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.

Streptococcus pneumoniae interplays with its environment by using 13 two-component regulatory systems and one orphan response regulator. These systems are involved in the sensing of environmental signals, thereby modulating pneumococcal pathophysiology. This study aimed to understand the functional role of genes subject to control by the TCS08. The identified genes play a role in transport of compounds such as sugars or amino acids. In addition, the intermediary metabolism and colonization factors are modulated by TCS08. Thus, TCS08 regulates genes involved in maintaining pneumococcal physiology, transport capacity, and adhesive factors to enable optimal colonization, which represents a prerequisite for invasive pneumococcal disease.

word count: 227 24 Importance word count: 101 25 Main text word count: 4743 26 2 ABSTRACT 27 Streptococcus pneumoniae two-component regulatory systems (TCS) enable 28 adaptation and ensure its maintenance in host environments. This study deciphers the 29 impact of the TCS08 on pneumococcal gene expression and its role in metabolic and 30 pathophysiological processes. Transcriptome analysis and real-time PCR demonstrated 31 a regulatory effect of the TCS08 on genes involved mainly in environmental information 32 processing, intermediary metabolism, and colonization by S. pneumoniae D39 and 33 TIGR4. Striking examples are genes of the fatty acid biosynthesis, arginine-deiminase 34 system, and psa operon encoding the manganese ABC transport system. In silico 35 analysis confirmed that TCS08 is homologous to Staphylococcus aureus SaeRS and a 36SaeR-like binding motif is displayed in the promotor region of pavB, the upstream gene 37 of the tcs08 operon encoding a surface-exposed adhesin. Indeed, PavB is regulated by 38 the TCS08 as confirmed by immunoblotting and surface abundance assays. Similarly, 39Pilus-1 of TIGR4 is regulated by TCS08. Finally, in vivo infections using the acute 40 pneumonia and sepsis models showed a strain dependent effect. Loss of function of 41 HK08 or TCS08 attenuated D39 virulence in lung infections. The RR08 deficiency 42 attenuated TIGR4 in pneumonia, while there was no effect on sepsis. In contrast, lack of 43 HK08 procured a highly virulent TIGR4 phenotype in both pneumonia and sepsis 44 infections. Taken together, these data indicate the importance of TCS08 in 45 pneumococcal fitness to adapt to the milieu of the respiratory tract during colonization. 46 47 48 IMPORTANCE 51 Streptococcus pneumoniae interplays with its environment by using 13 two-52 component regulatory systems and one orphan response regulator. These systems are 53 involved in the sensing of environmental signals thereby modulating pneumococcal 54 pathophysiology. This study aimed to understand the functional role of genes subject to 55 control by the TCS08. The identified genes play a role in transport of compounds such 56 as sugars or amino acids. In addition, the intermediary metabolism and colonization 57 factors are modulated by TCS08. Thus, TCS08 regulates genes involved in maintaining 58 pneumococcal physiology, transport capacity and adhesive factors to enable optimal 59 colonization, which represents a prerequisite for invasive pneumococcal disease.60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Regulatory systems are inherent features of living organisms, ensuring a rapid 76 response and adaptation to diverse environmental conditions and acting as on/off 77 switches for gene expression (1). Regulation in bacteria is predominantly conducted by 78 two-component regulatory systems (TCS), quorum sensing proteins and stand-alone 79 regulators (2-4). TCS are the most common and widespread sensing mechanisms in 80 prokaryotes, functioning by activation of effectors through the auto-phosphorylation of a 81 conserved ...

The two-component regulatory system 09 of Streptococcus pneumoniae has been shown to modulate resistance against oxidative stress as well as capsule expression. These data and the implication of TCS09 in cell wall integrity have been shown for serotype 2 strain D39. Other data have suggested strain-specific regulatory effects of TCS09. Contradictory data are known on the impact of TCS09 on virulence, but all have been explored using only the rr09-mutant. In this study, we have therefore deleted one or both components of the TCS09 (SP_0661 and SP_0662) in serotype 4 S. pneumoniae TIGR4. In vitro growth assays in chemically defined medium (CDM) using sucrose or lactose as a carbon source indicated a delayed growth of nonencapsulated tcs09-mutants, while encapsulated wild-type TIGR4 and tcs09-mutants have reduced growth in CDM with glucose. Using a set of antigen-specific antibodies, immunoblot analysis showed that only the pilus 1 backbone protein RrgB is significantly reduced in TIGR4ΔcpsΔhk09. Electron microscopy, adherence and phagocytosis assays showed no impact of TCS09 on the TIGR4 cell morphology and interaction with host cells. In contrast, in vivo infections and in particular competitive co-infection experiments demonstrated that TCS09 enhances robustness during dissemination in the host by maintaining bacterial fitness.

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