Heterogeneous nucleation is required for crystallization of the ZnuA domain of pneumococcal AdcA

Zn2+ is an essential nutrient for all known forms of life. In the major human pathogen Streptococcus pneumoniae, the acquisition of Zn2+ is facilitated by two Zn2+-specific solute-binding proteins: AdcA and AdcAII. To date, there has been a paucity of structural information on AdcA, which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn2+ acquisition. Native AdcA consists of two domains: an N-terminal ZnuA domain and a C-terminal ZinT domain. In this study, the ZnuA domain of AdcA was crystallized. The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent. No crystals were obtained in the absence of the heterogeneous nucleating agent. These initial crystals were subsequently used as seeds to produce diffraction-quality crystals. The crystals diffracted to 2.03 angstrom resolution and had the symmetry of space group P1. This study demonstrates the utility of heterogeneous nucleation. The solution of the crystal structures will lead to further understanding of Zn2+ acquisition by S. pneumoniae

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

Zinc is an essential element in all domains of life. Nonetheless, how pro-karyotes 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-do-main 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 bacte-rium. 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.</p

Sedentary Behaviors and Biomarkers Among Breast Cancer Survivors.

BackgroundSedentary behavior is associated with increased risk of poor outcomes in breast cancer survivors, but underlying mechanisms are not well understood. This pilot study explored associations between different aspects of sedentary behaviors (sitting, prolonged sitting, sit-to-stand transitions, and standing) and breast cancer risk-related biomarkers in breast cancer survivors (n = 30).MethodsSedentary behavior variables were objectively measured with thigh-worn activPALs. Breast cancer risk-related biomarkers assessed were C-reactive protein (CRP), insulin, and homeostatic model assessment of insulin resistance (HOMA-IR) and were measured in fasting plasma samples. Linear regression models were used to investigate associations between sedentary behavior variables and biomarkers (log CRP, insulin, and HOMA-IR).ResultsSit-to-stand transitions were significantly associated with insulin resistance biomarkers (P &lt; .05). Specifically, each 10 additional sit-to-stand transitions per day was associated with a lower fasting insulin concentration (β = -5.52; 95% CI, -9.79 to -1.24) and a lower HOMA-IR value (β = -0.22; 95% CI, -0.42 to -0.03). Sit-to-stand transitions were not significantly associated with CRP concentration (P = .08). Total sitting time, long sitting bouts, and standing time were not significantly associated with CRP, insulin, or HOMA-IR (P &gt; .05).ConclusionsSit-to-stand transitions may be an intervention target for reducing insulin resistance in breast cancer survivors, which may have favorable downstream effects on cancer prognosis

Sedentary Behaviors and Biomarkers Among Breast Cancer Survivors.

BackgroundSedentary behavior is associated with increased risk of poor outcomes in breast cancer survivors, but underlying mechanisms are not well understood. This pilot study explored associations between different aspects of sedentary behaviors (sitting, prolonged sitting, sit-to-stand transitions, and standing) and breast cancer risk-related biomarkers in breast cancer survivors (n = 30).MethodsSedentary behavior variables were objectively measured with thigh-worn activPALs. Breast cancer risk-related biomarkers assessed were C-reactive protein (CRP), insulin, and homeostatic model assessment of insulin resistance (HOMA-IR) and were measured in fasting plasma samples. Linear regression models were used to investigate associations between sedentary behavior variables and biomarkers (log CRP, insulin, and HOMA-IR).ResultsSit-to-stand transitions were significantly associated with insulin resistance biomarkers (P &lt; .05). Specifically, each 10 additional sit-to-stand transitions per day was associated with a lower fasting insulin concentration (β = -5.52; 95% CI, -9.79 to -1.24) and a lower HOMA-IR value (β = -0.22; 95% CI, -0.42 to -0.03). Sit-to-stand transitions were not significantly associated with CRP concentration (P = .08). Total sitting time, long sitting bouts, and standing time were not significantly associated with CRP, insulin, or HOMA-IR (P &gt; .05).ConclusionsSit-to-stand transitions may be an intervention target for reducing insulin resistance in breast cancer survivors, which may have favorable downstream effects on cancer prognosis

Host subversion of bacterial metallophore usage drives copper intoxication

ABSTRACT Microorganisms can acquire metal ions in metal-limited environments using small molecules called metallophores. While metals and their importers are essential, metals can also be toxic, and metallophores have limited ability to discriminate between metals. The impact of metallophore-mediated non-cognate metal uptake on bacterial metal homeostasis and pathogenesis remains to be defined. The globally significant pathogen Staphylococcus aureus uses the Cnt system to secrete the metallophore staphylopine in zinc-limited host niches. Here, we show that staphylopine and the Cnt system facilitate bacterial copper uptake, potentiating the need for copper detoxification. During in vivo infection, staphylopine usage increased S. aureus susceptibility to host-mediated copper stress, indicating that the innate immune response can harness the antimicrobial potential of altered elemental abundances in host niches. Collectively, these observations show that while the broad-spectrum metal-chelating properties of metallophores can be advantageous, the host can exploit these properties to drive metal intoxication and mediate antibacterial control. IMPORTANCE During infection, bacteria must overcome the dual threats of metal starvation and intoxication. This work reveals that the zinc-withholding response of the host sensitizes S. aureus to copper intoxication. In response to zinc starvation, S. aureus utilizes the metallophore staphylopine. The current work revealed that the host can leverage the promiscuity of staphylopine to intoxicate S. aureus during infection. Significantly, staphylopine-like metallophores are produced by a wide range of pathogens, suggesting that this is a conserved weakness that the host can leverage to toxify invaders with copper. Moreover, it challenges the assumption that the broad-spectrum metal binding of metallophores is inherently beneficial to bacteria

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

The structure and metal binding properties of Chlamydia trachomatis YtgA

The obligate intracellular pathogen 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 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 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 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. 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 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 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