Biology subjects

Mackenzie, J.

Publications and source records attributed to Mackenzie, J..

8 recordsLinked to original sources

bioRxiv2025

A novel reporter to visualize and quantify endogenous inflammasomes and caspase-1 recruitment

Inflammasomes are signaling complexes that coordinate inflammation by inducing rapid cytokine secretion and that protect against invading pathogens by initiating death of infected cells. Yet, current methods do not allow us to specifically monitor inflammasome activation in real-time at endogenous protein levels. To overcome this shortcoming, we developed and characterized a novel fluorescent reporter that visualizes inflammasome assembly and reports on the recruitment of the effector protein caspase-1 without impairing downstream signaling. The reporter permits the analysis of inflammasome assembly over time as well as precise quantification of cells with assembled inflammasomes. We have successfully applied the reporter in lentivirus-transduced human and murine cell lines and primary cells but have also applied recombinant viruses that encode the reporter in their genome to detect inflammasome responses in primary cells. Mouse intestinal enteroids expressing the reporter allowed us to visualize how Salmonella infection triggers NAIP/NLRC4 inflammasome assembly, cell death, and expulsion in complex tissues. Lastly, we apply the new tool to gain mechanistic insights and prove that caspase-1 activation on inflammasomes relies on the assembly of filaments of caspase-1CARD, which can be terminated by the CARD-only protein CARD17 to shut down cytokine secretion. We anticipate broad application of the reporter in fundamental and applied research, as it permits the quantitative assessment of inflammasome assembly at both high temporal and spatial resolution or in high throughput.

immunology↗

bioRxiv2023

Influenza infected macrophages release viral ribonucleoproteins that shape the local host response

Airway epithelial cells and macrophages (M{Phi}) represent cellular targets of infection by influenza A virus (IAV). Epithelial cells support IAV infection and replication by enabling the generation and release of new viral particles (productive replication). In contrast, M{Phi} are susceptible to initial IAV infection but the release of infectious viral particles is inhibited through abortive replication. Despite the lack of infectious virions released from infected M{Phi}, we detected newly synthesised viral RNA and nucleoprotein (NP) in M{Phi} supernatants. We show that viral RNA is released from infected M{Phi} as viral ribonucleoprotein (vRNP) complexes which elicits potent inflammatory responses when exposed to uninfected cells. These vRNPs specifically induced IL-1{beta}, CXCL13, IL-32, CCL4 and CXCL10 in uninfected cells and were at least partially sensed through the RIG-I/MDA5 pathway. While M{Phi} represent a dead-end for IAV infection through abortive replication, the release of vRNPs shapes immune repsonses of uninfected cells in the local microenvironment.

microbiology↗

bioRxiv2023

Antiviral Wolbachia strains associate with Aedes aegypti endoplasmic reticulum membranes and disturb host cell lipid distribution to restrict dengue virus replication.

The insect endosymbiotic bacterium Wolbachia pipientis is being utilised as a biocontrol tool to reduce the incidence of Aedes aegypti-transmitted viral diseases like dengue. However, the precise mechanisms underpinning Wolbachias antiviral activity are not well defined. Here we generated a panel of Ae. aegypti-derived cell lines infected with antiviral strains wMel and wAlbB or the non-antiviral strain wPip to understand host cell morphological changes specifically induced by antiviral strains. Antiviral strains were frequently found to be entirely wrapped by the host endoplasmic reticulum (ER) membrane, while wPip bacteria clustered separately in the host cell cytoplasm. ER-derived lipid droplets (LDs) increased in volume in wMel-and wAlbB-infected cell lines and mosquito tissues compared to cells infected with wPip or Wolbachia-free controls. Inhibition of fatty acid synthase (required for triacylglycerol biosynthesis) reduced LD formation and significantly restored ER-associated dengue virus replication in cells occupied by wMel. Together, this suggests that antiviral Wolbachia strains may specifically alter the lipid composition of the ER to preclude the establishment of DENV replication complexes. Defining Wolbachias antiviral mechanisms will support the application and longevity of this effective biocontrol tool that is already being used at scale. ImportanceAedes aegypti transmits a range of important human pathogenic viruses like dengue. However, infection of Ae. aegypti with the insect endosymbiotic bacterium, Wolbachia, reduces the risk of mosquito to human viral transmission. Wolbachia is being utilized at field sites across more than 13 countries to reduce the incidence of viruses like dengue, but it is not well understood how Wolbachia induces its antiviral effects. To examine this at the subcellular level, we compared how different strains of Wolbachia with varying antiviral strengths, associate with and modify host cell structures. Strongly antiviral strains were found to specifically associate with the host endoplasmic reticulum and induce striking impacts on host cell lipid distribution. Inhibiting Wolbachia-induced lipid redistribution partially restored dengue virus replication demonstrating this is a contributing role for Wolbachias antiviral activity. These findings provide new insights into how antiviral Wolbachia strains associate with and modify Ae. aegypti host cells.

microbiology↗

bioRxiv2021

Opposing dominance within a color gene block is associated with maladaptive hybrid signal discordance

The divergence of plumage color genes is increasingly recognized as important for speciation in songbirds through its influence on social signaling. However, the behavioral mechanisms underlying the eco-evolutionary feedback that acts across species boundaries is poorly understood. The hybrid zone between Setophaga occidentalis (SOCC) and S. townsendi (STOW) in the Cascade Mountain range provides a natural observatory to test the interplay between genetics, plumage signals, and territoriality in maintaining nascent species boundaries. Recently, we found that selection within a gene block underpinning color variation (ASIP-RALY) has maintained a stable and narrow hybrid zone. Here we investigated the roles of cheek darkness and flank streaking as plumage signals during simulated territorial intrusion, two melanin-based traits underpinned by ASIP-RALY that reflect opposing dominance of SOCC and STOW alleles. We found that both plumage traits act as honest signals of body size in the territorial sex (i.e., males). The opposing dominance effects of ASIP-RALY resulted in plumage signal discordance for heterozygotes, which in turn was associated with reduced territorial performance, a fitness proxy quantified by vocal and physical responses to a decoy intruder. Taken together, this study highlights a behavioral mechanism underlying selection acting on a simple genetic architecture that shapes species boundary despite gene flow.

evolutionary biology↗

bioRxiv2020

Liquid chalk is an antiseptic against SARS-CoV-2 and influenza A respiratory viruses

The COVID-19 pandemic has impacted and enforced significant restrictions within our societies, including the attendance of the public and professional athletes in gyms. Liquid chalk is a commonly used accessories in gyms and is comprised of magnesium carbonate and alcohol that quickly evaporates on the hands to leave a layer of dry chalk. We investigated whether liquid chalk is an antiseptic against highly pathogenic human viruses including, SARS-CoV-2, influenza virus and noroviruses. Chalk was applied before or after virus inoculum and recovery of infectious virus was determined to mimic the use in the gym. We observed that addition of chalk before or after virus contact lead to a significant reduction on recovery of infectious SARS-CoV-2 and influenza but had little impact on norovirus. These observations suggest that the use and application of liquid chalk can be an effective and suitable antiseptic for major sporting events, such as the Olympic Games.

microbiology↗

bioRxiv2020

Host immunity increases Mycobacterium tuberculosis reliance on cytochrome bd oxidase

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFN{gamma}), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that{Delta} cydA mutants were hypersusceptible to the low pH encountered in IFN{gamma}-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs. Author SummaryTuberculosis, caused by Mycobacterium tuberculosis (Mtb) is a serious global health problem that is responsible for over one million deaths annually, more than any other single infectious agent. In the host, Mtb can adapt to a wide variety of immunological and environmental pressures which is integral to its success as a pathogen. Accordingly, the respiratory capacity of Mtb is flexible. The electron transport chain of Mtb has two terminal oxidases, the cytochrome bc1/aa3 super complex and cytochrome bd, that contribute to the proton motive force and subsequent production of energy in the form of ATP. The bc1/aa3 super complex is required for optimal growth during infection but the role of cytochrome bd is unclear. Here we report that the cytochrome bd oxidase is required for resisting the adaptive immune response, in particular, acidification of the phagosome induced by lymphocyte-derived IFN{gamma}. We found that the cytochrome bd oxidase is specifically required under acidic conditions, where the bc1/aa3 complex is preferentially inhibited. Additionally, we show that acidic conditions increased the potency of Q203, a cytochrome bc1/aa3 inhibitor and candidate tuberculosis therapy. This work defines a new link between the host immune response and the respiratory requirements of Mtb that affects the potency of a potential new therapeutic.

microbiology↗

bioRxiv2019

Selection on a pleiotropic color gene block underpins early differentiation between two warbler species

When one species gradually splits into two, divergent selection on specific traits can cause peaks of differentiation in the genomic regions encoding those traits. Whether speciation is initiated by strong selection on a few genomic regions with large effects or by more diffused selection on many regions with small effects remains controversial. Differentiated phenotypes between differentiating lineages are commonly involved in reproductive isolation, thus their genetic underpinnings are key to the genomics architecture of speciation. When two species hybridize, recombination over multiple generations can help reveal the genetic regions responsible for the differentiated phenotypes against a genomic background that has been homogenized via backcrossing and introgression. We used admixture mapping to investigate genomic differentiation and the genetic basis of differentiated plumage features (relative melanin and carotenoid pigment) between hybridizing sister species in the early stage of speciation: Townsends (Setophaga townsendi) and Hermit warblers (S. occidentalis). We found a few narrow and dispersed divergent regions between allopatric parental populations, consistent with the divergence with gene flow model of speciation. One of the divergent peaks involves three genes known to affect pigmentation: ASIP, EIF2S2, and RALY (the ASIP-RALY gene block). After controlling for population substructure, we found that a single nucleotide polymorphism (SNP) inside the intron of RALY displays a strong pleiotropic association with cheek, crown, and breast coloration. In addition, we detect selection on the ASIP-RALY gene block, as the geographic cline of the RALY marker of this gene block has remained narrower than the plumage cline, which remained narrower than expected under neutral diffusion over two decades. Despite extensive gene flow between these species across much of the genome, the selection on ASIP-RALY gene block maintains stable genotypic and plumage difference between species allowing further differentiation to accumulate via linkage to its flanking genetic region or linkage-disequilibrium genome-wide.

evolutionary biology↗

bioRxiv2019

Mouse Norovirus infection arrests host cell translation uncoupled from the stress granule-PKR-eIF2α axis

The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2) resulting in the suppression of host translation, yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signalling cytokines such as IFN{beta}, IL-6 and TNF. Our subsequent analyses revealed that the phosphorylation of eIF2 was mediated via Protein kinase-R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2 and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection, and MNV has the capacity to restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears, MNV utilises G3BP1 to enhance replication, but equally to prevent SG formation, intimating an anti-MNV property of SGs. Overall, thus study highlights MNV manipulation of SGs, PKR and translational control to regulate cytokine translation and to promote viral replication. ImportanceViruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate and cause disease. In retaliation, cells possess various defence mechanisms to detect, destroy and clear infecting viruses as well as signal to neighbouring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and pro-inflammatory cytokines. However, virus replication and protein translation still ensues. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SG), by recruiting the key host protein G3BP1 to the MNV replication complex; a recruitment that is crucial to establishing and maintaining virus replication. Thus MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.

microbiology↗