Genetic and Anatomic Determinants of Enzootic Venezuelan Equine Encephalitis Virus Infection of Culex (Melanoconion) taeniopus

Venezuelan equine encephalitis (VEE) is a re-emerging, mosquito-borne viral disease with the potential to cause fatal encephalitis in both humans and equids. Recently, detection of endemic VEE caused by enzootic strains has escalated in Mexico, Peru, Bolivia, Colombia and Ecuador, emphasizing the importance of understanding the enzootic transmission cycle of the etiologic agent, VEE virus (VEEV). The majority of work examining the viral determinants of vector infection has been performed in the epizootic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus. Based on the fundamental differences between the epizootic and enzootic cycles, we hypothesized that the virus-vector interaction of the enzootic cycle is fundamentally different from that of the epizootic model. We therefore examined the determinants for VEEV IE infection in the enzootic vector, Culex (Melanoconion) taeniopus, and determined the number and susceptibility of midgut epithelial cells initially infected and their distribution compared to the epizootic virus-vector interaction. Using chimeric viruses, we demonstrated that the determinants of infection for the enzootic vector are different than those observed for the epizootic vector. Similarly, we showed that, unlike A. taeniorhynchus infection with subtype IC VEEV, C. taeniopus does not have a limited subpopulation of midgut cells susceptible to subtype IE VEEV. These findings support the hypothesis that the enzootic VEEV relationship with C. taeniopus differs from the epizootic virus-vector interaction in that the determinants appear to be found in both the nonstructural and structural regions, and initial midgut infection is not limited to a small population of susceptible cells

The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins

The CopC proteins are periplasmic copper binding proteins believed to play a role in bacterial copper homeostasis. Previous studies have focused on CopCs that are part of seven-protein Cop or Pco systems involved in copper resistance. These canonical CopCs contain distinct Cu­(I) and Cu­(II) binding sites. Mounting evidence suggests that CopCs are more widely distributed, often present only with the CopD inner membrane protein, frequently as a fusion protein, and that the CopC and CopD proteins together function in the uptake of copper to the cytoplasm. In the methanotroph <i>Methylosinus trichosporium</i> OB3b, genes encoding a CopCD pair are located adjacent to the particulate methane monooxygenase (pMMO) operon. The CopC from this organism (<i>Mst</i>-CopC) was expressed, purified, and structurally characterized. The 1.46 Å resolution crystal structure of <i>Mst</i>-CopC reveals a single Cu­(II) binding site with coordination somewhat different from that in canonical CopCs, and the absence of a Cu­(I) binding site. Extensive bioinformatic analyses indicate that the majority of CopCs in fact contain only a Cu­(II) site, with just 10% of sequences corresponding to the canonical two-site CopC. Accordingly, a new classification scheme for CopCs was developed, and detailed analyses of the sequences and their genomic neighborhoods reveal new proteins potentially involved in copper homeostasis, providing a framework for expanded models of CopCD function

The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins

The CopC proteins are periplasmic copper binding proteins believed to play a role in bacterial copper homeostasis. Previous studies have focused on CopCs that are part of seven-protein Cop or Pco systems involved in copper resistance. These canonical CopCs contain distinct Cu­(I) and Cu­(II) binding sites. Mounting evidence suggests that CopCs are more widely distributed, often present only with the CopD inner membrane protein, frequently as a fusion protein, and that the CopC and CopD proteins together function in the uptake of copper to the cytoplasm. In the methanotroph <i>Methylosinus trichosporium</i> OB3b, genes encoding a CopCD pair are located adjacent to the particulate methane monooxygenase (pMMO) operon. The CopC from this organism (<i>Mst</i>-CopC) was expressed, purified, and structurally characterized. The 1.46 Å resolution crystal structure of <i>Mst</i>-CopC reveals a single Cu­(II) binding site with coordination somewhat different from that in canonical CopCs, and the absence of a Cu­(I) binding site. Extensive bioinformatic analyses indicate that the majority of CopCs in fact contain only a Cu­(II) site, with just 10% of sequences corresponding to the canonical two-site CopC. Accordingly, a new classification scheme for CopCs was developed, and detailed analyses of the sequences and their genomic neighborhoods reveal new proteins potentially involved in copper homeostasis, providing a framework for expanded models of CopCD function

Repurposed HisC Aminotransferases Complete the Biosynthesis of Some Methanobactins

Methanobactins (Mbns) are ribosomally produced, post-translationally modified bacterial natural products with a high affinity for copper. MbnN, a pyridoxal 5′-phosphate-dependent aminotransferase, performs a transamination reaction that is the last step in the biosynthesis of Mbns produced by several <i>Methylosinus</i> species. Our bioinformatic analyses indicate that MbnNs likely derive from histidinol-phosphate aminotransferases (HisCs), which play a key role in histidine biosynthesis. A comparison of the HisC active site with the predicted MbnN structure suggests that MbnN’s active site is altered to accommodate the larger and more hydrophobic substrates necessary for Mbn biosynthesis. Moreover, we have confirmed that MbnN is capable of catalyzing the final transamination step in Mbn biosynthesis in vitro and in vivo. We also demonstrate that without this final modification, Mbn exhibits significantly decreased stability under physiological conditions. An examination of other Mbns and Mbn operons suggests that N-terminal protection of this family of natural products is of critical importance and that several different means of N-terminal stabilization have evolved independently in Mbn subfamilies

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT