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Journal articleSchuster CF, Wiedemann DM, Kirsebom FCM, et al., 2020,
High‐throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors
, Molecular Microbiology, Vol: 113, Pages: 699-717, ISSN: 0950-382XStaphylococcus aureus is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long‐term exposure. In this study, we used TN‐seq to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long‐term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene SAUSA300_0957 (gene 957) as essential under salt stress. Interestingly, a 957 mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.
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Conference paperXu X, van Sorge N, van der Lans S, et al., 2020,
Structural and Interaction Insight in <it>Streptococcal</it> beta C Proteins
, Annual Meeting on Experimental Biology, Publisher: WILEY, ISSN: 0892-6638 -
Journal articleSanchez Garrido J, Slater SL, Clements A, et al., 2020,
Vying for the control of inflammasomes: the cytosolic frontier of enteric bacterial pathogen - host interactions
, Cellular Microbiology, Vol: 22, Pages: 1-19, ISSN: 1462-5814Enteric pathogen-host interactions occur at multiple interfaces,includingthe intestinal epitheliumand deeper organsof the immune system. Microbial ligands and activities are detected by host sensorsthat elicit a range of immune responses. Membrane-bound Toll-Like Receptors (TLRs) and cytosolic inflammasomepathways are key signal transducers that trigger production of pro-inflammatory molecules such as cytokines and chemokinesand regulate cell deathin response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tusslebetween bacterial pathogens and the host. Inflammasomes are complexes that activate caspase-1and are regulated by related caspases, such as caspase-11, -4, -5 and -8.Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasomeactivation(e.g. Listeria monocytogenes, Helicobacter pylori), others(e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system (T3SS) effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasomepathwaysand their immune functions and discuss how enteric bacterial pathogens interact with them.These studies have not only shed light on the inflammasome-mediated immunity, but also the exciting area of mammalian cytosolic immune
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Journal articleLarrouy-Maumus G, Shahrezaei V, tang W, et al., 2020,
Discrimination of bovine milk from non-dairy milk by lipids fingerprinting using routine matrix-assisted laser desorption ionization mass spectrometry
, Scientific Reports, Vol: 10, ISSN: 2045-2322An important sustainable development goal for any country is to ensure food security by producing a sufficient and safe food supply. This is the case for bovine milk where addition of non-dairy milks such as vegetables (e.g., soya or coconut) has become a common source of adulteration and fraud. Conventionally, gas chromatography techniques are used to detect key lipids (e.g., triacylglycerols) has an effective read-out of assessing milks origins and to detect foreign milks in bovine milks. However, such approach requires several sample preparation steps and a dedicated laboratory environment, precluding a high throughput process. To cope with this need, here, we aimed to develop a novel and simple method without organic solvent extractions for the detection of bovine and non-dairy milks based on lipids fingerprint by routine MALDI-TOF mass spectrometry (MS). The optimized method relies on the simple dilution of milks in water followed by MALDI-TOF MS analyses in the positive linear ion mode and using a matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (super-DHB) solubilized at 10 mg/mL in 70% ethanol. This sensitive, inexpensive, and rapid method has potential for use in food authenticity applications.
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Journal articleWalter A, Unsleber S, Rismondo J, et al., 2020,
Phosphoglycerol-type wall- and lipoteichoic acids are enantiomeric polymersdifferentiated by the stereospecific glycerophosphodiesterase GlpQ
, Journal of Biological Chemistry, Vol: 12, Pages: 4024-4034, ISSN: 0021-9258The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.
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Journal articleSingh Khara J, Mojsoska B, Mukherjee D, et al., 2020,
Ultra-short antimicrobial peptoids show propensity for membrane activity against multi-drug resistant Mycobacterium tuberculosis
, Frontiers in Microbiology, Vol: 11, Pages: 1-11, ISSN: 1664-302XTuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.
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Journal articleMatthews S, McKenna S, Malito E, et al., 2020,
Structure, dynamics and immunogenicity of a catalytically inactive CXC Chemokine-degrading Protease SpyCEP from Streptococcus pyogenes
, Computational and Structural Biotechnology Journal, Vol: 18, Pages: 650-660, ISSN: 2001-0370Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.
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Journal articleMichael DR, Jack AA, Masetti G, et al., 2020,
A randomised controlled study shows supplementation of overweight and obese adults with lactobacilli and bifidobacteria reduces bodyweight and improves well-being
, Scientific Reports, Vol: 10<jats:title>Abstract</jats:title><jats:p>In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25–34.9 kg/m<jats:sup>2</jats:sup> received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, <jats:italic>p</jats:italic> < 0.0001), BMI (0.045 kg/m<jats:sup>2</jats:sup>, <jats:italic>p</jats:italic> < 0.0001), WC (0.94 cm, <jats:italic>p</jats:italic> < 0.0001) and WtHR (0.006, <jats:italic>p</jats:italic> < 0.0001) were in favour of the probiotic. Stratification identified greater body weight reductions in overweight subjects (1.88%, <jats:italic>p</jats:italic> < 0.0001) and in females (1.62%, <jats:italic>p</jats:italic> = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (−2.5%, <jats:italic>p</jats:italic> < 0.0001) alongside a significant between group reduction in small dense LDL-cholesterol (0.2 mmol/L, <jats:italic>p</jats:italic> = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, <jats:italic>p</jats:italic> <&thins
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ReportOtter J, Brophy K, Palmer J, et al., 2020,
Smart surfaces to tackle infection and antimicrobial resistance
, Briefing Paper -
Journal articleOvadia C, Perdones-Montero A, Fan HM, et al., 2020,
Ursodeoxycholic acid enriches intestinal bile salt hydrolase-expressing Bacteroidetes in cholestatic pregnancy
, Scientific Reports, Vol: 10<jats:title>Abstract</jats:title><jats:p>Ursodeoxycholic acid (UDCA) treatment can reduce itch and lower endogenous serum bile acids in intrahepatic cholestasis of pregnancy (ICP). We sought to determine how it could influence the gut environment in ICP to alter enterohepatic signalling. The gut microbiota and bile acid content were determined in faeces from 35 pregnant women (14 with uncomplicated pregnancies and 21 with ICP, 17 receiving UDCA). Faecal bile salt hydrolase activity was measured using a precipitation assay. Serum fibroblast growth factor 19 (FGF19) and 7α-hydroxy-4-cholesten-3-one (C4) concentrations were measured following a standardised diet for 21 hours. Women with a high ratio of<jats:italic>Bacteroidetes</jats:italic>to<jats:italic>Firmicutes</jats:italic>were more likely to be treated with UDCA (Fisher’s exact test p = 0.0178) than those with a lower ratio. Bile salt hydrolase activity was reduced in women with low<jats:italic>Bacteroidetes</jats:italic>:<jats:italic>Firmicutes</jats:italic>. Women taking UDCA had higher faecal lithocholic acid (p < 0.0001), with more unconjugated bile acids than women with untreated ICP or uncomplicated pregnancy. UDCA-treatment increased serum FGF19, and reduced C4 (reflecting lower bile acid synthesis). During ICP, UDCA treatment can be associated with enrichment of the gut microbiota with<jats:italic>Bacteroidetes</jats:italic>. These demonstrate high bile salt hydrolase activity, which deconjugates bile acids enabling secondary modification to FXR agonists, enhancing enterohepatic feedback via FGF19.</jats:p>
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