The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation.

A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + ß barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.

Efficient in situ screening of and data collection from microcrystals in crystallization plates.

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

Geogenic particles induce bronchial susceptibility to non-typeable Haemophilus influenzae.

Exposure to geogenic (earth-derived) particulate matter (PM) is linked to an increased prevalence of bronchiectasis and other respiratory infections in Australian Indigenous communities. Experimental studies have shown that the concentration of iron in geogenic PM is associated with the magnitude of respiratory health effects, however, the mechanism is unclear. We investigated the effect of geogenic PM and iron oxide on the invasiveness of non-typeable Haemophilus influenzae (NTHi). Peripheral blood mononuclear cell-derived macrophages or epithelial cell lines (A549 & BEAS-2B) were exposed to whole geogenic PM, their primary constituents (haematite, magnetite or silica) or diesel exhaust particles (DEP). The uptake of bacteria was quantified by flow cytometry and whole genome sequencing (WGS) was performed on NTHi strains. Geogenic PM increased the invasiveness of NTHi in bronchial epithelial cells. Of the primary constituents, haematite also increased NTHi invasion with magnetite and silica having significantly less impact. Furthermore, we observed varying levels of invasiveness amongst NTHi isolates. WGS analysis suggested isolates with more genes associated with heme acquisition were more virulent in BEAS-2B cells. The present study suggests that geogenic particles can increase the susceptibility of bronchial epithelial cells to select bacterial pathogens in vitro, a response primarily driven by haematite content in the dust. This demonstrates a potential mechanism linking exposure to iron-laden geogenic PM and high rates of chronic respiratory infections in remote communities in arid environments.

Iron Oxide Particles Alter Bacterial Uptake and the LPS-Induced Inflammatory Response in Macrophages.

Exposure to geogenic (earth-derived) particulate matter (PM) is linked to severe bacterial infections in Australian Aboriginal communities. Experimental studies have shown that the concentration of iron in geogenic PM is associated with the magnitude of respiratory health effects, however, the mechanism is unclear. We investigated the effect of silica and iron oxide on the inflammatory response and bacterial phagocytosis in macrophages. THP-1 and peripheral blood mononuclear cell-derived macrophages were exposed to iron oxide (haematite or magnetite) or silica PM with or without exposure to lipopolysaccharide. Cytotoxicity and inflammation were assessed by LDH assay and ELISA respectively. The uptake of non-typeable Haemophilus influenzae by macrophages was quantified by flow cytometry. Iron oxide increased IL-8 production while silica also induced significant production of IL-1ß. Both iron oxide and silica enhanced LPS-induced production of TNF-α, IL-1ß, IL-6 and IL-8 in THP-1 cells with most of these responses replicated in PBMCs. While silica had no effect on NTHi phagocytosis, iron oxide significantly impaired this response. These data suggest that geogenic particles, particularly iron oxide PM, cause inflammatory cytokine production in macrophages and impair bacterial phagocytosis. These responses do not appear to be linked. This provides a possible mechanism for the link between exposure to these particles and severe bacterial infection.

The respiratory health effects of geogenic (earth derived) PM10.

Inhalation of particulate matter less than 10 µm in diameter (PM10) has a range of implications for respiratory health. In order to mitigate these effects regulatory bodies have set ambient air quality guidelines based on the known dose-response relationships between PM10 exposure and health outcomes. However, these data are based almost entirely on PM10 from urban regions, which are typically dominated by particulates from combustion sources. In contrast, there are limited data on the respiratory health effects of particles from nonurban regions that often contain a high geogenic (earth derived) component. In this narrative review, we summarize the existing evidence for the respiratory health effects of inhalation of geogenic PM10. We outline the impact of physicochemical properties on the lung response, with a view to identifying gaps in the field.