Figure 1.
Comparison of genome-wide transcriptomic data for exponential (e) and early stationary phase (s) cultures of S. suis grown in pullulan (Pul) or glucose (Glc).
A. Venn diagram of S. suis genes differentially regulated during growth in pullulan (Pul) vs glucose (Glc) in the exponential (e) or stationary (s) phase. In each sector the numbers of shared or unique differentially expressed genes is indicated. B. GO term distribution of S. suis genes differentially regulated in pullulan vs glucose in early exponential and early stationary phase. GO Enrichment analysis was performed using BLAST2GO (P = 0.05, two-tailed Fisher's Exact test).
Figure 2.
Transcriptome analysis of S. suis metabolism in presence of pullulan.
A. Heatmap showing the effect of starch/pullulan on the transcription of genes involved in carbohydrate metabolism and capsule production. Expression (ratios) of genes participating in different pathways (indicated at the right of the heatmap) are shown for 6 different comparisons (indicated at the top of the heatmap). At the top of the Figure 2A, a color scale depicts the ratio of expression during growth in pullulan vs. glucose. Red indicates induction (upregulation) of the respective genes and blue indicates repression (downregulation) of the respective genes for each comparison. For each gene, the S. suis P1/7 locus tag and the gene name is depicted on the right. B. Schematic representation of S. suis metabolic pathways differentially regulated in pullulan vs glucose. α-glucans (i.e. starch/pullulan) are degraded by extracellular amylopullulanase (apuA) and the end degradation products, maltose/maltotriose and maltodextrins, are transported by PTS for maltose/maltotriose (malT) and maltodextrin ABC transport inside the bacteria (malX, malC and malD). Maltodextrins and maltose are most lilkely converted to glucose-1-phosphate (Glc1P) or α-glucose by 4-α-glucanotransferase and maltodextrin phosphorylase (malQ1 and glgP1 respectively). Glc1P can be metabolized in different pathways: phosphoglucomutase (pgm) isomerize glc1P to glucose-6-phosphate (glc6P) which may enter glycolysis (violet box) where it is consequently oxidated to pyruvate (pyr). Homolactic fermentation reduces pyruvate into lactate, whereas heterofermentative growth leads to other products, such as formate, acetate and ethanol (pyruvate metabolism, yellow box). The excess of glc1P that cannot enter in glycolysis may be used for synthesis of glycogen as an energy reserve (light blue box). The genome of S. suis is predicted to encode the enzymes sucrose phosphorylase gtfA, α-fructofuranosidase (interconvertase) invrtsC, and raffinose galactohydrolase, rafgH for the interconversion of raffinose- like sugars. These enzymes participate in the starch and galactose Leloir pathway. Part of Leloir pathway (e.g. galactose-1-phosphate uridylyltransferase, galT, and galactokinase, galK) was induced more strongly in starch/pullulan. GalT interconverts galactose-1-phosphate (gal1-P) and UDP-Glucose (UDP-glc) to UDP-galactose (UDP-gal) and glc1P. Alternatively, UDP-glc may be converted into glucuronic acid (glcur) by UDP-D-glucuronate (UDP-glcur) to enter in an alternative (to glycolysis) pathway for pyruvate (pyr) production. Pathway predictions were reconstructed based on genome information, literature and database surveys (KEGG, MetaCyc). The following gene annotation was downloaded from NCBI: galM, aldose 1-epimerase; galK, galactokinase; galE, UDP-glucose 4-epimerase; galT, galactose 1-phosphate uridylyltransferase; pgm, Phosphoglucomutase/phosphomannomutase; pfkA, 6-phosphofructokinase; fba, fructose bisphosphate aldolase; tpiA, triosephosphate isomerase; gapA, glyceraldehyde-3-phosphate dehydrogenase; pgk, phosphoglycerate kinase; gpmA, phosphoglyceromutase; eno, phosphopyruvate hydratase; pyk, pyruvate kinase; ldh, L-lactate dehydrogenase; pyroX, pyruvate oxidase; ackA, acetate kinase; pfl, pyruvate formate-lyase; adlE acetaldehyde-CoA dehydrogenase; adhE alcohol dehydrogenase; glgB, glgA glycogen synthase; glgC glucose-1-phosphate adenylyltransferase; glgP glycogen phosphorylase.
Table 1.
Confirmed and proposed S. suis virulence factors differentially expressed in pullulan (Pul) compared to glucose (Glc).
Figure 3.
Transcriptional regulation of apuA and sly grown in the presence of different carbon sources.
A. S. suis S10 growth curve at 37°C in CM containing 1% (w/v) of different sugars as indicated. The graph shows the means and standard deviations from two independent experiments. B. and C. Relative expression of apuA and sly in S. suis grown in CM containing 1% (w/v) different sugars was determined by qPCR. The transcript levels of apuA were measured after 2.5 hours and 4 hours of growth relative to the reference gene proS, which is constitutively expressed at similar levels during growth in different sugars (data not shown). The height of the bars represent mean values for the relative expression data ± SEM from 2 independent experiments (n = 3). Statistical significance was calculated using a two-way ANOVA test followed by Bonferroni's post hoc test (* p<0.05; ** p<0.01; *** p<0.001.).
Figure 4.
Induction of apuA expression by putative inducers in the presence of glucose or lactose.
A. Growth curves of S. suis S10 in CM containing 1% w/v lactose or B. 1% w/v glucose before and after addition of 0.25% w/v putative inducers (arrow) i.e. maltotriose, pullulan or glucose. The graphs show the means and standard deviations from two independent experiments. C. Relative expression of apuA genes following addition of putative inducers to S. suis growing in CM plus lactose. D. Relative expression of apuA genes following addition of putative inducers. The relative expression of apuA, was measured by qPCR 10, 30, 60 and 90 minutes after addition of the putative inducers, The height of the bars shows the mean (n = 3) fold change in expression ± SEM from two independent experiments. Statistical significance was calculated using a two-way ANOVA test followed by Bonferroni's post hoc test (* p<0.05; ** p<0.01; *** p<0.001.).
Figure 5.
Identification of conserved operator binding motifs for ApuR (OM1) and CcpA (OM2) in S. suis P1/7, B. subtilis 168 and L. monocytogenes EDG-e.
A. The 6 kb amylopullulanase gene apuA is located downstream of apuR which encodes a putative transcriptional regulator of the LacI/GalR family. Located downstream of apuA are a cluster of genes predicted to be involved in uptake and fermentation of ascorbate (sgaT, sgaB). For each gene, the direction of transcription is indicated by an arrow, the size of which is proportional to the length of the corresponding open reading frame. Putative promoters are represented by arrows and transcription terminators by loops. The operator motifs OM1 and OM2/cre (shaded sequences in B, C and D) were determined using the MEME software suite and their relative probability p-values are indicated. B. The apuA promoter based on the experimentally determined transcription start site (Ts arrow) C. The B. subtilis mdxE (BSU34610) promoter D. The L. monocytogenes lmo2125 promoter E. and F. Sequence alignment of the DNA binding domains of the ApuR and CcpA proteins of S. suis, L. monocytogenes and B. subtilis. Conserved amino acid sequences are indicated in black.
Figure 6.
Comparison of adherence A. and invasion B. of S. suis after growth in CM+1% w/v pullulan (black bars) vs. CM+1% w/v glucose (white bars).
NPTr confluent monolayers were co-cultivated for 2 h with S. suis S10 bacteria grown in CM plus pullulan or glucose. Adherence and invasion are shown as mean % values of the initial inoculum from two independent experiments in triplicate. Error bars indicate the SD.
Figure 7.
Hemolysis assay of S. suis growing in two different carbon sources.
A. The hemolysis production was measured by analyzing the supernatant of S. suis grown in CM plus 1% w/v of glucose or pullulan in the lag, exponential and stationary phase (OD600 values 0 to 0.56). B. Deep-well titer plate showing hemolytic activity of supernatants collected from S. suis grown in CM supplemented with glucose or pullulan.
Figure 8.
Expression of apuA and sly in S. suis recovered from blood and tissues of experimentally infected piglets.
The relative expression of apuA and sly in S. suis blood, joints, heart and brain recovered from intravenously infected piglets calculated using the GeNorm method [38] using three housekeeping genes for data normalization. A. The relative expression of apuA (×107) are shown for S. suis recovered from blood and different organs. B. Relative expression of sly (×105) in blood and different organs.
Figure 9.
Links between carbohydrate metabolism and virulence in Streptococcus suis.
At the mucosal surfaces a high ratio of a-glucans to glucose upregulates expression of several sugar transport systems and metabolic pathways associated with starch metabolism. Additionally, several virulence factors involved in adherence to host cells, degradation of connective tissue (spreading factors), and avoidance of phagocytic killing, including ApuA and suilysin are upregulated when glucose is diminished. Suilysin may facilitate dispersion of bacteria in mucosal tissues due to loss of barrier integrity. Once S. suis reaches the bloodstream metabolism is adapted for optimal growth on glucose and the expression of virulence factors is reduced by CcpA mediated-repression. In infected organs glucose levels are lower than in the blood and are further reduced by inflammation and utilization by S. suis leading to upregulation of ApuA, suilysin and other virulence factors. In the organs and tissues, glycogen released from damaged cells is degraded by ApuA to generate maltodextrins which sustain growth of S. suis.