Disruption of Cell-Cell Adhesion Codes Underlies the Unique X-linked Inheritance Pattern of Protocadherin 19 Girls Clustering Epilepsy

Epilepsy is a disease of the central nervous system (CNS) caused by increased neuronal activity resulting in seizures and often loss of consciousness. Epilepsy can result from both traumas to the brain or a genetic predisposition. Recent advances in DNA sequencing technology has identified many forms of epilepsy caused by mutations in single genes. Although such monogenic epilepsies are rare, investigating their underlying molecular mechanisms provides important insights into pathways and processes that cause seizures and assists in the application of pharmacotherapy and surgical strategies. The second most common form of monogenic epilepsy is Protocadherin 19 Girls Clustering Epilepsy (PCDH19-GCE) caused by mutation of the X-linked gene PCDH19. This disorder is characterised by clusters of febrile seizures beginning in early childhood that is often accompanied by variable intellectual disability and autism spectrum disorder. The most striking feature of PCDH19-GCE is its unique X-linked inheritance pattern; heterozygous females with PCDH19 mutations are affected whereas hemizygous males are not. It is hypothesised that the mixture of PCDH19-WT and PCDH19-mutant neurons (generated by random X-inactivation in female brains) causes abnormal neuronal connections leading to disease. However, there is no experimental evidence supporting this hypothesis and the cellular and molecular mechanisms underpinning this unique inheritance pattern are unknown. To better understand how mutation of PCDH19 leads to the unique X-linked inheritance pattern this thesis uses a Pcdh19 null mouse, cell culture assays and unique CRISPR-Cas9 engineered mouse models. It is shown that heterozygous and homozygous deletion of Pcdh19 in mice does not cause any gross brain morphological abnormalities and that Pcdh19 null neurons are present within the correct layers in the cortex despite their slight increase in migration potential in vitro. Using cultured K562 cells it is shown that PCDH19 and other non-clustered (NC) PCDH members contribute to combinatorial adhesion codes that dictate specific cell-cell interactions. Similarly, mosaic expression of Pcdh19 in heterozygous mice leads to abnormal cell sorting in the developing cortex such that cells separate into PCDH19 positive and negative patches, correlating with altered brain network activity consistent with changes that can underlie seizures in adult mice. Deletion of Pcdh19 in heterozygous embryos using CRISPR-Cas9 technology eliminates this incompatibility of adhesion codes and prevents abnormal cell sorting from occurring. In addition, variable cortical folding malformations in PCDH19-GCE epilepsy patients were identified. Collectively these results highlight the role of PCDH19 in determining specific adhesion codes during brain development and how disruption of these codes is associated with the unique X-linked inheritance pattern of PCDH19-GCE. Importantly, a framework is provided for investigating how this abnormal neuronal segregation phenotype leads to seizures and cognitive deficits.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

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

Zinc stress induces copper depletion in Acinetobacter baumannii

Background: The first row transition metal ions zinc and copper are essential to the survival of many organisms, although in excess these ions are associated with significant toxicity. Here, we examined the impact of zinc and copper stress on Acinetobacter baumannii, a common opportunistic pathogen. Results: We show that extracellular zinc stress induces a copper-specific depletion phenotype in A. baumannii ATCC 17978. Supplementation with copper not only fails to rescue this phenotype, but further exacerbates the copper depletion. Extensive analysis of the A. baumannii ATCC 17978 genome identified 13 putative zinc/copper resistance efflux pumps. Transcriptional analyses show that four of these transporters are responsive to zinc stress, five to copper stress and seven to the combination of zinc and copper stress, thereby revealing a likely foundation for the zinc-induced copper starvation in A. baumannii. In addition, we show that zinc and copper play crucial roles in management of oxidative stress and the membrane composition of A. baumannii. Further, we reveal that zinc and copper play distinct roles in macrophage-mediated killing of this pathogen. Conclusions: Collectively, this study supports the targeting of metal ion homeostatic mechanisms as an effective antimicrobial strategy against multi-drug resistant bacterial pathogens.Karl A. Hassan, Victoria G. Pederick, Liam D. H. Elbourne, Ian T. Paulsen, James C. Paton, Christopher A. McDevitt and Bart A. Eijkelkam

Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae.

Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes

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

Arachidonic acid stress impacts pneumococcal fatty acid homeostasis

Published: 11 May 2018Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.Bart A. Eijkelkamp, Stephanie L. Begg, Victoria G. Pederick, Claudia Trapetti, Melissa K. Gregory, Jonathan J. Whittall, James C. Paton and Christopher A. McDevit

A Trap-Door Mechanism for Zinc Acquisition by Streptococcus pneumoniae AdcA.

Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes. IMPORTANCE Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined. We report the first structure of a two-domain lipoprotein, AdcA from S. pneumoniae, and use computational, spectroscopic, and microbiological approaches to provide new insights into the functional basis of zinc recruitment. Our findings reveal that AdcA employs a novel mechanism for zinc binding that we have termed the “trap-door” mechanism, and we show how the static metal-binding site of the protein, which confers its selectivity for zinc ions, is combined with a dynamic surface element to facilitate zinc recruitment and import into the bacterium. Together, these findings expand our understanding of how bacteria acquire zinc from the environment and provide a foundation for inhibiting this process, through antimicrobial targeting of the dynamic structural elements to block bacterial zinc scavenging

The role of the copA copper efflux system in acinetobacter baumannii virulence

Acinetobacter baumannii has emerged as one of the leading causative agents of nosocomial infections. Due to its high level of intrinsic and adapted antibiotic resistance, treatment failure rates are high, which allows this opportunistic pathogen to thrive during infection in immune-compromised patients. A. baumannii can cause infections within a broad range of host niches, with pneumonia and bacteraemia being associated with the greatest levels of morbidity and mortality. Although its resistance to antibiotics is widely studied, our understanding of the mechanisms required for dealing with environmental stresses related to virulence and hospital persistence, such as copper toxicity, is limited. Here, we performed an in silico analysis of the A. baumannii copper resistome, examining its regulation under copper stress. Using comparative analyses of bacterial P-type ATPases, we propose that A. baumannii encodes a member of a novel subgroup of P1B-1 ATPases. Analyses of three putative inner membrane copper efflux systems identified the P1B-1 ATPase CopA as the primary mediator of cytoplasmic copper resistance in A. baumannii. Using a murine model of A. baumannii pneumonia, we reveal that CopA contributes to the virulence of A. baumannii. Collectively, this study advances our understanding of how A. baumannii deals with environmental copper toxicity, and it provides novel insights into how A. baumannii combats adversities encountered as part of the host immune defence.Saleh F. Alquethamy, Marjan Khorvash , Victoria G. Pederick, Jonathan J. Whittall, James C. Paton, Ian T. Paulsen, Karl A. Hassan, Christopher A. McDevitt and Bart A. Eijkelkam

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

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