Cellular Mn/Zn Ratio Influences Phosphoglucomutase Activity and Capsule Production in Streptococcus pneumoniae D39

Abstract

Capsular polysaccharide (CPS) is a major virulence determinant for human pathogenic bacteria. Although the essential functional roles for CPS in bacterial virulence have been established, knowledge of how CPS production is regulated remains limited. Streptococcus pneumoniae (pneumococcus) CPS expression levels and overall thickness change in response to available oxygen and carbohydrate. These nutrients in addition to transition metal ions can vary significantly between host environmental niches and infection stage. Since pneumococcus must modulate CPS expression among various host niches during disease progression, we examined the impact of nutritional transition metal availability of manganese (Mn) and zinc (Zn) on CPS production. We demonstrate that increased Mn/Zn ratio increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway in a transcription-independent manner. Further, we find that the downstream CPS protein CpsB, a Mn-dependent phosphatase, does not promote aberrant dephosporylation of its target capsule-tyrosine kinase CpsD during Mn-stress. Together, these data reveal a direct role for cellular Mn/Zn ratios in the regulation of CPS biosynthesis via direct activation of Pgm. We propose a multilayer mechanism used by the pneumococcus in regulating CPS levels across various host niches. IMPORTANCE Evolving evidence strongly indicates that maintenance of metal homeostasis is essential for establishing colonization and continued growth of bacterial pathogens in the vertebrate host. In this study, we demonstrate the impact of cellular manganese/zinc (Mn/Zn) ratios on bacterial capsular polysaccharide (CPS) production, an important virulence determinant of many human pathogenic bacteria, including Streptococcus pneumoniae. We show that higher Mn/Zn ratios increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway. The findings provide a direct role for Mn/Zn homeostasis in the regulation of CPS expression levels and further support the ability for metal cations to act as important cellular signaling mediators in bacteria.

Regulation of Bacterial Manganese Homeostasis and Usage During Stress Responses and Pathogenesis

“…Deletion of cvfD caused increased transcription of the Mn-specific ABC-importer psaBCA and the Zn exporter czcD. Mn supplementation rescued the cvfD growth defect and restored capsule formation, which is dependent on the Mn:Zn ratio (McFarland et al, 2021), suggesting that CvfD helps maintain intracellular Mn:Zn balance. It is not yet known whether CvfD affects mRNA levels directly or by mediating sRNA base-pairing to target mRNAs.…”

“…For some enzymes (Fe, Mg, or apoenzymes), mismetallation with Mn can lead to inactivation or poor activity (e.g., ferrochelatase, SodA, NrdAB (Cotruvo and Stubbe, 2012;Martin et al, 2015)). For other enzymes, binding with Mn can cause inappropriate activation or hyperactivation (e.g., PhpP, CpsB, Pgm (Martin et al, 2017b;McFarland et al, 2021)), which can lead to increased capsular polysaccharide (CPS) and cell elongation. (Right) During oxidative stress, Mn uptake is stimulated (Imlay, 2014).…”

“…Fluctuating intracellular Mn levels serve as an important role post-translationally, after nascent Mn-using proteins are synthesized. 7) The intracellular labile metal pool can influence the metallation status and activity of Mn-enzymes, such as PhpP, CspB, and Pgm (Martin et al, 2017b;McFarland et al, 2021). Such enzymes are active when bound to Mn, but become inactive with loss of metal or when replaced with Zn; reduced activity is observed with other metal cations.…”

“…Historically, Mn:Fe balance has been studied in Fe-centric bacteria like E. coli where the intracellular Mn:Fe ratio is ≤ 0.1 and there is a larger number of Fe-using proteins (Bosma et al, 2021). S. pneumoniae, and possibly other Mn-centric bacteria with an intracellular Mn:Fe ratio ≥1, is also toxified by excess Mn, but via hyperactivation of Mn-enzymes (Martin et al, 2017b;McFarland et al, 2021). In S. pneumoniae, elevated Mn increases intracellular Fe levels during microaerophilic growth but significantly decreases Fe levels during anaerobiosis (Martin et al, 2017b).…”

Metal Homeostasis in Pathogenic Streptococci

“…A specific Mn-dependent enzyme called phosphoglucomutase (Pgm) is involved in capsular polysaccharide production and is influenced by not only Mn, but also Zn concentrations [ 125 ]. An example of this was shown in S. pneumoniae , where a low Mn/high Zn ratio resulted in inactive Pgm and a thinner capsule [ 126 ]. Fe is used by most organisms for a wide range of metabolic and informational cellular pathways including electron transport, peroxide reduction, and amino acid synthesis.…”