Biology subjects

Gibson, A. J.

Publications and source records attributed to Gibson, A. J..

4 recordsLinked to original sources

bioRxiv2024

M. bovis PPD Enhances Respiratory Bioenergetics of Human vs. Bovine Macrophages

The role of macrophage (MO) cellular metabolism and reprogramming during TB infection is of great interest due to the influence of Mycobacterium spp. on MO bioenergetics. Recent studies have shown that M. tuberculosis induces a TLR2-dependent shift towards aerobic glycolysis and metabolic reprogramming, comparable to the established LPS induced pro-inflammatory M1 MO polarisation. Distinct differences in the metabolic profile of murine and human MO indicates species-specific differences in bioenergetics. So far, studies examining the metabolic potential of cattle are lacking, thus the basic bioenergetics of bovine and human MO were explored in response to a variety of innate immune stimuli. Cellular energy metabolism kinetics were measured concurrently for both species on a Seahorse XFe96 platform to generate bioenergetic profiles for the response to the bona-fide TLR2 and TLR4 ligands, FSL-1 and LPS respectively. Despite previous reports of species-specific differences in TLR signalling and cytokine production between human and bovine MO, we observed similar respiratory profiles for both species. Basal respiration remained constant between stimulated MO and controls, whereas addition of TLR ligands induced increased glycolysis. In contrast to MO stimulation with M. tuberculosis PPD, another TLR2 ligand, M. bovis PPD treatment significantly enhanced basal respiration rates and glycolysis only in human MO. Respiratory profiling further revealed significant elevation of ATP-linked OCR and maximal respiration suggesting a strong OXPHOS activation upon M. bovis PPD stimulation in human MO. Our results provide an exploratory set of data elucidating the basic respiratory profile of bovine vs. human MO that will not only lay the foundation for future studies to investigate host-tropism of the M. tuberculosis complex but may explain inflammatory differences observed for other zoonotic diseases. HighlightsO_LISimilar baseline respiratory profiles for human and bovine macrophages C_LIO_LIM. bovis PPD treatment altered metabolic profile only in human MO C_LIO_LIStrong OXPHOS activation upon M. bovis PPD stimulation only in human MO C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/582730v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@e833fborg.highwire.dtl.DTLVardef@acdda6org.highwire.dtl.DTLVardef@10bcbfforg.highwire.dtl.DTLVardef@17ddf31_HPS_FORMAT_FIGEXP M_FIG Created with BioRender (www.biorender.com) by A. Gibson C_FIG

immunology↗

bioRxiv2023

Characterisation of the bovine C-type lectin receptor Mincle and potential evidence for an endogenous ligand

Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6'-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognize endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

immunology↗

bioRxiv2022

Defining the genes required for survival of Mycobacterium bovis in the bovine host offers novel insights into the genetic basis of survival of pathogenic mycobacteria

Tuberculosis has severe impacts in both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human adapted species, and Mycobacterium bovis, the animal adapted species is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models, however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study we utilise transposon insertion sequencing in M. bovis to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, PDIM synthesis, mycobactin synthesis and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis, phoPR is required by M. bovis in vivo despite the known defect in signalling through this system. Comparison to studies performed in glycerol adapted species such as M. bovis BCG and M. tuberculosis suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon), oxidation (hsd) and detoxification (cyp125). We report good correlation with existing mycobacterial virulence functions, but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics. ImportanceThis is the first report of the genetic requirements of an animal adapted member of the MTBC in a natural host. M. bovis has devastating impacts in cattle and bovine tuberculosis is a considerable economic, animal welfare and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identifies several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.

microbiology↗

bioRxiv2021

Probing differences in gene essentiality between the human and animal adapted lineages of the Mycobacterium tuberculosis complex using TnSeq

Members of the Mycobacterium tuberculosis complex (MTBC) show distinct host adaptations, preferences and phenotypes despite being >99% identical at the nucleic acid level. Previous studies have explored gene expression changes between the members, however few studies have probed differences in gene essentiality. To better understand the functional impacts of the nucleic acid differences between Mycobacterium bovis and Mycobacterium tuberculosis we used the Mycomar T7 phagemid delivery system to generate whole genome transposon libraries in laboratory strains of both species and compared the essentiality status of genes during growth under identical in vitro conditions. Libraries contained insertions in 54% of possible TA sites in M. bovis and 40% of those present in M. tuberculosis, achieving similar saturation levels to those previously reported for the MTBC. The distributions of essentiality across the functional categories were similar in both species. 527 genes were found to be essential in M. bovis whereas 477 genes were essential in M. tuberculosis and 370 essential genes were common in both species. CRISPRi was successfully utilised in both species to determine the impacts of silencing genes including wag31, a gene involved in peptidoglycan synthesis and Rv2182c/Mb2204c, a gene involved in glycerophospholipid metabolism. We observed species specific differences in the response to gene silencing, with the inhibition of expression of Mb2204c in M. bovis showing significantly less growth impact than silencing its ortholog (Rv2182c) in M. tuberculosis. Given that glycerophospholipid metabolism is a validated pathway for antimicrobials, our observations suggest that target vulnerability in the animal adapted lineages cannot be assumed to be the same as the human counterpart. This is of relevance for zoonotic tuberculosis as it implies that the development of antimicrobials targeting the human adapted lineage might not necessarily be effective against the animal adapted lineage. The generation of a transposon library and the first reported utilisation of CRISPRi in M. bovis will enable the use of these tools to further probe the genetic basis of survival under disease relevant conditions.

microbiology↗