The First Histidine Triad Motif of PhtD Is Critical for Zinc Homeostasis in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination of in vitro growth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the amino-and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence.

Structural characterisation of the HT3 motif of the polyhistidine triad protein D from Streptococcus pneumoniae

“…Examination of the PhtD solution structure and the homology models of HT2, HT4 and HT5 regions reveals that, similar to PhtD 269-339 , all these fragments possess charged surfaces and would suggest protein surface localization. This structural composition would be consistent with the contribution of these motifs to Zn 2+ acquisition at the pathogen-host interface [11,15]. Notably, the histidine triad residues in HT3 are on a b-hairpin, and as such it would be expected that the conformation of this region would be largely stable in the absence of Zn 2+ .…”

“…Recent work by Eijkelkamp et al . revealed a hierarchy for the five HT motifs of PhtD with respect to their importance for in vitro Zn 2+ acquisition. That work showed that HT1 of PhtD, which is closest to the pneumococcal cell membrane, was the most important, whereas HT5 had the smallest contribution.…”

“…However, in a complex protein, such as PhtD, it is not unreasonable to expect cooperativity between the HT motifs and allosteric interactions between sites that may influence Zn 2+ binding. Recent work by Eijkelkamp et al [15] revealed a hierarchy for the five HT motifs of PhtD with respect to their importance for in vitro Zn 2+ acquisition. That work showed that HT1 of PhtD, which is closest to the pneumococcal cell membrane, was the most important, whereas HT5 had the smallest contribution.…”

Comparative genomics and transcriptomics depict ericoid mycorrhizal fungi as versatile saprotrophs and plant mutualists

“…methyltransferases, zinc finger transcriptional factors and deoxycytidylate deaminases). Proteins specific to ERM fungi included enzymes involved in resistance to environmental stress, such as isochorismate synthase (Sadeghi et al, 2013) and histidine triad domain-containing proteins (Eijkelkamp et al, 2016).…”

Opening the OPK Assay Gatekeeper: Harnessing Multi-Modal Protection by Pneumococcal Vaccines

“…The difficulty of implementing this requirement was illustrated by a recent Phase II clinical trial of a combination of 10 polysaccharides with the histidine triad protein PhtD and a pneumolysin toxoid [12]. PhtD is believed to participate in zinc homeostasis in the nasopharynx, and the toxin pneumolysin causes damage to respiratory epithelial cells during pneumonia; both vaccine antigens decreased nasopharyngeal carriage in preclinical models [13,14]. However, the only functional assay developed to test sera after this combined vaccine was not directed to mucosal activity, but rather focused on assessment of complement-mediated killing by leukocytes [15].…”