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. 2017 Feb 27:7:43587.
doi: 10.1038/srep43587.

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

Firas A Y Al-Bayati  1   2 , Hasan F H Kahya  1   2 , Andreas Damianou  1 , Sulman Shafeeq  3 , Oscar P Kuipers  3 , Peter W Andrew  1 , Hasan Yesilkaya  1
Affiliations

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

Firas A Y Al-Bayati et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
EMSA analysis showing the direct interaction of CcpA (a and b), GlnR (c and d), with PpflB or PpflA, respectively. Each lane contains approximately 30 ng PpflB or PpflA. CcpA and GlnR were used between 0.1 to 0.5 μM. The coding sequence of gyrB (30 ng) was used as a negative control. Gels were stained with SYBR Green EMSA for visualizing DNA.
Figure 2
Figure 2. EMSA analysis showing the impact of formate on binding affinity of CcpA and GlnR.
Sodium formate enhanced CcpA- and decreased GlnR affinity for PpflB (b and d, respectively) compared to CcpA (a), and GlnR (c) interaction without sodium formate. Each lane contains approximately 30 ng PpflB; 0.2–1.5 μM of CcpA, or GlnR with or without 10 mM sodium formate. The experiment was repeated three times, and a representative image is shown.
Figure 3
Figure 3
EMSA analysis showing sequence specificity of CcpA, and GlnR for the cre1 sites in (a) PpflB and (b) PpflA. Each lane contains approximately 30 ng of promoter probes, and 0.5 μM of CcpA or GlnR.
Figure 4
Figure 4
Expression levels (in Miller Units) of pneumococcal transcriptional lacZ-fusions to the promoters of pflA (a and c) and pflB (b and d) in different backgrounds grown anaerobically in CDM supplemented with 55 mM of galactose (a and b) or glucose (c and d). The activity is expressed as nmol p-nitrophenol/min/ml. Error bars show the standard error of the mean for three individual measurements each with three replicates. ***p < 0.001, ****p < 0.0001.
Figure 5
Figure 5
(a) Schematic representation showing the analysis of predicted promoter region and binding sites of PccpA, and (b) EMSA analysis showing the direct interaction of CcpA and GlnR with PccpA. (a) The core promoter region containing the −10 and −35 elements is indicated. The putative cre sequences are indicated in red. F: indicates forward primer while R: refers to the reverse primer used for amplifying the promoter probe. T: potential terminator structure. The black arrow presents the direction of transcription. (b) Each lane contains approximately 30 ng PccpA; 0.5 μM of CcpA or GlnR as appropriate.
Figure 6
Figure 6. Pneumococcal growth and fermentation end products after culture anaerobically in CDM supplemented with different sugars.
(a) shows growth in 55 mM glucose and (b) in galactose. (c) and (d) show fermentation end product profiles after growth on glucose and galactose, respectively. Error bars show the standard error of the mean for three individual measurements each with three replicates. Significant differences were seen comparing the growth rates, and the fermentative profile of mutant strains to the wild type D39 using ANOVA followed by Dunnett’s multiple comparison test. **p < 0.01 and ****p < 0.0001.
Figure 7
Figure 7. Contribution of CcpA and GlnR to pneumococcal virulence and colonization.
(a) Survival time of mice infected intranasally with approximately 2 × 106 CFU pneumococci. Each dot represents the survival time of individual animal, and the horizontal bars mark the median survival times derived from 10 animals. (b) Progression of bacteraemia in mice infected intranasally with ΔccpA, ΔglnR and their derivatives at 24 h and 36 h post-infection. Each point is the mean of data from ten mice. (c) Pneumococcal strains defective in ccpA and glnR were less able to colonize nasopharynx. Mice were infected approximately with 1 × 105 CFU pneumococci. At day 0 and day 7, five mice were culled, and bacterial CFU/mg were determined by serial dilutions of nasopharyngeal homogenates. Each column represents the mean of data from five mice. Error bars show the standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 8
Figure 8. Schematic model of pflB and pflA regulation.
On galactose, formate production by PFL activity decreases GlnR affinity for PpflB and GlnR increases ccpA transcription. The increased formate production increases CcpA affinity for PpflB, and CcpA either alone or after interacting with GlnR binds to PpflB and increases pflB transcription. However, CcpA also binds to PpflA and represses its expression to fine-tune the level of active PFL. Increased formate production then creates a positive feed back loop, and CcpA self-regulates its own expression. Glu: glucose, Gal: galactose, SF: sodium formate. The green arrow indicates increase in expression, whereas the red arrow is for decreased expression. *Posttranslational activation of PFL by PFL-AE has been previously reported.
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