Interacting with Hemoglobin: Paracoccidioides spp. Recruits hsp30 on Its Cell Surface for Enhanced Ability to Use This Iron Source

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.

Using the hemoglobin-binding Staphylococcus aureus protein IsdH to enable plasma analysis of hemolyzed blood samples

Background Intravascular hemolysis and in vitro hemolysis are prevalent contributors to failed blood sample analysis in the routine hospital laboratory. Interferences by hemoglobin in spectrophotometric and certain enzyme activity assays is the major causative factor. Methods By exploiting the hemoglobin-binding properties of the iron-regulated surface determinant H (IsdH) protein from Staphylococcus aureus we have developed a new method to instantly remove hemoglobin and hemoglobin-haptoglobin complexes from plasma in vitro thereby enabling the measurement of hemoglobin-sensitive analytes in hemolyzed plasma. In the present study we used an engineered IsdH mutant form conjugated to Sepharose for the efficient removal of plasma hemoglobin in concentrations up to 15 mg/mL. The high abundance of haptoglobin, which forms a tight complex with hemoglobin in plasma, did not affect the hemoglobin removal by IsdH Sepharose. Results Applying the method on plasma samples that beforehand were spiked with blood hemolysate re-enabled measurement of the hemolysis sensitive parameters: alkaline phosphatase, conjugated bilirubin, iron, ferritin, γ-glutamyltransferase, total thyroxine and troponin T. IsdH Sepharose-mediated hemoglobin removal also enabled measurement of hemolysis sensitive parameters in hemolyzed samples from anonymized patients. Conclusions In conclusion, IsdH Sepharose is a simple cost-effective pretreatment of hemolyzed samples correcting and enabling the measurement of several important hemoglobin-sensitive parameters in a way compatible with standard procedures in routine laboratories.

Systems-level physiology of the human red blood cell is computed from metabolic and macromolecular mechanisms

ABSTRACTThe human red blood cell has served as a starting point for the application and development of systems biology approaches due to its simplicity, intrinsic experimental accessibility, and importance in human health applications. Here, we present a multi-scale computational model of the human red blood cell that accounts for the full metabolic network, key proteins (>95% of proteome mass fraction), and several macromolecular mechanisms. Proteomics data are used to place quantitative constraints on individual protein complexes that catalyze metabolic reactions, as well as a total proteome capacity constraint. We explicitly describe molecular mechanisms—such as hemoglobin binding and the formation and detoxification of reactive oxygen species—and takes standard hematological variables (e.g., hematocrit, hemoglobin concentration) as input, allowing for personalized physiological predictions. This model is built from first principles and allows for direct computation of physiologically meaningful quantities such as the oxygen dissociation curve and an accurate computation of the flux state of the metabolic network. More broadly, this work represents an important step toward including the proteome and its function in whole-cell models of human cells.

Toward a Proteome-Complete Computational Model of the Human Red Blood Cell

The human red blood cell has served as a starting point for the application and development of systems biology approaches due to its simplicity, intrinsic experimental accessibility, and importance in human health applications. Here, we present a multi-scale computational model of the human red blood cell that accounts for metabolism and macromolecules. Proteomics data are used to place quantitative constraints on individual protein complexes that catalyze metabolic reactions, as well as a total proteome capacity constraint. We explicitly describe molecular mechanisms-such as hemoglobin binding and the formation and detoxification of reactive oxygen species-and account for sequence variations between individuals, allowing for personalized physiological predictions. This model allows for direct computation of the oxyhemoglobin curve as a function of model species and a more accurate computation of the flux state of the metabolic network. More broadly, this work represents another step toward building increasingly more complete systems biology whole-cell models. Disclosures No relevant conflicts of interest to declare.

Acute phase proteins in bitches subjected to conventional and minimally invasive ovariohysterectomy

ABSTRACT: The aim of this study was to evaluate and to compare the possible inflammatory changes by screening acute phase proteins concentrations in healthy bitches subjected to ovariohysterectomy. Minimally invasive and conventional (laparotomy) ovariohysterectomies were performed in 17 client-owned adult female mixed breed dogs. Nine animals were subjected to minimally invasive and eight animals to conventional ovariohysterectomy. Blood samples were taken before surgery, 24, 48 hours, and seven days postoperatively. Serum C-reactive concentration was determined by a commercial ELISA kit and serum haptoglobin concentration was measured via hemoglobin binding assay, both previously validated for use in dogs. As the data did not meet the normal distribution criteria, the nonparametric Kruskall-Wallis was performed to compare quantitative variables between groups. One-way ANOVA and the Friedman test were used for multiple comparisons between time points, with a P<0.05 considered significant. C-reactive protein concentration was significantly different (P<0.0001) at 24 hours postoperatively between groups. There was no significant difference in haptoglobin concentration between groups. C-reactive protein and haptoglobin concentrations were significantly different at 24 and 48 hours postoperatively for minimally invasive and conventional ovariohisterectomies. These findings provided an overview of the short-term inflammatory effects produced by minimally invasive and conventional ovariohysterectomies.