The Two-Component System 09 Regulates Pneumococcal Carbohydrate Metabolism and Capsule Expression

Abstract

Streptococcus pneumoniae two-component regulatory systems (TCSs) are important systems that perceive and respond to various host environmental stimuli. In this study, we have explored the role of TCS09 on gene expression and phenotypic alterations in S. pneumoniae D39. Our comparative transcriptomic analyses identified 67 differently expressed genes in total. Among those, agaR and the aga operon involved in galactose metabolism showed the highest changes. Intriguingly, the encapsulated and nonencapsulated hk09-mutants showed significant growth defects under nutrient-defined conditions, in particular with galactose as a carbon source. Phenotypic analyses revealed alterations in the morphology of the nonencapsulated hk09- and tcs09-mutants, whereas the encapsulated hk09- and tcs09-mutants produced higher amounts of capsule. Interestingly, the encapsulated D39∆hk09 showed only the opaque colony morphology, while the D39∆rr09- and D39∆tcs09-mutants had a higher proportion of transparent variants. The phenotypic variations of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 are in accordance with their higher numbers of outer membrane vesicles, higher sensitivity against Triton X-100 induced autolysis, and lower resistance against oxidative stress. In conclusion, these results indicate the importance of TCS09 for pneumococcal metabolic fitness and resistance against oxidative stress by regulating the carbohydrate metabolism and thereby, most likely indirectly, the cell wall integrity and amount of capsular polysaccharide.

The Two-Component System 09 of Streptococcus pneumoniae Is Important for Metabolic Fitness and Resistance during Dissemination in the Host

“…Pneumococcal single mutants (RR09 or HK09) or double mutants (RR09 and HK09), were generated by insertion-deletion mutagenesis in S. pneumoniae TIGR4, TIGR4Δ cps and TIGR4 lux using the protocol and plasmid constructs described in a previous study [ 23 ].…”

“…RNA isolation, purification and qPCR were performed as described [ 23 ]. In brief, pneumococcal strain TIGR4Δ cps and isogenic ∆ rr09 -, ∆ hk09 - and ∆ tcs09 -mutants were cultivated in glucose supplemented CDM up to an OD 600nm of 0.6.…”

“…Bacteria were cultivated in CDM with glucose until OD 600nm 0.3 (encapsulated strains) or 0.6 (nonencapsulated strains) at 37 °C. Samples were prepared for FESEM and TEM as described [ 23 ]. In brief, encapsulated strains were fixed first with 2.5% glutardialdehyde, 2% paraformaldehyde, 0.075% ruthenium red and 75 mM L-lysine acetate salt and stained with 1% osmium solution containing ruthenium red.…”

“…Pneumococci express 13 TCSs and one orphan response regulator. Their role in pneumococcal physiology, fitness and virulence has been extensively discussed recently [ 13 , 17 , 23 ]. For TCS09, the impact on virulence has already been demonstrated by several studies [ 11 , 24 , 25 , 26 ].…”

“…In a previous study, we deciphered the role of TCS09 for S. pneumoniae D39 metabolic fitness, autolysis and oxidative stress. Using transcriptomics, we indicated differential gene expression of genes involved in carbohydrate metabolism and illustrated an altered CPS amount and phenotype in tcs09 -mutants [ 23 ]. Because of these changes, we hypothesized that the TCS09 contributes at least indirectly to pneumococcal colonization and virulence.…”