The first histidine triad motif of phtd is critical for zinc homeostasis in Streptococcus pneumoniae

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

Pcdh19 loss-of-function increases neuronal migration in vitro but is dispensable for brain development in mice

Protocadherin 19 (Pcdh19) is an X-linked gene belonging to the protocadherin superfamily, whose members are predominantly expressed in the central nervous system and have been implicated in cell-cell adhesion, axon guidance and dendrite self-avoidance. Heterozygous loss-of-function mutations in humans result in the childhood epilepsy disorder PCDH19 Girls Clustering Epilepsy (PCDH19 GCE) indicating that PCDH19 is required for brain development. However, understanding PCDH19 function in vivo has proven challenging and has not been studied in mammalian models. Here, we validate a murine Pcdh19 null allele in which a β-Geo reporter cassette is expressed under the control of the endogenous promoter. Analysis of β-Geo reporter activity revealed widespread but restricted expression of PCDH19 in embryonic, postnatal and adult brains. No gross morphological defects were identified in Pcdh19(+/β-Geo) and Pcdh19(Y/β-Geo) brains and the location of Pcdh19 null cells was normal. However, in vitro migration assays revealed that the motility of Pcdh19 null neurons was significantly elevated, potentially contributing to pathogenesis in patients with PCDH19 mutations. Overall our initial characterization of Pcdh19(+/β-Geo), Pcdh19(β-Geo/β-Geo) and Pcdh19(Y/β-Geo)mice reveals that despite widespread expression of Pcdh19 in the CNS, and its role in human epilepsy, its function in mice is not essential for brain development.Daniel T. Pederick, Claire C. Homan, Emily J. Jaehne, Sandra G. Piltz, Bryan P. Haines, Bernhard T. Baune, Lachlan A. Jolly, James N. Hughes, Jozef Gecz, Paul Q. Thoma

Dietary zinc and the control of Streptococcus pneumoniae infection

© 2019 Eijkelkamp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

ZnuA and zinc homeostasis in pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and a clinically significant opportunistic human pathogen. Central to the ability of P. aeruginosa to colonise both environmental and host niches is the acquisition of zinc. Here we show that P. aeruginosa PAO1 acquires zinc via an ATP-binding cassette (ABC) permease in which ZnuA is the high affinity, zinc-specific binding protein. Zinc uptake in Gram-negative organisms predominantly occurs via an ABC permease, and consistent with this expectation a P. aeruginosa ΔznuA mutant strain showed an ~60% reduction in cellular zinc accumulation, while other metal ions were essentially unaffected. Despite the major reduction in zinc accumulation, minimal phenotypic differences were observed between the wild-type and ΔznuA mutant strains. However, the effect of zinc limitation on the transcriptome of P. aeruginosa PAO1 revealed significant changes in gene expression that enable adaptation to low-zinc conditions. Genes significantly up-regulated included non-zinc-requiring paralogs of zinc-dependent proteins and a number of novel import pathways associated with zinc acquisition. Collectively, this study provides new insight into the acquisition of zinc by P. aeruginosa PAO1, revealing a hitherto unrecognized complexity in zinc homeostasis that enables the bacterium to survive under zinc limitation.Victoria G. Pederick, Bart A. Eijkelkamp, Stephanie L. Begg, Miranda P. Ween, Lauren J. McAllister, James C. Paton, Christopher A. McDevit

Functional equivalence of the SOX2 and SOX3 transcription factors in the developing mouse brain and testes

Gene duplication provides spare genetic material that evolution can craft into new functions. Sox2 and Sox3 are evolutionarily related genes with overlapping and unique sites of expression during embryogenesis. It is currently unclear whether SOX2 and SOX3 have identical or different functions. Here, we use CRISPR/Cas9-assisted mutagenesis to perform a gene-swap, replacing the Sox3 ORF with the Sox2 ORF to investigate their functional equivalence in the brain and testes. We show that increased expression of SOX2 can functionally replace SOX3 in the development of the infundibular recess/ventral diencephalon, and largely rescues pituitary gland defects that occur in Sox3 null mice. We also show that ectopic expression of SOX2 in the testes functionally rescues the spermatogenic defect of Sox3 null mice, and restores gene expression to near normal levels. Together, these in vivo data provide strong evidence that SOX2 and SOX3 proteins are functionally equivalent.Fatwa Adikusuma, Daniel Pederick, Dale McAninch, James Hughes and Paul Thoma

Crystal Structure of Bovine Alpha-Chymotrypsin in Space Group P6₅

Chymotrypsin is a protease that is commonly used as a standard for protein crystallization and as a model system for studying serine proteases. Unliganded bovine α-chymotrypsin was crystallized at neutral pH using ammonium sulphate as the precipitant, resulting in crystals that conform to P65 symmetry with unit cell parameters that have not been reported previously. Inspection of crystallographic interfaces revealed that the major interface between any two molecules in the crystal lattice represents the interface of the biological dimer, as previously observed for crystals of unliganded α-chymotrypsin grown at low pH in space group P21