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Journal articleHolmes AH, Gill SK, Hui K, et al., 2016,
Increased airway glucose increases airway bacterial load in hyperglycaemia
, Scientific Reports, Vol: 6, ISSN: 2045-2322Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection.We hypothesize that increased airway glucose caused by hyperglycaemia leads to increasedbacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisationis significantly more likely when blood glucose is high. We engineered mutants in genesaffecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa,strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented withglucose as the sole carbon source. The effect of glucose on growth in vivo was tested usingstreptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose.Bacterial burden in hyperglycaemic animals was greater than control animals when infected withwild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reducedboth airway glucose and bacterial load. These data support airway glucose as a criticaldeterminant of increased bacterial load during diabetes.
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Journal articleRolhion N, Furniss R, Grabe G, et al., 2016,
Inhibition of nuclear transport of NF-kB p65 by the Salmonella type III secretion system effector SpvD
, Plos Pathogens, Vol: 12, Pages: 1-26, ISSN: 1553-7374Salmonella enterica replicates in macrophages through the action of effector proteins translocated across the vacuolar membrane by a type III secretion system (T3SS). Here we show that the SPI-2 T3SS effector SpvD suppresses proinflammatory immune responses. SpvD prevented activation of an NF-ĸB-dependent promoter and caused nuclear accumulation of importin-α, which is required for nuclear import of p65. SpvD interacted specifically with the exportin Xpo2, which mediates nuclear-cytoplasmic recycling of importins. We propose that interaction between SpvD and Xpo2 disrupts the normal recycling of importin-α from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-ĸB regulated promoters. SpvD down-regulated pro-inflammatory responses and contributed to systemic growth of bacteria in mice. This work shows that a bacterial pathogen can manipulate host cell immune responses by interfering with the nuclear transport machinery.
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Journal articleHergott CB, Roche AM, Tamashiro E, et al., 2016,
Detection of peptidoglycan from the gut microbiota governs the lifespan of circulating phagocytes at homeostasis
, Blood, Vol: 127, Pages: 2460-2471, ISSN: 1528-0020Maintenance of myeloid cell homeostasis requires continuous turnover of phagocytes from the bloodstream, yet whether environmental signals influence phagocyte longevity in the absence of inflammation remains unknown. Here, we show that the gut microbiota regulates the steady-state cellular lifespan of neutrophils and inflammatory monocytes, the two most abundant circulating myeloid cells and key contributors to inflammatory responses. Treatment of mice with broad-spectrum antibiotics, or with the gut-restricted aminoglycoside neomycin alone, accelerated phagocyte turnover and increased the rates of their spontaneous apoptosis. Metagenomic analyses revealed that neomycin altered the abundance of intestinal bacteria bearing γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a ligand for the intracellular peptidoglycan sensor Nod1. Accordingly, signaling through Nod1 was both necessary and sufficient to mediate the stimulatory influence of the flora on myeloid cell longevity. Stimulation of Nod1 signaling increased the frequency of lymphocytes in the murine intestine producing the pro-inflammatory cytokine interleukin 17A (IL-17A), and liberation of IL-17A was required for transmission of Nod1-dependent signals to circulating phagocytes. Together, these results define a mechanism through which intestinal microbes govern a central component of myeloid homeostasis and suggest perturbations of commensal communities can influence steady-state regulation of cell fate.
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Journal articlePercy M, Karinou E, Webb A, et al., 2016,
Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications
, Journal of Bacteriology, Vol: 198, Pages: 2029-2042, ISSN: 1098-5530Listeria monocytogenes is a food-borne Gram-positive bacterial pathogen and many of its virulence factors are either secreted proteins, or proteins covalently or non-covalently-attached to the cell wall. Previous work has indicated that non-covalently-attached proteins with GW domains are retained in the cell wall by binding to the cell wall polymer lipoteichoic acid (LTA). LTA is a glycerolphosphate polymer, which is modified in L. monocytogenes with galactose and D-alanine residues. We identified Lmo0933 as the cytoplasmic glycosyltransferase required for the LTA glycosylation process and renamed the protein GtlA for glycosyltransferase LTA A. Using L. monocytogenes mutants lacking galactose or D-alanine modifications or the complete LTA polymer, we show that GW-domain proteins are still retained within the cell wall, indicating that other cell wall polymers are involved in the retention of GW-domain proteins. Further experiments reveal peptidoglycan as the binding receptor as a purified GW domainfusion protein can bind to L. monocytogenes cells lacking wall teichoic acid (WTA) as well as purified peptidoglycan derived from a wild-type or WTA-negative strain. With this, we not only identified the first enzyme involved in the LTA glycosylation process, but we also provide new insight into the binding mechanism of non-covalently attached cell wall proteins.
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Journal articleSantos AJM, Durkin C, Helaine S, et al., 2016,
Clustered intracellular Salmonella Typhimurium Blocks Host Cell Cytokinesis
, Infection and Immunity, Vol: 84, Pages: 2149-2158, ISSN: 1098-5522Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonise the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonisation. We found that intracellular Salmonella enterica Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 hours post invasion, which was dependent on an intact Salmonella pathogenicity island-2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organising centre of infected epithelial cells. During host cell division these clustered microcolonies prevented the correct localisation of members of the chromosomal passenger complex and mitotic kinesin-like protein 1, and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, either resulting in chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S. Typhimurium delays epithelial cell turnover in the intestine.
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Journal articleLund-Palau H, Turnbull AR, Bush A, et al., 2016,
Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches
, Expert Review of Respiratory Medicine, Vol: 10, Pages: 685-697, ISSN: 1747-6348Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.
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Journal articleChambonnier G, Roux L, Redelberger D, et al., 2016,
The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa.
, PLOS Genetics, Vol: 12, ISSN: 1553-7390In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.
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Journal articleArmstrong-James DPH, 2016,
Calcineurin Orchestrates Lateral Transfer of Aspergillus fumigatus during Macrophage Cell Death
, American Journal of Respiratory and Critical Care Medicine, Vol: 194, Pages: 1127-1139, ISSN: 1535-4970Rationale: Pulmonary aspergillosis is a lethal mold infection in the immunocompromised host. Understanding initial control of infection and how this is altered in the immunocompromised host are key goals for comprehension of the pathogenesis of pulmonary aspergillosis.Objectives: To characterize the outcome of human macrophage infection with Aspergillus fumigatus and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants.Methods: We defined the outcome of human macrophage infection with A. fumigatus, as well as the impact of calcineurin inhibitors, through a combination of single-cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies.Measurements and Main Results: Macrophage phagocytosis of A. fumigatus enabled control of 90% of fungal germination. However, fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell–cell transfer of A. fumigatus between macrophages, which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein envelope. Its relevance to the control of fungal germination was also shown by direct visualization in our zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506 (tacrolimus) reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and also resulted in hyphal escape.Conclusions: These observations identify programmed, necrosis-dependent lateral transfer of A. fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. Our studies provide fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.
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Journal articleKhara JS, Priestman M, Uhia I, et al., 2016,
Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability
, Journal of Antimicrobial Chemotherapy, Vol: 71, Pages: 2181-2191, ISSN: 1460-2091Objectives The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities.Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy.Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm.Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.
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Journal articleValentini M, Filloux A, 2016,
Biofilms and cyclic di-GMP (c-di-GMP) signaling: lessons from Pseudomonas aeruginosa and other bacteria
, Journal of Biological Chemistry, Vol: 291, Pages: 12547-12555, ISSN: 1083-351XThe cyclic-di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation and transmission.
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