ChBp-I2 enhance biofilm formation of S. pneumoniae in vitro
In previous studies, we reported the binding capacity of LytA-derived choline binding peptides (ChBp) to pneumococcal cells. Similar as the choline binding domains in CBP, ChBps are supposed with stronger binding capacity to choline residues on the cell surface for they get smaller spatial obstacles. During the analysis of ChBp-I2 we found it can promote the biofilm formation of S. pneumoniae in vitro on a dose dependent manner (Fig.1). And this promotion ability is much stronger than the two rest homologous sequence (ChBp-I1 and ChBp-I3). After analysis of these three peptides, we found ChBp-I2 got a higher isoelectric point (7.66) than the rest twos (7 and 5.09). Considering the biofilm formation promotion activity of ChBp-I2, we further analyzed the content of extracellular CBPs during the growth of S. pneumoniae in vitro. As a result, the choline binding proteins and high molecular weight protein were only detected during the stationary phase. Low molecular weight proteins (<10Kd) that can be enriched by DEAE were detected at both time points (Fig.2). These proteins may derive from the medium for the proteins are enriched at 2th hour, when the cell dense is low. These indicate the content of extracellular CBPs is low during the logarithmic phase, and extracellular CBPs may mainly function at late logarithmic phase and stationary phase.
ChBp-I2 influences membrane transport carbohydrate metabolism pathways
Beside biofilm formation promotion, we found ChBp-I2 can enhance the growth of pneumococcal cells with a small extent at the initial culture period (not shown). Thus, we infer ChBp-I2 may influence the gene expression of S. pneumoniae. After analysis, a total of 81 genes were identified as down regulated and 138 genes were up regulated (|log2 fold change|≥1.5). The differentially expressed genes are mainly enriched in membrane transport and carbohydrate metabolism related pathways. The membrane transport pathways include carbohydrate, lipid, protein, cation and phosphate. Meanwhile, the carbohydrate metabolism pathways include fructose, mannose, galactose, starch, sucrose, amino sugar and nucleotide sugar metabolism (Figure 3). By Q-PCR we found the regulations of these genes are mostly dose dependent (Fig.S2). Considering the only binding activity of ChBp-I2, we infer the regulations of these DE genes may relate to the physical changes on cells surface. Considering the increased extracellular CBPs at late logarithmic phase and stationary phase, the DE genes were analyzed to genes regulations at late logarithmic phase to analyze the potential regulations of extracellular CBPs. There are 12 genes were co-up regulated and 37 genes were regulated conversely. 45 genes were co-down regulated and 21 were regulated conversely (Figure 4). This indicates most of these DE genes are down regulated at late logarithmic phase, which may relate to the decrease of growth rate caused by nutrition exhaustion.
CBDs function differently on growth, biofilm formation and gene expression of S. pneumoniae
According to gene expressions at late logarithmic phase, the expression of LytA, LytC and CbpD were characterized as up-regulated. Thus we further analyzed some growth phenotype under the CBDs of these three proteins. When culture in mediums supplemented with the CBDs of different concentrations, CBD-A and CBD-D of high concentration (200μg/mL) exhibits a certain inhibitory effect on growth rate. Similar with ChBp, both of three CBDs can promote the growth of S. pneumoniae at different degrees, the optimum concentrations are both about 25μg/mL, which is lower than ChBp-I2 (100μg/mL) but higher than ChBp (3μg/mL) (Figure 5A). Meanwhile, CBD-C and CBD-D of high concentrations (100-200μg/mL) can promote the formation of biofilm (Figure 5B). Through phenotypic observation, we found the cells under CBD-C are easy to form aggregation when place at room temperature, and both CBD-A, CBD-C and CBD-D show inhibition to autolysis. Under microscope the biofilm under CBD-C form large area of membranous region, which is different to the ones with CBD-A and CBD-D (Figure 6, Figure S3). By PI analysis we found the CBRs in LytA are tending to be negative charged, the CBRs in CbpD are positive charged under neutral environment. CBD-C contains similar number of negative charged and positive charged CBRs (not shown). A higher PI may be helpful in in explaining the biofilm formation promotion activities of CBD-C and CBD-D.
CBDs are involved in the regulation of extracellular nucleic acid and protein
For the similar phenotypes of pneumococcal cells under CBDs and ChBp-I2, we further analyzed the expression of DE genes (12 down regulated and 14 up regulated genes) under the existence of CBDs. After analysis we found most of the DE genes are up-regulated under CBDs, indicating the CBDs may functions differently (Figure S2). To further define the function of extracellular CBDs, four kind of components were tested (cation, phosphate, protein and nucleic acid) in the culture medium which related to the regulated pathways. As indicated, the most significant feature is the increased extracellular protein. After ultrafiltration concentration, the proteins under CBD-A, CBD-D and ChBp are more than 4mg/mL, the proteins in the control group is only about 0.5mg/mL (Figure 7A). When being precipitated by TCA, the high molecular weight proteins (HMWp) were mainly detected as insoluble form in the mediums of CBD-A and CBD-D (Figure S4). Meanwhile, the extracellular nucleic acid is increased with CBD-A, but decreased under CBD-C and CBD-D (Figure 7B). A slight increase in extracellular phosphate (CBD-C and ChBp) and cation (CBD-A and CBD-D) was detected (Figure 7C, 7D). Taken together, CBD-A is supposed to promote extracellular transport of nucleic acid participate in the formation of HMWp. CBD-C can increase the concentration of extracellular phosphate and decrease extracellular nucleic acid. CBD-D can promote the formation of HMWp, decrease extracellular nucleic acid, and increase extracellular cation.