Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.     

Release of Enterococcus mundtii Bacteriocin ST4SA from Self-Setting Brushite Bone Cement

Maxillofacial and craniofacial surgery is on the increase, which exposes more patients at risk of acquiring microbial infections. The use of antibiotic-loaded calcium phosphate bone cements has been shown to reduce the incidence of infection. A marked increase in antibiotic-resistant pathogens, including multidrug-resistant pathogens, has been reported. This has led to the investigation of various compounds as alternatives to conventional treatments. In this paper, we report on the incorporation and release of a broad-spectrum class II antimicrobial peptide, bacteriocin ST4SA produced by Enterococcus mundtii, into a calcium orthophosphate-based bone cement. Our results suggest class II bacteriocins may be incorporated into self-setting bone cements to produce implants with antimicrobial activity over extended periods of time.     

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron [German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 h Aspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.     

The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection

Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN‐γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN‐γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN‐γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock‐out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.     

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied—one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.     

Encapsulation of Lactobacillus plantarum 423 and its Bacteriocin in Nanofibers

Plantaricin 423, produced by Lactobacillus plantarum 423, was encapsulated in nanofibers that were produced by the electrospinning of 18% (w/v) polyethylene oxide (200 000 Da). The average diameter of the nanofibers was 288 nm. Plantaricin 423 activity decreased from 51 200 AU/ml to 25 600 AU/ml and from 204 800 AU/ml to 51 200 AU/ml after electrospinning, as determined against Lactobacillus sakei DSM 20017 and Enterococcus faecium HKLHS, respectively. Cells of L. plantarum 423 encapsulated in nanofibers decreased from 2.3 × 10¹⁰ cfu/ml before electrospinning to 4.7 × 10⁸ cfu/ml thereafter. Cells entrapped in the nanofibers continued to produce plantaricin 423. This is the first report on the encapsulation of a bacteriocin and cells of L. plantarum in nanofibers. The method may be used to design a drug delivery system for bacteriocins and the encapsulation of probiotic lactic acid bacteria. The technology is currently being optimized.     

Does dust result in mite outbreaks in apple orchards?

The effect of dust on phytophagous mite numbers was examined in five apple orchards situated in the dry, inland apple producing Ceres area, South Africa. The study was conducted over three seasons. The season with the most dust had the least number of mites. There was no relationship between the amount of dust on leaves and mite numbers from different orchards. Of the 15 correlations between the amount of dust on individual trees and the number of mites on these trees, two were marginally not significant and one was highly significant, but negative. Therefore, seasons during which there is a lot of dust did not result in mite outbreaks nor did dusty orchards harbour elevated mite population levels, and trees with a lot of dust did not necessarily harbour more mites than trees with less dust. However, if there is enough dust to cause stress to the trees, phytophagous mite outbreaks could occur.     

TPL‐2 kinase induces phagosome acidification to promote macrophage killing of bacteria

Tumour progression locus 2 (TPL‐2) kinase mediates Toll‐like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase‐independent regulatory function for TPL‐2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL‐2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL‐2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V‐ATPase proton pump subunits. Furthermore, TPL‐2 stimulated the phosphorylation of DMXL1, a regulator of V‐ATPases, to induce V‐ATPase assembly and phagosome acidification. Consistent with these results, TPL‐2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL‐2 therefore controls innate immune responses of macrophages to bacteria via V‐ATPase induction of phagosome maturation.