Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.
Abstract Calcium and the proteins that bind to it play important roles in normal physiological processes and have been implicated in a variety of diseases. The importance of calcium is due mainly to its role as a second messenger in signal transduction. The calcium signal is mediated and controlled by many proteins, the majority of which belong to the EF‐hand superfamily of calcium‐binding proteins. EF‐hand proteins are classified into calcium signal sensors and modulators. The signal modulators fine‐tune the shape and duration of calcium signals. The calcium sensors undergo significant conformational changes when they bind calcium, which exposes new surfaces that interact with target proteins. Together, EF‐hand calcium‐binding proteins serve in the critical process of converting the ionic signal into activation of intracellular signalling pathways. Key Concepts: The EF‐hand is a helix–loop–helix structural motif. Calcium binds to oxygen atoms from the backbone and side‐chain atoms of specific amino acids in EF‐hand calcium‐binding proteins. The basic structural and functional unit of EF‐hand calcium‐binding proteins is a pair of EF‐hand motifs. EF‐hand calcium‐binding proteins can be classified as sensors or modulators of calcium signals. EF‐hand calcium senor proteins need to transition from an ‘off’ state at the resting level of calcium in the cell, to an ‘on’ (activated) state when calcium signals increase the concentration of calcium. The binding of calcium by EF hand calcium sensing proteins induces structural changes that activate the protein for interaction with other target proteins. EF‐hand calcium‐binding proteins play important roles in health and disease.
The need for resolving complex dynamic spatial relationships between and within cells has driven the development of super-resolution microscopic techniques [1.Glancy B. Visualizing mitochondrial form and function within the cell.Trends Mol. Med. 2020; 26: 58-70Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar, 3.De Castro O. et al.Magnetic sector secondary ion mass spectrometry on FIB-SEM instruments for nanoscale chemical imaging.Anal. Chem. 2022; 94: 10754-10763Crossref PubMed Scopus (3) Google Scholar]. The explosion of computational and data storage capabilities has propelled the evolution of fully automated, high-resolution microscopes capable of rendering fine cellular ultrastructure in 3D with high fidelity. Focused ion beam scanning electron microscopy (FIB-SEM) is a technique with resolution <10 nm in all planes, making it ideal for exploring organelle–organelle interactions such as endoplasmic reticulum–mitochondrial contacts sites or resolving fine synaptic features [2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar,4.Xu C.S. et al.Enhanced FIB-SEM systems for large-volume 3D imaging.eLife. 2017; 6e25916Crossref Scopus (174) Google Scholar]. FIB-SEM works by pairing a focused beam of ions to finely ablate the surface of heavily contrasted, resin-embedded samples with a scanning, low-voltage electron beam and backscatter electron detector for surface imaging [2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar]. An array of software are available for 3D reconstruction of features of interest (Table 1) [5.Garza-Lopez E. et al.Protocols for generating surfaces and measuring 3D organelle morphology using amira.Cells. 2022; 11: 65Crossref Scopus (8) Google Scholar,6.Lam J. et al.A universal approach to analyzing transmission electron microscopy with ImageJ.Cells. 2021; 10: 2177Crossref PubMed Scopus (32) Google Scholar]. For more versatility, FIB-SEM can be paired with cryo-capabilities and secondary detectors (Figure 1) [7.Vidavsky N. et al.Cryo-FIB-SEM serial milling and block face imaging: large volume structural analysis of biological tissues preserved close to their native state.J. Struct. Biol. 2016; 196: 487-495Crossref PubMed Scopus (54) Google Scholar,8.Hayles M.F. De Winter D.A.M. An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples.J. Microsc. 2021; 281: 138-156Crossref PubMed Scopus (20) Google Scholar].Table 1Examples of 3D reconstruction analysis softwareOpen-source or publicly availableCommercially availableMicroscopy Image BrowserThermo Scientific AmiraReconstructImarisIMODDragonfly Proilastik3D SlicerImageJ/FIJInapari Open table in a new tab Precise, fine sample removal along the z axis. Fine balance of ultra-resolution and volumetric sampling for 3D imaging. Amenable to pairing with a wide array of techniques that can yield localization, topography, and elemental composition with high spatial resolution. Easy access to image processing software. Fully automated. Adjustable, allowing for beam strength to be adjustable and multiple specimens in a small area to be surveyed to ensure efficiency. Cryo-capabilities may aid in fluorescence confocal imaging, the imaging of proteins and molecules in their native state, and avoiding potential damage and morphological changes which may occur with fixation and embedding of traditional FIB-SEM techniques. Unlike transmission electron microscopy (TEM), cannot resolve fine, nanostructural details, such as visualization of viral spike proteins or between intermembrane organellar connections [2.Biazik J. et al.The versatile electron microscope: an ultrastructural overview of autophagy.Methods. 2015; 75: 44-53Crossref PubMed Scopus (28) Google Scholar,9.Baena V. et al.FIB-SEM as a volume electron microscopy approach to study cellular architectures in SARS-CoV-2 and other viral infections: a practical primer for a virologist.Viruses. 2021; 13: 611Crossref PubMed Scopus (16) Google Scholar]. Longer acquisition time and smaller volume sampling capabilities than other volumetric EM techniques. Sample is consumed during image milling and acquisition. Needs large RAM and storage (~TB range) for efficient data transfer and processing for volumetric applications. Contamination may occur with gallium beam, affecting physical and electrical attributes. Learning curve for the analysis software, which varies across different software, may have separate user interfaces, require coding knowledge, or require manual segmentation. High-cost commitment (instrumentation, facility, maintenance, operating, and training costs) and expertise are required, inhibiting ancillary uses of FIB-SEM in many cases; furthermore, cryo-FIB-SEM requires further extensive specialized techniques and materials. We thank Trace A. Christensen, MBA, Mayo Microscopy and Cell Analysis Core, and Jian Shao, PhD, Central Microscopy Research Facility University of Iowa, for their valuable feedback on the manuscript. Funding by the UNCF/Bristol-Myers Squibb E.E. Just Faculty Fund, BWF Career Awards at the Scientific Interface Award, BWF Ad-hoc Award, NIH Small Research Pilot Subaward to 5R25HL106365-12 from the National Institutes of Health PRIDE Program, and DK020593, Vanderbilt Diabetes and Research Training Center for DRTC Alzheimer's Disease Pilot & Feasibility Program. CZI Science Diversity Leadership grant number 2022- 253529 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (to A.H.). NSF EES2112556, NSF EES1817282, NSF MCB1955975, and CZI Science Diversity Leadership grant number 2022-253614 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (to S.A.D.). Image created using BioRender. All representative images are original. The authors have no conflicts of interest to declare.
The complex [Ti(2,4-dimethyl-2,4-pentanediolate)2]2 (1) has been synthesized from [Ti(OiPr)4] by transesterification with a stoichiometric amount of 2,4-dimethyl-2,4-pentanediol. We have characterized complex 1 in the solid state by single-crystal X-ray diffraction and in the gas phase by desorption chemical ionization mass spectrometry (DCI-MS). The structural and mass spectrometric data show complex 1 to be stable as a dimer in both the solid and gas phases. The retention of dimeric nuclearity in the gas phase sets complex 1 apart from other simple titanium alkoxide complexes [Ti(OiPr)4] and [Ti(OMe)4]4 that give rise to respective families of molecular ions in the DCI-MS experiment. The highest mass molecular ions for Ti alkoxide complexes in the gas phase may reveal the highest nuclearity that these complexes achieve in condensed phases. According to this interpretation the complex [Ti(OiPr)4] is principally dimeric in the gas phase and probably also in the pure liquid phase and should be represented by the formula [Ti(OiPr)4]2.
Background:
The S100A8/S100A9 heterodimer calprotectin (CP) is expressed abundantly in neutrophils and plays a key role in innate immunity. In tissue abscesses, CP sequesters the transition metals manganese (Mn) and zinc (Zn) which serves to inhibit microbial growth and contributes to the control of pathogens. This activity is often referred to as nutritional immunity. We have shown previously that CP contains two transition metal binding sites, both of which have high affinity for Zn, however only one of which exhibits high affinity for Mn.
Methods:
Here we report an in-depth characterization of the biophysical and biochemical properties of CP-Mn and CP-Zn complexes using a combination of isothermal titration calorimetry (ITC), mutagenesis, and microbiology experiments. Atomic level details into the structural basis for function are provided by a high-resolution crystal structure of CP with bound Mn.
Results:
The crystal structure of CP with bound Mn has been determined to 1.8 A. ITC experiments show a stoichiometry of two Zn ions with dissociation constants of 1.4 nM and 5.6 nM, whereas only one Mn ion binds with high affinity (dissociation constant 1.3 nM). Mutants of CP with inactivated metal binding sites have been designed and characterized physically and functionally.
Conclusions:
CP binds two Zn ions, but only one Mn ion, with high affinity. The x-ray crystal structure reveals a single octahedrally coordinated Mn ion at the S100A8/S100A9 subunit interface that also involves the C-terminal tail of S100A9. The chelation of Mn by six histidine residues is unique and has not been observed in other Mn binding proteins. The biochemical and biophysical characterization of wild type and mutant CP variants that inhibit Mn binding show that sequestration of Mn is required for the antimicrobial activity of CP. The distinctive features of the Mn binding site explain why CP exhibits broad-spectrum anti-microbial activity via a nutritional immunity mechanism.
Abstract Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1.
Disability remains an underacknowledged and underdiscussed topic in science, technology, engineering, mathematics, and medicine (STEMM). Social stigma and fear of negative outcomes have resulted in a consistent lack of disclosure. Disabilities cause social and professional difficulties for those that have them. While some faculty can be allies, past literature shows that steps must be taken to make disabilities visible in STEMM at both student and faculty levels. Here, we offer suggestions to better support faculty and students in enhancing the outcomes of individuals who have invisible disabilities. Critically, techniques such as abolishing stigma, universal learning, and better mentoring may improve the challenges faced by those who self-identify as an individual with a disability.
