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Goulding, D.

Publications and source records attributed to Goulding, D..

13 recordsLinked to original sources

Anti-amyloid immunotherapy drives APOE4 specific increases in glial reactivity, perivascular immune activation, and ARIA-like events

Anti-amyloid antibodies represent the first disease modifying therapeutics for Alzheimers disease (AD). Adoption of these novel treatments has been slowed by the occurrence of amyloid related imaging abnormalities (ARIA) - treatment-associated edema (ARIA-E) or microhemorrhages (ARIA-H) that disproportionately affect carriers of the E4 allele of apolipoprotein E (APOE). With E4 carriers comprising nearly 70% of the AD population, there is a critical need to understand the unique vulnerability of E4 carriers to these events. To address this gap, we utilized the EFAD mouse model - which expresses human APOE isoforms on the 5xFAD background of amyloidosis - to directly compare the effects of anti-amyloid therapy across APOE genotypes. 9-month-old E2, E3, and E4FAD mice received weekly injections of chimeric Aducanumab (chAdu) or IgG control for 12 weeks, to assess APOE isoform-specific effects on amyloid dynamics, ARIA-H-like microhemorrhages, and underlying cellular and transcriptomic responses. E4FAD mice demonstrated plaque reductions with accompanying increases in microhemorrhages (measured on both MRI and histology), and increases in microglial and astrocyte reactivity - especially in the perivascular compartment. Additionally, vascular branching analysis and parallel single cell and spatial transcriptomics revealed a loss of vascular plasticity and increased inflammatory and immune signaling in the neurovascular units of E4FAD mice. Together, these findings suggest the cerebrovasculature of E4s is uniquely susceptible to antibody mediated vascular damage and provide immunological targets for the assessment or mitigation of ARIA risk in this highest need population

neuroscience↗

Slow-growing human cell lines are a suitable alternative to rabbit Sf1Ep cells for in vitro cultivation of Treponema pallidum

Treponema pallidum subsp. pallidum, the causative agent of syphilis, remains difficult to study owing to long-standing limitations in in vitro cultivation. Although a rabbit epithelial cell co-culture system (Sf1Ep) enabled major advances, the absence of human cell-based models limits clinical relevance and mechanistic understanding of host-pathogen interactions. Here, we established a co-culture system using human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six epithelial or epithelial-like cell lines from diverse tissues were evaluated under microaerophilic conditions using standard cultivation medium. CAL-39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviors, and long-term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility were quantified over extended culture. Using live-cell imaging, we defined two distinct host-cell interaction behaviors: surface-associated crawling and stable single-polar attachment. Both behaviors were observed across all tested cell lines but persisted only in those permissive for sustained growth. Together, our findings establish clinically relevant human epithelial co-culture models for T. pallidum, provide new insights into host-cell-dependent growth and motility, and create a platform for mechanistic studies of syphilis pathogenesis and vaccine target discovery.

microbiology↗

Trade-offs between phage resistance and conjugative ability shape the ecological and evolutionary response of a multidrug resistance plasmid to plasmid-dependent phage

Phage therapy is a promising alternative to antibiotics to treat multidrug resistant infections. Plasmid dependent phages (PDPs) are particularly attractive as therapeutics because they can both kill targeted pathogen cells, whilst also potentially preventing the further spread of antibiotic resistance genes encoded by plasmids. However, we lack experimental studies of the ecological and evolutionary response of multidrug resistance plasmids against plasmid dependent phage treatment under ecologically relevant scenarios allowing plasmid conjugation. We experimentally evolved populations of E. coli carrying the multidrug resistance RP4 plasmid with the PRD1 PDP under conditions where conjugation was associated with either strong or weak benefits. When opportunities for conjugation were rare, PRD1 only transiently suppressed the conjugative plasmid population due to the rapid evolution of PRD1 resistant plasmids that lacked conjugative ability. Increasing the ecological opportunity for conjugation enhanced plasmid suppression and delayed the evolution of PRD1 resistant plasmids. PRD1 resistance was associated with reduced conjugative ability, but this trade-off was complex due to the heterogeneous impacts of resistance mutations on pilus production and conjugative ability. Mutations and IS element insertions in conjugation genes caused a wide range of PRD1 resistance phenotypes, ranging from complete resistance (virB4) to partial resistance (trbB, trbL). Bioinformatic analysis of publicly available IncP plasmid sequences showed that truncated variants of VirB4 protein are common in natural populations, suggesting that plasmid-dependent phages are an important selective pressure in microbial communities. Our results demonstrate an evolutionary trade-off between conjugative ability and phage resistance that cannot be easily circumvented by plasmids. Targeting multidrug resistance plasmids with PDPs is likely to drive loss of conjugation limiting the transfer of antibiotic resistance genes in bacterial communities.

evolutionary biology↗

Hatching of whipworm eggs induced by bacterial contact is serine-protease dependent

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage and visualised the process of degradation of the polar plugs that leads to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing hatch-ready L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes. Author summaryHuman whipworms are parasites that cause the gastrointestinal disease trichuriasis in millions of people around the world. Infections occur when whipworm eggs, ingested in contaminated food and water, hatch in the intestine in response to gut bacteria (microbiota). The egg encloses a larva within an egg-shell and has a plug at each end. Hatching liberates the larva that burrows inside the cells that line the gut. Interactions between the microbiota of the gut and whipworm eggs are needed for hatching but are poorly understood. In this study, using the natural mouse whipworm as an infection model, we show that bacteria bind the whipworm egg plugs during the initial stages of hatching, resulting in their degradation and leading to larval exit. We further show that disintegration of the egg plugs is caused by protein-degrading enzymes produced by the bacteria and the larvae. The production of those enzymes by the parasite is dependent on the full development of the larva inside the whipworm egg. These new mechanistic insights pave the way for future studies to understand human whipworm infection and develop new tools to tackle these globally important parasites.

microbiology↗

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted Green Fluorescent Protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes. Graphical abstractBy employing genetic engineering, T. pallidum was modified to express GFP, and Sf1Ep cells to express mCherry on the cytoplasmic membrane and BFP in the nucleus. These new resources for syphilis research will facilitate experimental designs to better define the complex interplay between T. pallidum and the host during infection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/596454v1_ufig1.gif" ALT="Figure 1"> View larger version (114K): org.highwire.dtl.DTLVardef@1cc5bcborg.highwire.dtl.DTLVardef@19e8a12org.highwire.dtl.DTLVardef@17aafdborg.highwire.dtl.DTLVardef@1af6823_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

An invariant Trypanosoma vivax vaccine antigen inducing protective immunity

Trypanosomes are protozoan parasites that cause infectious diseases including human African trypanosomiasis (sleeping sickness), and nagana in economically-important livestock animals1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms including antigenic variation3 that present an apparently insurmountable barrier to vaccination. Here we show using a systematic genome-led vaccinology approach4 and a murine model of Trypanosoma vivax infection5 that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell surface protein localised to the flagellum membrane termed "invariant flagellum antigen from T. vivax" (IFX) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to IFX could induce sterile protection and revealed multiple mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained the socioeconomic development of sub-Saharan African countries6 and challenges long-held views that vaccinating against trypanosome infections cannot be achieved.

microbiology↗

Host adaptation in gut Firmicutes is associated with sporulation loss and altered colonisation patterns

Human-to-human transmission of symbiotic, anaerobic bacteria is a fundamental evolutionary adaptation essential for membership of the human gut microbiota. However, despite its importance, the genomic and biological adaptations underpinning symbiont transmission remain poorly understood. Here, we show that sporulation ability, which promotes transmission of anaerobic bacteria, has been independently lost in many distinct evolutionary lineages of gut bacteria belonging to the Firmicutes phyla. Analysis of 1358 genome-sequenced Firmicutes reveals loss of sporulation is associated with features of host-adaptation such as genome reduction and specialized metabolic capabilities. Consistent with these data, analysis of 28,000 gut metagenomes from people around the world demonstrates that bacteria now incapable of sporulation are more abundant but less prevalent in the human population compared to spore-forming bacteria. Our results suggest host adaptation in gut Firmicutes is an evolutionary trade-off between transmission range and colonisation abundance, leading to distinct transmission cycles. We reveal host transmission as an underappreciated process that shapes the evolution, assembly and functions of gut Firmicutes.

microbiology↗

Defining the early stages of intestinal colonisation by whipworms

Whipworms are large metazoan parasites that inhabit distinct multi-intracellular epithelial burrows described as syncytial tunnels, in the large intestine of their hosts. How first-stage larvae invade host epithelia and establish infection remains unclear. Here, we investigate early infection events both using Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection. We show that larvae degrade the mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular but woven through multiple live enterocytes and goblet cells. We also use single cell RNA sequencing for the first time to describe the mouse caecum. From infected caeca, the transcriptome data reveal the progression of infection results in cell damage and an expansion of enterocytes with a type-I interferon (IFN) signature, characterised by the expression of Isg15, instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal new specific interactions between the host and the parasite that allow the whipworm to establish its multi-intracellular niche.

immunology↗

Control of fluid flow by Adgrd1 is essential for mammalian oviductal embryo transport

Dysfunction of oviductal embryo transport can lead to ectopic pregnancy which affects 1 to 2% of all conceptions in the United States and Europe, and is the most common cause of pregnancy-related death in the first trimester1, 2. Ectopic pregnancies almost always occur in the Fallopian tube, emphasizing the critical role of oviductal transport in human reproduction3. Oviductal transit is regulated and involves a valve-like "tubal-locking" phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilization occurs4. Here, we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they are unable to unlock the restraining mechanism at the AIJ, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid production is dysregulated in Adgrd1-deficient mice. We identified Plxdc2 as an activating ligand for Adgrd1 displayed on the surface of cumulus cells. Our findings suggest that regulating oviductal luminal fluid production by Adgrd1 controls embryo transit, and provides important insights into the genetic regulation and molecular mechanisms involved embryo tubal transport.

molecular biology↗

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids to that of enteroids identifying differences in intestinal epithelial cell (IEC) populations that mimic those found in the caecum and small intestine. The remarkable similarity in the IECs composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system we investigated the responses of caecal IECs to extracellular vesicles (EVs) secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm EVs on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon (IFN) signalling.

immunology↗

The capsule regulatory network of Klebsiella pneumoniae defined by density-TraDISort

Klebsiella pneumoniae infections affect infants and the immunocompromised, and the recent emergence of hypervirulent and multi-drug resistant K. pneumoniae lineages is a critical healthcare concern. Hypervirulence in K. pneumoniae is mediated by several factors, including the overproduction of extracellular capsule. However, the full details of how K. pneumoniae capsule biosynthesis is achieved or regulated are not known. We have developed a robust and sensitive procedure to identify genes influencing capsule production, density-TraDISort, which combines density gradient centrifugation with transposon-insertion sequencing. We have used this method to explore capsule regulation in two clinically-relevant Klebsiella strains; K. pneumoniae NTUH-K2044 (capsule type K1), and K. pneumoniae ATCC43816 (capsule type K2). We identified multiple genes required for full capsule production in K. pneumoniae, as well as putative suppressors of capsule in NTUH-K2044, and have validated the results of our screen with targeted knockout mutants. Further investigation of several of the K. pneumoniae capsule regulators identified - ArgR, MprA/KvrB, SlyA/KvrA and the Sap ABC transporter - revealed effects on capsule amount and architecture, serum resistance and virulence. We show that capsule production in K. pneumoniae is at the centre of a complex regulatory network involving multiple global regulators and environmental cues, and that the majority of capsule regulatory genes are located in the core genome. Overall our findings expand our understanding of how capsule is regulated in this medically-important pathogen, and provide a technology that can be easily implemented to study capsule regulation in other bacterial species.\n\nImportanceCapsule production is essential for K. pneumoniae to cause infections, but its regulation and mechanism of synthesis are not fully understood in this organism. We have developed and applied a new method for genome-wide identification of capsule regulators. Using this method, many genes that positively or negatively affect capsule production in K. pneumoniae were identified, and we use these data to propose an integrated model for capsule regulation in this species. Several of the genes and biological processes identified have not previously been linked to capsule synthesis. We also show that the methods presented here can be applied to other species of capsulated bacteria, providing the opportunity to explore and compare capsule regulatory networks in other bacterial strains and species.

microbiology↗

Exclusive dependence of IL-10Rα signalling on intestinal microbiota homeostasis and control of whipworm infection

The whipworm Trichuris trichiura is a soil-transmitted helminth that dwells in the epithelium of the caecum and proximal colon of their hosts causing the human disease, trichuriasis. Trichuriasis is characterized by colitis attributed to the inflammatory response elicited by the parasite while tunnelling through intestinal epithelial cells (IECs).\n\nThe IL-10 family of receptors, comprising combinations of subunits IL-10R, IL-10R{beta}, IL-22R and IL-28R, modulates intestinal inflammatory responses. Here we carefully dissected the role of these subunits in the resistance of mice to infection with T. muris, a mouse model of the human whipworm T. trichiura. Our findings demonstrate that whilst IL-22R and IL-28R are dispensable in the host response to whipworms, IL-10 signalling through IL-10R and IL-10R{beta} is essential to control caecal pathology, worm expulsion and survival during T. muris infections. We show that deficiency of IL-10, IL-10R and IL-10R{beta} results in dysbiosis of the caecal microbiota characterised by expanded populations of opportunistic bacteria of the families Enterococcaceae and Enterobacteriaceae. Moreover, breakdown of the epithelial barrier after whipworm infection in IL-10, IL-10R and IL-10R{beta}-deficient mice, allows the translocation of these opportunistic pathogens or their excretory products to the liver causing organ failure and lethal disease. Importantly, bone marrow chimera experiments indicate that signalling through IL-10R and IL-10R{beta} in haematopoietic cells, but not IECs, is crucial to control worm expulsion and immunopathology. These findings are supported by worm expulsion upon infection of conditional mutant mice for the IL-10R on IECs. Our findings emphasize the pivotal role of systemic IL-10R signalling on immune cells in promoting microbiota homeostasis and maintaining the intestinal epithelial barrier, thus preventing immunopathology during whipworms infections.\n\nAuthor summaryThe human gut is home to millions of bacteria, collectively called the microbiota, and also to parasites that include whipworms. The interactions between gut cells, the microbiota and whipworms define conditions for balanced parasitism. Cells lining the gut host whipworms but also interact with gut immune cells to deploy measures that control or expel whipworms whilst maintaining a barrier to prevent microbial translocation. Whipworms affect the composition of the microbiota, which in turn impacts the condition of the gut lining and the way in which immune cells are activated. In order to avoid tissue damage and disease, these interactions are tightly regulated. Here we show that signalling through a member of the IL-10 receptor family, IL-10R, in gut immune cells is critical for regulating of these interactions. Lack of this receptor on gut immune cells results in persistence of whipworms in the gut accompanied by an uncontrolled inflammation that destroys the gut lining. This tissue damage is accompanied by the overgrowth of members of the microbiota that act as opportunistic pathogens. Furthermore, the destruction of the gut barrier allows these bacteria to reach the liver where they cause organ failure and fatal disease.

immunology↗

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggests a differential effect on twitching motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

genomics↗