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Streptococcus pneumoniae colonizes asymptomatically the upper respiratory tract as a commensal, but has also a high virulence potential and can leave this ecological niche, thereby spreading to the lungs and blood. During this process, pneumococci must adapt to changing external environmental conditions and parameters such as nutrient availability, temperature, or oxygen levels. The transmission of these signals into the bacterial cell interior occurs via the process of signal transduction, which ultimately results in controlled differential gene expression. The most commonly strategy for signal transduction is the use of two-component regulatory systems (TCS), consisting of a membrane-bound histidine kinase as a sensor and a cytoplasmic response regulator that binds to the promoter region of its target genes and interferes with gene expression.
In this study the regulatory impact and influence of the TCS08 and TCS09 on the phenotype and pathophysiology of S. pneumoniae were investigated using two different serotypes
(serotype 2: D39 and serotype 4: TIGR4). For all functional assays, single (Δrr08/Δrr09 or Δhk08/Δhk09) and double (Δtcs08 or Δtcs09) mutants that were constructed by insertion-deletion mutagenesis, were applied.
In the first study a comparative transcriptome analysis using RNA-sequencing was conducted with our tcs09-mutants and the parental wild-type D39. The data indicated upregulation of the aga operon, which is related to galactose metabolism, and downregulation of the regulator AgaR, particularly in the absence of HK09. Interestingly, encapsulated and nonencapsulated hk09-mutants in D39 showed significant growth defects when galactose was used as sole carbohydrate source. Electron microscopy revealed morphological changes such as an increased number of membrane vesicles and cell wall degradation for the nonencapsulated hk09- and tcs09-mutants of strain D39. An increased capsule production was indicated for the encapsulated hk09- and tcs09-mutants in D39. The latter two mutants as well as the encapsulated rr09-mutant also showed altered colony morphology. While D39Δhk09 formed only opaque colonies, the mutants D39Δrr09 and D39Δtcs09 showed increased numbers of transparent colonies. In a Triton X-100 induced autolysis assay and in the presence of oxidative stress, a negative effect of the morphological changes of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 on their survivability was demonstrated. In conclusion, we observed that TCS09 in S. pneumoniae D39 is important for its fitness through regulation of carbohydrate metabolism. This indirectly influences cell wall integrity and capsular polysaccharide amount via other regulatory mechanisms, which ultimately affects stress tolerance.
In a second study, we investigated the virulence potential of TCS09 in pneumococcal strain TIGR4. In vitro growth analyses in complex medium showed no effect after loss of function of TCS09 on pneumococcal fitness. In contrast, using the disaccharides lactose and sucrose in chemically defined medium, an extended lag phase of tcs09-mutants was monitored. To assess changes of virulence factor expression, immunoblots were applied to demonstrate the abundance of various essential virulence factors of S. pneumoniae. The results revealed a decreased amount for RrgB, which is the backbone pilus component of type 1 pili, in the hk09-mutant. Field emission scanning electron microscopy and transmission electron microscopy images were applied to study alterations of the bacterial cell shape. The illustrations by FESEM and TEM showed no effect of TCS09-deletion on pneumococcal cell morphology. Cell culture-based infection analyses revealed a similar adhesion capacity of the parental strain and isogenic mutants to lung epithelial cells. However, phagocytosis assays indicated a significantly increased killing rate of intracellular TIGR4ΔcpsΔtcs09, when compared to the isogenic parental strain. In experimental mouse infection models of acute pneumonia and systemic infection the tcs09-mutants were not attenuated. However, to decipher in more detail differences between the wild-type and tcs09-mutants, in vivo co-infection were performed, which highlighted a significantly lower bacterial load of TIGR4luxΔhk09 and TIGR4luxΔtcs09 especially in the lungs, blood, and brain after 48 h. In conclusion, the TCS09 in TIGR4 is necessary for maintaining metabolic fitness, which in turn contributes to dissemination in the host.
In the third study, the influence of TCS08 on gene expression and metabolic and pathophysiological processes of S. pneumoniae was analyzed. In particular, differential gene expression in the hk08-mutant of TIGR4 was detected using microarray and qPCR. The transcriptome analysis revealed a downregulation of cellobiose specific phosphotransferase systems as well as an upregulation of the fab operon, arc operon, and psa operon. These operons encode proteins involved in fatty acid biosynthesis, arginine catabolism, and manganese uptake, respectively. Furthermore, we measured a downregulation of pilus 1 genes in TIGR4ΔcpsΔtcs08 and an increased expression of pavB in TIGR4ΔcpsΔhk08. These data were confirmed by immunoblotting and surface localization studies. Using in silico analysis, a SaeR-like binding motif was identified in the promoter region of pavB. Furthermore, the impact of TCS08 on pneumococcal virulence was investigated in vivo using the acute pneumonia and sepsis models. These models showed a strain-dependent effect of the single TCS08 component deletions between D39 and TIGR4 pneumococci. Whereas loss of HK08 or TCS08 in D39 attenuated the mutants in the pneumonia model, loss of RR08 in TIGR4 was responsible for a similar effect. In contrast, loss of HK08 in TIGR4 promoted increased virulence in the pneumonia and sepsis model. Overall, these data indicate that TCS08 is involved as key player in bacterial fitness during host colonization.