Regulation of neuraminidase expression in Streptococcus pneumoniae

Abstract

Background: Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung.

Results: In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells.

Conclusions: The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.

Figure 1

Structure of the neuraminidase locus in different streptococci. A. The schematic maps of the nanAB operon of S. pneumoniae G54 and the orthologous locus in its close relatives, including S. gordonii V288 (NC_009785.1), S. sanguinis SK36 (NC_009009.1) [46], S. mitis B6 (NC_013853.1) [47] and S. oralis Uo5 (NC_015291.1) [48] are shown. In S. pneumoniae the complete locus includes 18 ORFs, some of them conserved in the other species [23]. The two neuraminidases (NanA and NanB) are in pink, while the three different transporters (two ABC transporters and one PTS) are in blue. The phosphosugar binding transcriptional regulator is shown in grey and the metabolic enzymes involved in sialic acid metabolism are in orange. The homologous regions in green refer to DNA identity above 50% and represent orthology of genes. The black arrows placed upstream of SPG1601, SPG1599, SPG1593, and SPG1583 represent the promoters of the regulon [21]. The gene numeration is detailed in Table 1. B. Schematic representation of the first steps in sialic acid catabolism. The first step involves the N-acetylneuraminate lyase SPG1585 which removes a pyruvate group from sialic acid, yielding N-acetylmannosamine (ManNAc). Subsequently, an N-acetylmannosamine kinase (SPG1584) adds a phosphate group to ManNAc, resulting in the formation of N-acetylmannosamine-6-phosphate (ManNAc-6P). SPG1593 encodes an N-acetylmannosamine-6-phosphate 2-epimerase, which transforms ManNAc-6P into N-acetylglucosamine-6-phosphate (GlcNAc-6P) [15,16].

Figure 2

Metabolic utilisation 0f ManNAc and NeuNAc by S. gordonii, S. mitis and S. pneumoniae . S. gordonii V288 (A), S. pneumoniae G54 (B), and S. mitis NCTC12661 (C) were grown in CAT medium (200 U catalase) supplemented with 2 g/L glucose (black line) 2 g/L ManNAc (red line) and 1.5 g/L NeuNAc (blue line). CAT medium alone as a source of carbon is in grey line. All strains were grown for 38 hours at 37°C in 200 μl of medium in a 96 well microplate with reading intervals of 10 min. For the fermentation assay (panel D) bacteria were incubated for 24 and 48 h with serial dilutions of either ManNAc (left columns) or NeuNAc (right columns) as sole carbon sources in microtiter plates containing phenol red as a pH indicator. Sugar fermentation is evidenced by a yellow colour change due to acidification of the culture medium. Carbohydrate concentrations (% w/v) are shown on the right.

Figure 3

Growth and induction of gene expression by ManNAc. (A) Growth of S. pneumoniae strains on CAT medium supplemented with 10 g/L of ManNAc: FP65 (open squares), nanAB-deficient mutant (open triangles), and SPG1583-regulator deletion mutant (closed circles). (B) Growth of FP65 on CAT medium without added sugar (closed squares) and supplemented with ManNAc 10 g/L (open squares). The white and black arrows indicate samples taken for quantitative Real Time-PCR. Gene expression analysis of the genes coding for NanA the ABC transporter SPG1598, the PTS transporter SPG1592, and the ABC transporter SPG1591 is shown in panel C and D. Panel C refers to fold changes in transcriptional levels at OD 0.02 in medium with or without ManNAc (for sampling see closed arrows in panel 3B). Panel D refers to analysis of sequential samples (OD₅₉₀ = 0.02 and OD₅₉₀ = 0.05) of bacteria grown in ManNAc (for sampling see open arrows in panel 3B). The fold changes are reported as mean from independent triplicate or quadruplicate experiments. Two-tailed Student t test was used for analyse statistical significance (*, p < 0.05; **, p < 0.01). Generation time on unsuplemented CAT medium is 40 min and on ManNAc 140 min.

Figure 4

Repression of nanAB locus by glucose. (A) Growth curves of FP65 in medium supplemented with glucose (closed circles), ManNAc (open triangles), and glucose plus ManNAc (open squares). (B) Growth curves of PF65 in medium supplemented with glucose (open circles), NeuNAc (open triangles), and glucose plus NeuNAc (open squares). The arrows indicated sampling. (C) Gene expression of SPG1598, SPG1592, and SPG1591 in medium supplemented with amino sugars are compared to growth in glucose. Variation of gene expression is shown for genes of bacteria grown in ManNAc (open bars), glucose plus ManNAc (open striped bars), NeuNAc (grey bars), and glucose plus NeuNAc (grey striped bars). Results are represented as fold changes ± SD of gene expression from 3 to 4 independent experiments. Statistical analysis was carried out using Tukey’s Multiple Comparison Test (ns non significant; *, p < 0.05; **, p < 0.01). Generation time on glucose containing media is 38–45 min, 90 min on NeuNAc and 140 min on ManNAc.

Figure 5

Neuraminidase protein production and activity on whole cells. A cytofluorimetric assay with an anti-NanA serum was performed on pneumococci grown on different carbohydrates (panel A). The presence of NanA at the bacterial surface was tested in samples cultivated in glucose (open bar), glucose + ManNAc, ManNAc alone (grey bar), and NeuNAc alone (black bar) (all carbohydrates were at 1 g/L). Data are represented as mean values ± SD of percent bacterial population positive for NanA production and derived from quadruplicates experiments performed independently. Asterisks (*, p < 0.05; **, p < 0.001) indicated statistical significance. Panel B shows the hydrolysis of 2’-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-Neu5Ac) in the presence of 40 μl S. pneumoniae FP65 cell samples grown in CAT medium with either glucose (white circles) or N-acetylmannosamine (black circles). The neuraminidase activity was computed as the variation of fluorescence vs time using a linear regression of the data (dashed lines). Inlet. Hydrolysis of 4MU-Neu5Ac by purified NanA neuraminidase, showing the proportionality between enzyme concentration and rate of fluorescence variation. Enzyme concentrations were 10 nM (black circles), 20 nM (triangles), 30 nM (diamonds) and 40 nM (squares). The empty circles show the variation of fluorescence vs time for the substrate alone.