biofilms in supplemented media with those in unsupplemented medium using Student's t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤0.001; ***, P ≤ 0.0001. Experiments were repeated a minimum of three times. Mean results are shown, and error bars represent the standard error of the mean.

FIG 6

Metabolic activity of pneumococci during biofilm formation. (A) TIGR4 orthologs of 6A-10 genes determined to be differentially expressed by DESeq analysis of RNA-seq data were subjected to a pathway enrichment analysis performed with GAGE, KEGG orthology terms, and pathway maps for S. pneumoniae TIGR4. Heat maps represent the log₂ fold change of biofilm (BF) expression over planktonic (PK) expression. Each subpanel harbors three columns corresponding to the three paired replicates for each condition (3 replicates of BF versus PK). Values of log₂ fold change are indicated for each replicate pair. Compared growth conditions include S. pneumoniae strain 6A-10 grown as biofilm in unsupplemented medium (THB BF) or medium supplemented with galactose (GAL BF) versus the planktonic growth phenotype in unsupplemented medium (THB PK) or medium supplemented with glucose (GLU PK) or galactose (GAL PK). Pathways differentially regulated across multiple growth conditions are highlighted with the same background color (e.g., pyruvate metabolism in green). (B) Differentially regulated genes involved in pyruvate metabolism in S. pneumoniae were painted on the KEGG pyruvate metabolism reference pathway (SPN00620) using the packages GAGE and Pathview. Each gene/enzyme box is divided into three colored thirds corresponding to the three paired replicates for each condition (3 replicates of THB BF versus THB PK). Differential expression is depicted according to the heat map provided in the upper right corner of panel B. Values are log₂ fold change of biofilm (THB BF) expression over planktonic (THB PK) expression.