TprA/PhrA Quorum Sensing System Has a Major Effect on Pneumococcal Survival in Respiratory Tract and Blood, and Its Activity Is Controlled by CcpA and GlnR.

Streptococcus pneumoniae is able to cause deadly diseases by infecting different tissues, each with distinct environmental and nutritional compositions. We hypothesize that the adaptive capabilities of the microbe is an important facet of pneumococcal survival in fluctuating host environments. Quorum-sensing (QS) mechanisms are pivotal for microbial host adaptation. We previously demonstrated that the TprA/PhrA QS system is required for pneumococcal utilization of galactose and mannose, neuraminidase activity, and virulence. We also showed that the system can be modulated by using linear molecularly imprinted polymers. Due to being a drugable target, we further studied the operation of this QS system in S. pneumoniae. We found that TprA controls the expression of nine different operons on galactose and mannose. Our data revealed that TprA expression is modulated by a complex regulatory network, where the master regulators CcpA and GlnR are involved in a sugar dependent manner. Mutants in the TprA/PhrA system are highly attenuated in their survival in nasopharynx and lungs after intranasal infection, and growth in blood after intravenous infection.

Pneumococcal galactose catabolism is controlled by multiple regulators acting on pyruvate formate lyase.

Catabolism of galactose by Streptococcus pneumoniae alters the microbe's metabolism from homolactic to mixed acid fermentation, and this shift is linked to the microbe's virulence. However, the genetic basis of this switch is unknown. Pyruvate formate lyase (PFL) is a crucial enzyme for mixed acid fermentation. Functional PFL requires the activities of two enzymes: pyruvate formate lyase activating enzyme (coded by pflA) and pyruvate formate lyase (coded by pflB). To understand the genetic basis of mixed acid fermentation, transcriptional regulation of pflA and pflB was studied. By microarray analysis of ΔpflB, differential regulation of several transcriptional regulators were identified, and CcpA, and GlnR's role in active PFL synthesis was studied in detail as these regulators directly interact with the putative promoters of both pflA and pflB, their mutation attenuated pneumococcal growth, and their expression was induced on host-derived sugars, indicating that these regulators have a role in sugar metabolism, and multiple regulators are involved in active PFL synthesis. We also found that the influence of each regulator on pflA and pflB expression was distinct in terms of activation and repression, and environmental condition. These results show that active PFL synthesis is finely tuned, and feed-back inhibition and activation are involved.

Lactate dehydrogenase is the key enzyme for pneumococcal pyruvate metabolism and pneumococcal survival in blood.

Streptococcus pneumoniae is a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenic ldh mutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed by in vivo nuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of the ldh mutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.