Papers by Richard Strugnell
Mucosal Immunology, 2016
Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (... more Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (Ags), the precise requirements for in vivo MAITcell responses to infection remain unclear. Using major histocompatibility complex-related protein 1 (MR1) tetramers, the MAIT cell response was investigated in a model of bacterial lung infection employing riboflavin gene-competent and -deficient bacteria. MAIT cells were rapidly enriched in the lungs of C57BL/6 mice infected with Salmonella Typhimurium, comprising up to 50% of ab-T cells after 1 week. MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long-term accumulation in the lungs, draining lymph nodes and spleen. Lung MAITcells from infected mice displayed an activated/memory phenotype, and most expressed the transcription factor retinoic acid-related orphan receptor ct. T-bet expression increased following infection. The majority produced interleukin-17 while smaller subsets produced interferon-c or tumor necrosis factor, detected directly ex vivo. Thus the activation and expansion of MAIT cells coupled with their pro-inflammatory cytokine production occurred in response to Ags derived from microbial riboflavin synthesis and was augmented by co-stimulatory signals.
Molecular & Cellular Proteomics, 2019
Strains of Salmonella utilise two distinct type three secretion systems to deliver effector prote... more Strains of Salmonella utilise two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, while ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg 376 and Arg 293 respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.
Cytokine
Infection with Helicobacter pylori causes chronic inflammation and damage to the gastric epitheli... more Infection with Helicobacter pylori causes chronic inflammation and damage to the gastric epithelium. This leads to chronic gastritis, and in a proportion of patients, ulcer, or gastric cancer. Candidate H. pylori vaccines tested in volunteers to date have proved poorly immunogenic and/or to induce adverse reactions. Indeed there is some evidence that “post immunisation gastritis” occurs because the tightly controlled inflammatory response in the gastric mucosa is unbalanced [1] . Vaccination is effective in animal models, and CD4 + T cells, and both the adipokine leptin (Ob) [2] and interleukin 17 (IL-17) [3] have been show to be essential for vaccine-induced protection against H. pylori . Leptin is pro-inflammatory, and impacts CD4+ T cell, regulatory CD4+ T cell (T reg ), and even neutrophil function. IL-17 promotes neutrophil recruitment into tissues, and inflammatory cytokine production by macrophages. Aims To investigate the roles of leptin receptor (ObR) signalling and IL-17 in the vaccine- induced immune responses against H. pylori . Methods We studied responses in vaccinated wild-type C57BL/6 and ObR-signalling deficient C57BL/6 CgObR db/db mice ( db/db ). Results Vaccinated db/db mice are not protected from H. pylori challenge, we find also that gastric IL-17 production was reduced in these mice. In the stomachs of wild-type mice, both leptin and ObR are expressed on epithelial, CD4 + T cells, CD4 + T reg , macrophages and neutrophils. Further, bone marrow chimaera studies revealed that functional ObR on BM-derived cells was essential for vaccine- induced protection. Notably, Th17 cells in the gastric mucosa of vaccinated, protected WT mice also secrete leptin. Conclusions Our data support a link between leptin and Th-17 responses in gastric inflammation. We hypothesise that T-cell derived leptin impacts both T reg function and IL-17 production, regulating protective responses in the vaccinated stomach.
PLoS Pathogens, 2010
Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from ... more Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans worldwide. S. typhimurium (S. tm att , sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRb 2/2 d 2/2 , J H 2/2 , IgA 2/2 , pIgR 2/2). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using 'L-mice' which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm att from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance (= pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most ''classical'' immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.
EcoSal, 2006
The best-characterized mucosa-associated lymphoid tissue (MALT), and also the most relevant for t... more The best-characterized mucosa-associated lymphoid tissue (MALT), and also the most relevant for this review, is the gastrointestinal-associated lymphoid tissue (GALT). The review reviews our understanding of the importance of mucosal immune responses in resisting infections caused by E. coli and Salmonella spp. It focuses on the major human E. coli infections and discusses whether antigen-specific mucosal immune responses are important for resistance against primary infection or reinfection by pathogenic E. coli . It analyzes human data on mucosal immunity against E. coli , a growing body of data of mucosal responses in food production animals and other natural hosts of E. coli , and more recent experimental studies in mice carrying defined deletions in genes encoding specific immunological effectors, to show that there may be considerable conservation of the effective host mucosal immune response against this pathogen. The species Salmonella enterica contains a number of serovars that include pathogens of both humans and animals; these bacteria are frequently host specific and may cause different diseases in different hosts. Ingestion of various Salmonella serovars, such as Typhimurium, results in localized infections of the small intestine leading to gastroenteritis in humans, whereas ingestion of serovar Typhi results in systemic infection and enteric fever. Serovar Typhi infects only humans, and the review discusses the mucosal immune responses against serovar Typhi, focusing on the responses in humans and in the mouse typhoid fever model.
Microbial Pathogenesis, 1993
Immunologic Research, 2004
The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antib... more The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antibody (Ab), CD4 knockout (CD4-/-) and major histocompatibility complex (MHC) class II knockout (class II-/-) mice. We recently described the anti-CD4 Ab transgenic mouse (GK) as an improved CD4 cell-deficient model. This review compares this new GK mouse model with the widely available class II-/and CD4-/mice, when exposed to complex antigens (foreign grafts and during bacterial or viral infection). We highlight here the cytometric and functional differences (including Ab isotype, viral or bacterial clearance, and graft survival) among these CD4 cell-deficient models. For example, whereas grafts are generally rejected in class II-/and CD4-/mice as quickly as in wild-type mice, they survive longer in GK mice. Also, CD4-/mice produce IgG against both simple model and complex antigens, but class II-/and GK mice produce small amounts of IgG2a against complex antigens but not simple model antigens. These differences harbinger the caveats in the use of these various mice.
Towards a Global Core Value System in Doctoral Education
Figure S1 IgA and IgG levels in the serum and faeces indicate selective depletion of SIgA in pIgR... more Figure S1 IgA and IgG levels in the serum and faeces indicate selective depletion of SIgA in pIgRâ /â mice. (DOCX 576 kb)
Molecular Medicine, 2019
Background: The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial ba... more Background: The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial barriers by transporting polymeric antibodies and antigens through the epithelial mucosa into the lumen. In this study, we examined the role of pIgR in maintaining gut barrier integrity, which is important for the normal development in mice. Methods: Cohorts of pIgR -/-mice and their wildtype controls were housed under Specific Pathogen Free (SPF) conditions and monitored for weight gain as an indicator of development over time. The general physiology of the gastrointestinal tract was analysed using immunohistochemistry in young (8-12 weeks of age) and aged mice (up to 18 months of age), and the observed immunopathology in pIgR -/-mice was further characterised using flow cytometry. Urinary metabolites were analysed using gas chromatography-mass spectrometry (GC-MS), which revealed changes in metabolites that correlated with age-related increase in gut permeability in pIgR -/-mice. We observed that pIgR -/-mice exhibited delayed growth, and this phenomenon is associated with lowgrade gut inflammation that increased with ageing. The gross intraepithelial lymphocytic (IEL) infiltration characteristic of pIgR -/-mice was redefined as CD8α + αβ + T cells, the majority of which expressed high levels of CD103 and CD69 consistent with tissue resident memory T cells (T RM ). Comparison of the urinary metabolome between pIgR -/-and wild-type mice revealed key changes in urinary biomarkers fucose, glycine and Vitamin B5, suggestive of altered mucosal permeability. A significant increase in gut permeability was confirmed by analysing the site-specific uptake of sugar probes in different parts of the intestine. Conclusion: Our data show that loss of the secretory antibody system in mice results in enhanced accumulation of inflammatory IELs in the gut, which likely reflects ongoing inflammation in reaction to gut microbiota or food antigens, leading to delayed growth in pIgR -/-mice. We demonstrate that this leads to the presence of a unique urinary metabolome profile, which may provide a biomarker for altered gut permeability.
Medical Journal of Indonesia
Keberhasilan uji serta peredaran vaksin baru berasaL dari mutan S. typhi yang dilemahkan telah me... more Keberhasilan uji serta peredaran vaksin baru berasaL dari mutan S. typhi yang dilemahkan telah membuka kesempatan untuk mengembangkan vaksin-vaksin bivalen atau multivalen yang baru. Pengembangan vaksin semacam ini perlu memperhatikan analisis yang teliti terhadap sistem optimal ekspresi gen yang dipakm (closis gen, kekuatan prometer dsb) untuk menjamin bahwa rekombinan s' typhi menimbulkan respons maksimal dibandingknn antigen heterolog yang diekpresi bersamanya. Penelitian ini mengembangkan model ,àkri, biuol", S. typhimurium yang dicoba pada mencit tifuid. seb gai model antigen digunakan fragmen c toksin tetanus (TT) ltang ternyata sangat imunogenik pa'da mencit biitt diekspresikan dengan rekombinan protein dari s. typhimvitm yang dilemahkan' Pada penelitian ni aitetiti berbagai parameter yang penting seperti mutasi yang mungkin ada, jumlah plasmid dan stabilitastrya, kekuatnn prometer dan efek translasi. Mutan gen yang isogenik seperrr purA, aroA, aroA"/aroD, htrA dan ompR darl S. typhimurium SL 1344 akan clibandingkan tlengan mutan cyalcrp dari SR-lI dalam' hal pertumbuhan it vivo dart penetrasi ke organ linrfuid dan dibantlingknn pula clengan imurtisasi fragmen C. Efek protektif dari antibodi spesifik-TT tlitemuknn pada seluruh rekombinan Salmonella kecuali mutan purA. Reaksl immtogenisitas terbukti tidak ada hubungannya dengan kemampuan kuman melnkukan penetrasi ke limpa dan tidak berhubungan pukt lengan respons spesifi.k sel T; namun berkorelasi tlengan koLonisasi kuman daLam plak Peyer Imunogenitas maksimaL dari konstruksi plasmid yang'mengandung fragmen C berkorelasi dengan stabilitas plasmid; dan replikon dengan jumlah)tang rendah, namun stabil (mis. pRSF NIT) akàn sangat efektif sebagai wahana pembawavaksin. Fragmen c yang diekspresiknn oleh col El plasmid pBR 322 clm derivatnya pAT 153 dapat menginduksi antibodi spesifik TT dalam level yang tinggi, sedangkan clerivat plttsmid pIC21H yang jumlah plnsmid/sel sangat tiuggi ternyata justru tidak memacu respon imun spesifik-TT, dan juga tidak stabil in vivo. Gen ,pagC, ,nirB clan ,kaLG yang *"rr1roko,, pio*ot"r yorrg diregulasi in vivo dibandingkan dengan promoter constitutive ,trc tlalam hal ekspresi ln vivo antigen reporter firefly luciferase, dan dalam hal imunogenitas konstruksifragmen C tnvivo. Promoter in vivo yang terkuat (ptrc) tentyàa kurang efektif pada imunisasi dibandingkan dengan promoter yatxg diregulasi in vivo (opagC)' Informasi ini akan digunakan untuk secara optimàl mengekspresikan antigen heterolog lain pada mtûan rekombinan S. typhimurium, dengan tujuan akhir mengembangknn vaksin bivalen S. typhi yang baru.
MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many... more MHC class II haplotypes control the specificity of Th immune responses and susceptibility to many autoimmune diseases. Understanding the role of HLA class II haplotypes in immunity is hampered by the lack of animal models expressing these genes as authentic cis-haplotypes. In this study we describe transgenic expression of the autoimmune prone HLA DR3-DQ2 haplotype from a yeast artificial chromosome (YAC) containing an intact ϳ320-kb region from HLA DRA to DQB2. In YAC-transgenic mice HLA DR and DQ gene products were expressed on B cells, macrophages, and dendritic cells, but not on T cells indicating cell-specific regulation. Positive selection of the CD4 compartment by human class II molecules was 67% efficient in YAC-homozygous mice lacking endogenous class II molecules (A null/null) and expressing only murine CD4. A broad range of TCR V families was used in the peripheral T cell repertoire, which was also purged of V5-, V11-, and V12-bearing T cells by endogenous mouse mammary tumor virus-encoded superantigens. Expression of the HLA DR3-DQ2 haplotype on the A null/null background was associated with normal CD8-dependent clearance of virus from influenza-infected mice and development of CD4-dependent protection from otherwise lethal infection with Salmonella typhimurium. HLA DR-and DQ-restricted T cell responses were also elicited following immunization with known T cell determinants presented by these molecules. These findings demonstrate the potential for human MHC class II haplotypes to function efficiently in transgenic mice and should provide valuable tools for developing humanized models of MHC-associated autoimmune diseases.
Virology Journal
Introduction The sudden arrival of the COVID-19 pandemic placed significant stresses on supply ch... more Introduction The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. Methods VTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. Results The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM...
mSphere
O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of ... more O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However...
Genome Biology
Understanding the secretion of an enterotoxin by enterotoxigenic Escherichia coli strains paves t... more Understanding the secretion of an enterotoxin by enterotoxigenic Escherichia coli strains paves the way to the development of new drugs Significance and context To date, four classes of enterovirulent Escherichia coli strains have been described, all of which cause gastroenteritis in humans. Among these are the enterotoxigenic E. coli (ETEC) strains that have been associated with diarrheal illness in all age groups from diverse locations. Infection with ETEC causes a cholera-like diarrhea in infants in less-developed countries and in visitors there from industrialized countries. (A better known name of the disease is traveler's diarrhea.) Every year, approximately 380,000 children less than 5 years old die from illness caused by infection with ETEC. It has been demonstrated that ETEC strains produce and secrete one or both types of enterotoxins, namely heatstable enterotoxin (ST) and heat-labile enterotoxin (LT); the ST form survives boiling for 30 minutes, in contrast to the LT form. A better understanding of the toxin-secretion systems could improve prevention and treatment of ETEC-induced illness. Although detailed studies on the action of LT have been performed, how it is secreted by ETEC remains largely unknown. LT and the cholera toxin (CT), produced by Vibrio cholerae, are structurally and biologically related. V. cholerae secretes both plasmid-encoded LT and CT, a process which takes place in two steps. First, two subunits are translocated towards the periplasm, where they undergo folding and assembly. Second, the holotoxin is transported across the outer membrane via a type-II protein secretion pathway. Upon introduction of the plasmids into the nonpathogenic E. coli K-12 strain, however, LT and CT accumulate mainly in the periplasm, in contrast to ETEC LT-secreting isolates from humans. Tauschek et al. characterized a type-II protein secretion pathway present in ETEC but absent from E. coli K-12, and demonstrated that this pathway is required for the secretion of the LT. Key results Previously, a type-II secretion system was identified in a rabbit-specific enteropathogenic E. coli strain. This sequence was used as a template to design primers that were used to isolate a similar genetic locus in the human ETEC strain H10407. A locus of 11,765 bp was isolated that contained 13 open
International Journal of Environmental Research and Public Health
Proximal characteristics and conditions in the residential setting deserve greater attention for ... more Proximal characteristics and conditions in the residential setting deserve greater attention for their potential to influence typhoid transmission. Using a case-control design in Central Division, Republic of Fiji, we examined bacterial (coliform and Escherichia coli) contamination and chemical composition of water and soil as potential vehicles of exposure to Salmonella Typhi, combining observational analysis of residential living conditions, geospatial analysis of household locations, and factor analysis to explore multivariate associations with the risk of developing typhoid fever. Factors positively associated with typhoid infection related to drainage [phosphate (OR 4.235, p = 0.042) and E. coli concentrations (OR 2.248, p = 0.029) in toilet drainage soil, housing [external condition (OR 3.712, p < 0.001)], drinking water contamination (OR 2.732, p = 0.003) and sanitary condition (OR 1.973, p = 0.031). These five factors explained 42.5% of the cumulative variance and were si...
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Papers by Richard Strugnell