14 Search Results

To successfully mount infections, nearly all bacterial pathogens must acquire iron, a key metal cofactor that primarily resides within human hemoglobin. Corynebacterium diphtheriae causes the life-threatening respiratory disease diphtheria and captures hemoglobin for iron scavenging using the surface-displayed receptor HbpA. Here, we show using X-ray crystallography, NMR, and in situ binding measurements that C. diphtheriae selectively captures iron-loaded hemoglobin by partially ensconcing the heme molecules of its α subunits. Quantitative growth and heme release measurements are compatible with C. diphtheriae acquiring heme passively released from hemoglobin’s β subunits. We propose a model in which HbpA and heme-binding receptors collectively functionmore » on the C. diphtheriae surface to capture hemoglobin and its spontaneously released heme. Acquisition mechanisms that exploit the propensity of hemoglobin’s β subunit to release heme likely represent a common strategy used by bacterial pathogens to obtain iron during infections.« less

Introduction: Blood source is a known preanalytical factor affecting hemoglobin (Hb) concentrations, and there is evidence that capillary and venous blood may yield disparate Hb levels and anemia prevalence. However, data from adolescents are scarce. Objective: To compare Hb and anemia prevalence measured by venous and individual pooled capillary blood among a sample of girls aged 10–19 years from 232 schools in four regions of Ghana in 2022. Methods: Among girls who had venous blood draws, a random subsample was selected for capillary blood. Hb was measured using HemoCue® Hb-301. We used Lin’s concordance correlation coefficient (CCC) to quantify themore » strength of the bivariate relationship between venous and capillary Hb and a paired t-test for difference in means. We used McNemar’s test for discordance in anemia cases by blood source and weighted Kappa to quantify agreement by anemia severity. A multivariate generalized estimating equation was used to quantify adjusted population anemia prevalence and assess the association between blood source and predicted anemia risk. Results: We found strong concordance between Hb measures (CCC = 0.86). The difference between mean venous Hb (12.8 g/dL, ± 1.1) and capillary Hb (12.9 g/dL, ± 1.2) was not significant (p = 0.26). Crude anemia prevalence by venous and capillary blood was 20.6% and 19.5%, respectively. Adjusted population anemia prevalence was 23.5% for venous blood and 22.5% for capillary ( = 0.45). Blood source was not associated with predicted anemia risk (risk ratio: 0.99, 95% CI: 0.96, 1.02). Discordance in anemia cases by blood source was not significant (McNemar p = 0.46). Weighted Kappa demonstrated moderate agreement by severity (k =0.67). Among those with anemia by either blood source (n = 111), 59% were identified by both sources. Conclusion: In Ghanaian adolescent girls, there was no difference in mean Hb, anemia prevalence, or predicted anemia risk by blood source. However, only 59% of girls with anemia by either blood source were identified as having anemia by both sources. These findings suggest that pooled capillary blood may be useful for estimating Hb and anemia at the population level, but that caution is needed when interpreting individual-level data.« less

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparationmore » method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540–660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390–540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5–25 RBCs obtained from N = 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.« less

Iron is an important micronutrient that is required by bacteria to proliferate and to cause disease. Many bacterial pathogens forage iron from human hemoglobin (Hb) during infections, which contains this metal within heme (iron–protoporphyrin IX). Several clinically important pathogenic species within the Firmicutes phylum scavenge heme using surface-displayed or secreted NEAr Transporter (NEAT) domains. In this review, we discuss how these versatile proteins function in the Staphylococcus aureus Iron-regulated surface determinant system that scavenges heme-iron from Hb. S. aureus NEAT domains function as either Hb receptors or as heme-binding chaperones. In vitro studies have shown that heme-binding NEAT domains canmore » rapidly exchange heme amongst one another via transiently forming transfer complexes, leading to the interesting hypothesis that they may form a protein-wire within the peptidoglycan layer through which heme flows from the microbial surface to the membrane. In Hb receptors, recent studies have revealed how dedicated heme- and Hb-binding NEAT domains function synergistically to extract Hb’s heme molecules, and how receptor binding to the Hb-haptoglobin complex may block its clearance by macrophages, prolonging microbial access to Hb’s iron. The functions of NEAT domains in other Gram-positive bacteria are also reviewed.« less

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group. Here, we report the 2.15 Å resolution X-ray crystal structure of the AmphNOmore » adduct of adult human hemoglobin as the Hb [α-Fe^III(H₂O)][β-Fe^II(AmphNO)] derivative. We show that the binding of AmphNO to the β subunit is enabled by an E helix movement and stabilization of ligand binding by H-bonding with the distal His63 residue. We also observe an AmphNHOH group in the Xe2 pocket in close proximity to the α heme site in this derivative. Additionally, UV–vis spectroscopy was used to characterize this and related wt and mutant Mb adducts. Importantly, our X-ray crystal structure of this Hb-nitrosoamphetamine complex represents the first crystal structure of a wild-type heme protein adduct of any amphetamine metabolite. Our results provide a framework for further studies of AmphNHOH/AmphNO interactions with Hb and Mb as viable processes that potentially contribute to the overall biological inorganic chemistry of amphetamine drugs.« less

In order to proliferate and mount an infection, many bacterial pathogens need to acquire iron from their host. The most abundant iron source in the body is the oxygen transporter hemoglobin (Hb). Streptococcus pyogenes, a potentially lethal human pathogen, uses the Shr protein to capture Hb on the cell surface. Shr is an important virulence factor, yet the mechanism by which it captures Hb and acquires its heme is not well-understood. In this work, we show using NMR and biochemical methods that Shr binds Hb using two related modules that were previously defined as domains of unknown function (DUF1533). Thesemore » hemoglobin-interacting domains (HIDs), called HID1 and HID2, are autonomously folded and independently bind Hb. The 1.5 Å resolution crystal structure of HID2 revealed that it is a structurally unique Hb-binding domain. Mutagenesis studies revealed a conserved tyrosine in both HIDs that is essential for Hb binding. Our biochemical studies indicate that HID2 binds Hb with higher affinity than HID1 and that the Hb tetramer is engaged by two Shr receptors. NMR studies reveal the presence of a third autonomously folded domain between HID2 and a heme-binding NEAT1 domain, suggesting that this linker domain may position NEAT1 near Hb for heme capture.« less

Background: Understanding the mechanisms of attenuation and virulence of foot-and-mouth disease virus (FMDV) in the natural host species is critical for development of next-generation countermeasures such as live-attenuated vaccines. Functional genomics analyses of FMDV have identified few virulence factors of which the leader proteinase (Lpro) is the most thoroughly investigated. Previous work from our laboratory has characterized host factors in cattle inoculated with virulent FMDV and attenuated mutant strains with transposon insertions within Lpro. Methods: In the current study, the characteristics defining virulence of FMDV in cattle were further investigated by comparing the pathogenesis of a mutant, attenuated strain (FMDV-Mut)more » to the parental, virulent virus from which the mutant was derived (FMDV-WT). The only difference between the two viruses was an insertion mutation in the inter-AUG region of the leader proteinase of FMDV-Mut. All cattle were infected by simulated-natural, aerosol inoculation. Results: Both viruses were demonstrated to establish primary infection in the nasopharyngeal mucosa with subsequent dissemination to the lungs. Immunomicroscopic localization of FMDV antigens indicated that both viruses infected superficial epithelial cells of the nasopharynx and lungs. The critical differences between the two viruses were a more rapid establishment of infection by FMDV-WT and quantitatively greater virus loads in secretions and infected tissues compared to FMDV-Mut. The slower replicating FMDV-Mut established a subclinical infection that was limited to respiratory epithelial sites, whereas the faster replication of FMDV-WT facilitated establishment of viremia, systemic dissemination of infection, and clinical disease. Conclusion: The mutant FMDV was capable of achieving all the same early pathogenesis landmarks as FMDV-WT, but was unable to establish systemic infection. The precise mechanism of attenuation remains undetermined; but current data suggests that the impaired replication of the mutant is more responsible for attenuation than differences in host immunological factors. These results complement previous studies by providing data of high-granularity describing tissue-specific tropism of FMDV and by demonstrating microscopic localization of virulent and attenuated clones of the same field-strain FMDV.« less

Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetramericmore » hemoglobin molecules. The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α₁β₂/α₂β₁ intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.« less

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H₂S but, lacking organelles, cannot dispose of H₂S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H₂S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. Here, the structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized againstmore » it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H₂S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H₂S is postulated to signal.« less