Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Maternal selenium dietary supplementation alters sociability and reinforcement learning deficits induced by in utero exposure to maternal immune activation in mice.

Maternal immune activation (MIA) during pregnancy increases the risk for the unborn foetus to develop neurodevelopmental conditions such as autism spectrum disorder and schizophrenia later in life. MIA mouse models recapitulate behavioural and biological phenotypes relevant to both conditions, and are valuable models to test novel treatment approaches. Selenium (Se) has potent anti-inflammatory properties suggesting it may be an effective prophylactic treatment against MIA. The aim of this study was to determine if Se supplementation during pregnancy can prevent adverse effects of MIA on offspring brain and behaviour in a mouse model. Selenium was administered via drinking water (1.5 ppm) to pregnant dams from gestational day (GD) 9 to birth, and MIA was induced at GD17 using polyinosinic:polycytidylic acid (poly-I:C, 20 mg/kg via intraperitoneal injection). Foetal placenta and brain cytokine levels were assessed using a Luminex assay and brain elemental nutrients assessed using inductively coupled plasma- mass spectrometry. Adult offspring were behaviourally assessed using a reinforcement learning paradigm, the three-chamber sociability test and the open field test. MIA elevated placental IL-1ß and IL-17, and Se supplementation successfully prevented this elevation. MIA caused an increase in foetal brain calcium, which was prevented by Se supplement. MIA caused in offspring a female-specific reduction in sociability, which was recovered by Se, and a male-specific reduction in social memory, which was not recovered by Se. Exposure to poly-I:C or selenium, but not both, reduced performance in the reinforcement learning task. Computational modelling indicated that this was predominantly due to increased exploratory behaviour, rather than reduced rate of learning the location of the food reward. This study demonstrates that while Se may be beneficial in ameliorating sociability deficits caused by MIA, it may have negative effects in other behavioural domains. Caution in the use of Se supplementation during pregnancy is therefore warranted.

Spatial distribution of trace metals and associated transport proteins during bacterial infection.

Innate immune systems alter the concentrations of trace elements in host niches in response to invading pathogens during infection. This work reports the interplay between d-block metal ions and their associated biomolecules using hyphenated elemental techniques to spatially quantify both elemental distributions and the abundance of specific transport proteins. Here, lung tissues were collected for analyses from naïve and Streptococcus pneumoniae-infected mice fed on a zinc-restricted or zinc-supplemented diet. Spatiotemporal distributions of manganese (55Mn), iron (56Fe), copper (63Cu), and zinc (66Zn) were determined by quantitative laser ablation-inductively coupled plasma-mass spectrometry. The murine transport proteins ZIP8 and ZIP14, which are associated with zinc transport, were also imaged by incorporation of immunohistochemistry techniques into the analytical workflow. Collectively, this work demonstrates the potential of a single instrumental platform suitable for multiplex analyses of tissues and labelled antibodies to investigate complex elemental interactions at the host-pathogen interface. Further, these methods have the potential for broad application to investigations of biological pathways where concomitant measurement of elements and biomolecules is crucial to understand the basis of disease and aid in development of new therapeutic approaches.

Comparison of contemporary invasive and non-invasive Streptococcus pneumoniae isolates reveals new insights into circulating anti-microbial resistance determinants.

Streptococcus pneumoniae is a major human pathogen with a high burden of disease. Non-invasive isolates (those found in non-sterile sites) are thought to be a key source of invasive isolates (those found in sterile sites) and a reservoir of anti-microbial resistance (AMR) determinants. Despite this, pneumococcal surveillance has almost exclusively focused on invasive isolates. We aimed to compare contemporaneous invasive and non-invasive isolate populations to understand how they interact and identify differences in AMR gene distribution. We used a combination of whole-genome sequencing and phenotypic anti-microbial susceptibility testing and a data set of invasive (n = 1,288) and non-invasive (n = 186) pneumococcal isolates, collected in Victoria, Australia, between 2018 and 2022. The non-invasive population had increased levels of antibiotic resistance to multiple classes of antibiotics including beta-lactam antibiotics penicillin and ceftriaxone. We identified genomic intersections between the invasive and non-invasive populations and no distinct phylogenetic clustering of the two populations. However, this analysis revealed sub-populations overrepresented in each population. The sub-populations that had high levels of AMR were overrepresented in the non-invasive population. We determined that WamR-Pneumo was the most accurate in silico tool for predicting resistance to the antibiotics tested. This tool was then used to assess the allelic diversity of the penicillin-binding protein genes, which acquire mutations leading to beta-lactam antibiotic resistance, and found that they were highly conserved (≥80% shared) between the two populations. These findings show the potential of non-invasive isolates to serve as reservoirs of AMR determinants.

Copper(II) Complexes of 2,2'-Bisdipyrrins: Synthesis, Characterization, Cell Uptake, and Radiolabeling with Copper-64.

Complexes prepared with positron-emitting copper-64 are of interest as imaging agents for positron emission tomography (PET). This work investigates the potential of using acyclic tetrapyrrolic 2,2'-bisdipyrrins as ligands to prepare charge-neutral, lipophilic, cell-permeable, redox active complexes with positron-emitting copper-64. The synthesis and characterization of a series of tetrapyrrolic 2,2'-bisdipyrrin copper(II) complexes are reported. Four 2,2'-bisdipyrrin copper(II) complexes were prepared with different functional groups in the meso-position of the ligands. Two of the new copper(II) complexes, one palladium(II) complex, and one nickel(II) complex were characterized by X-ray crystallography, which demonstrated that the copper(II) is in a distorted square planar environment. An investigation of the electrochemical properties of the complexes by cyclic voltammetry revealed that the complexes undergo multiple quasi-reversible processes. A comparison of the cyclic voltammetry of the copper complexes with their palladium(II) analogues suggests that these redox processes are ligand-based and not metal-based. The copper(II) complexes are cell-permeable in A431 mammalian cells and are nontoxic at concentrations of 50 µM. The ligands can be radiolabeled with copper-64 at room temperature.

Dietary zinc and the control of Streptococcus pneumoniae infection.

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.

The Molecular Basis of Acinetobacter baumannii Cadmium Toxicity and Resistance.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii, which is notorious for persisting in harsh environments. Here, we show that A. baumannii utilizes a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

Intracellular Accumulation of Staphylopine Can Sensitize Staphylococcus aureus to Host-Imposed Zinc Starvation by Chelation-Independent Toxicity.

The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant.IMPORTANCEStaphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.

Disruption of Phosphate Homeostasis Sensitizes Staphylococcus aureus to Nutritional Immunity.

To control infection, mammals actively withhold essential nutrients, including the transition metal manganese, by a process termed nutritional immunity. A critical component of this host response is the manganese-chelating protein calprotectin. While many bacterial mechanisms for overcoming nutritional immunity have been identified, the intersection between metal starvation and other essential inorganic nutrients has not been investigated. Here, we report that overexpression of an operon encoding a highly conserved inorganic phosphate importer, PstSCAB, increases the sensitivity of Staphylococcus aureus to calprotectin-mediated manganese sequestration. Further analysis revealed that overexpression of pstSCAB does not disrupt manganese acquisition or result in overaccumulation of phosphate by S. aureus However, it does reduce the ability of S. aureus to grow in phosphate-replete defined medium. Overexpression of pstSCAB does not aberrantly activate the phosphate-responsive two-component system PhoPR, nor was this two-component system required for sensitivity to manganese starvation. In a mouse model of systemic staphylococcal disease, a pstSCAB-overexpressing strain is significantly attenuated compared to wild-type S. aureus This defect is partially reversed in a calprotectin-deficient mouse, in which manganese is more readily available. Given that expression of pstSCAB is regulated by PhoPR, these findings suggest that overactivation of PhoPR would diminish the ability of S. aureus to resist nutritional immunity and cause infection. As PhoPR is also necessary for bacterial virulence, these findings imply that phosphate homeostasis represents a critical regulatory node whose activity must be precisely controlled in order for S. aureus and other pathogens to cause infection.

Structure and Metal Binding Properties of Chlamydia trachomatis YtgA.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-Å resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCEChlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

Synergy between Nutritional Immunity and Independent Host Defenses Contributes to the Importance of the MntABC Manganese Transporter during Staphylococcus aureus Infection.

During infection, the host utilizes a diverse array of processes to combat invaders, including the restriction of availability of essential nutrients such as manganese. Similarly to many other pathogens, Staphylococcus aureus possesses two manganese importers, MntH and MntABC. Several infection models have revealed a critical role for MntABC during staphylococcal infection. However, culture-based studies have suggested parity between the two transporters when cells are resisting manganese starvation imposed by the manganese binding immune effector calprotectin. In this investigation, initial elemental analysis revealed that MntABC is the primary transporter responsible for obtaining manganese in culture in the presence of calprotectin. MntABC was also necessary to maintain wild-type levels of manganese-dependent superoxide dismutase activity in the presence of calprotectin. Building on this framework, we investigated if MntABC enabled S. aureus to resist the synergistic actions of nutritional immunity and other host defenses. This analysis revealed that MntABC critically contributes to staphylococcal growth when S. aureus is subjected to manganese limitations and exposed to oxidative stress. This transporter was also important for growth in manganese-limited environments when S. aureus was forced to consume glucose as an energy source, which occurs when it encounters nitric oxide. MntABC also expanded the pH range conducive for S. aureus growth under conditions of manganese scarcity. Collectively, the data presented in this work provide a robust molecular basis for the crucial role of MntABC in staphylococcal virulence. Further, this work highlights the importance of synergy between host defenses and the necessity of evaluating the contribution of virulence factors to pathogenesis in the presence of multiple stressors.

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.

The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity.

Zinc is an essential trace element that serves as a catalytic cofactor in metalloenzymes and a structural element in proteins involved in general metabolism and cellular defenses of pathogenic bacteria. Despite its importance, high zinc levels can impair cellular processes, inhibiting growth of many pathogenic bacteria, including the major respiratory pathogen Streptococcus pneumoniae. Zinc intoxication is prevented in S. pneumoniae by expression of the zinc exporter CzcD, whose expression is activated by the novel TetR-family transcriptional zinc-sensing regulator SczA. How zinc bioavailability triggers activation of SczA is unknown. It is shown here through functional studies in S. pneumoniae that an unannotated homodimeric TetR from S. agalactiae (PDB 3KKC) is the bona fide zinc efflux regulator SczA, and binds two zinc ions per protomer. Mutagenesis analysis reveals two metal binding sites, termed A and B, located on opposite sides of the SczA C-terminal regulatory domain. In vivo, the A- and B-site SczA mutant variants impact S. pneumoniae resistance to zinc toxicity and survival in infected macrophages. A model is proposed for S. pneumoniae SczA function in which both A- and B-sites were required for transcriptional activation of czcD expression, with the A-site serving as the evolutionarily conserved intracellular sensing site in SczAs.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin.

Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus. Unlike E. coli that could fulfill its biotin requirements through de novo synthesis, S. aureus showed improved growth rates in media supplemented with 10 nM biotin. S. aureus also accumulated more radiolabeled biotin from the media highlighting its ability to efficiently scavenge exogenous material. These data are consistent with S. aureus colonizing low biotin microhabitats. We also demonstrate that the S. aureus BirA protein is a transcriptional repressor of BioY, a subunit of the biotin transporter, and an operon containing yhfT and yhfS, the products of which have a putative role in fatty acid homeostasis. Increased expression of bioY is proposed to help cue S. aureus for efficient scavenging in low biotin environments.

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.

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.

Imperfect coordination chemistry facilitates metal ion release in the Psa permease.

The relative stability of divalent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other divalent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for divalent cations during competition from zinc ions, which are more abundant in biological systems.

Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids.

ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein.

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae.

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.