ORCID Profile
0000-0002-9255-9033
Current Organisation
Leica Microsystems GmbH
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Microbiology | Microbial Genetics | Enzymes | Bacteriology | Biological Physics | Bacteriology
Expanding Knowledge in the Chemical Sciences | Infectious diseases | Expanding Knowledge in the Biological Sciences | Preventive medicine | Higher education | Expanding Knowledge in the Medical and Health Sciences |
Publisher: Hindawi Limited
Date: 03-2010
DOI: 10.1111/J.1462-5822.2009.01404.X
Abstract: Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kappaB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.
Publisher: Springer Science and Business Media LLC
Date: 03-2015
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-0473-0_9
Abstract: Twitching motility is a mode of solid surface translocation that occurs under humid conditions on semisolid or solid surfaces, is dependent on the presence of retractile type IV pili and is independent of the presence of a flagellum. Surface translocation via twitching motility is powered by the extension and retraction of type IV pili and can manifest as a complex multicellular collective behavior that mediates the active expansion of colonies cultured on the surface of solidified nutrient media, and of interstitial colonies that are cultured at the interface between solidified nutrient media and an abiotic material such as the base of a petri dish or a glass coverslip. Here we describe two methods for assaying twitching motility mediated interstitial colony expansion in P. aeruginosa. The first method, the "Macroscopic Twitching Assay," can be used to determine if a strain is capable of twitching motility mediated interstitial colony expansion and can also be used to quantitatively assess the influence of mutation or environmental signals on this process. The second method, the "Microscopic Twitching Assay," can be used for detailed interrogation of the movements of in idual cells or small groups of bacteria during twitching motility mediated colony expansion.
Publisher: Cold Spring Harbor Laboratory
Date: 28-10-2020
DOI: 10.1101/2020.10.28.358747
Abstract: Bacterial biofilms are comprised of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.VETMIC.2015.03.025
Abstract: Dichelobacter nodosus is the essential causative agent of footrot in sheep and type IV fimbriae-mediated twitching motility has been shown to be essential for virulence. We have identified a two-component signal transduction system (TwmSR) that shows similarity to chemosensory systems from other bacteria. Insertional inactivation of the gene encoding the response regulator, TwmR, led to a twitching motility defect, with the mutant having a reduced rate of twitching motility when compared to the wild-type and a mutant complemented with the wild-type twmR gene. The reduced rate of twitching motility was not a consequence of a reduced growth rate or decreased production of surface located fimbriae, but video microscopy indicated that it appeared to result from an overall loss of twitching directionality. These results suggest that a chemotactic response to environmental factors may play an important role in the D. nodosus-mediated disease process.
Publisher: American Society for Microbiology
Date: 04-2014
DOI: 10.1128/AAC.01901-13
Abstract: The Gram-negative human pathogen Pseudomonas aeruginosa tolerates high concentrations of β-lactam antibiotics. Despite inhibiting the growth of the organism, these cell wall-targeting drugs exhibit remarkably little bactericidal activity. However, the mechanisms underlying β-lactam tolerance are currently unclear. Here, we show that P. aeruginosa undergoes a rapid en masse transition from normal rod-shaped cells to viable cell wall-defective spherical cells when treated with β-lactams from the widely used carbapenem and penicillin classes. When the antibiotic is removed, the entire population of spherical cells quickly converts back to the normal bacillary form. Our results demonstrate that these rapid population-wide cell morphotype transitions function as a strategy to survive antibiotic exposure. Taking advantage of these findings, we have developed a novel approach to efficiently kill P. aeruginosa by using carbapenem treatment to induce en masse transition to the spherical cell morphotype and then exploiting the relative fragility and sensitivity of these cells to killing by antimicrobial peptides (AMPs) that are relatively inactive against P. aeruginosa bacillary cells. This approach could broaden the repertoire of antimicrobial compounds used to treat P. aeruginosa and serve as a basis for developing new therapeutic agents to combat bacterial infections.
Publisher: Wiley
Date: 12-07-2013
DOI: 10.1111/MMI.12306
Abstract: The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.
Publisher: Public Library of Science (PLoS)
Date: 28-02-2013
Publisher: Public Library of Science (PLoS)
Date: 13-02-2013
Publisher: Hindawi Limited
Date: 22-11-2014
DOI: 10.1111/CMI.12377
Abstract: Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells.
Publisher: Informa UK Limited
Date: 09-11-2013
DOI: 10.4161/CIB.27331
Publisher: MyJove Corporation
Date: 29-09-2014
DOI: 10.3791/51469
Publisher: Wiley
Date: 28-03-2021
DOI: 10.1111/EJSS.13105
Abstract: Biochar is recommended as a soil amendment for its positive influence on soil hydrological properties, which results in improved soil fertility and crop yield. Much research in the last decade has been conducted in field and laboratory conditions on the effect of biochar on the hydraulic properties of soil. However, reported results in the literature are substantially inconsistent. Here we performed a meta‐analysis to capture the variations in change in hydraulic properties of arable soils after application of different rates of biochar. The meta‐analysis revealed that high biochar rates ( t ha −1 ) compared to low rates ( t ha −1 ) significantly improved dry bulk density in sandy and clay soils, in field and laboratory experiments. However, field capacity only improved in laboratory experiments on sandy soils. The plant available water, permanent wilting point and saturated hydraulic conductivity did not significantly increase at high rates of biochar application compared to the low rates when applied to different types of soils in both field and laboratory experiments. We discuss possible reasons for this, including hydrophobicity of the biochar with future research directions. We concluded that the current evidence does not support the notion that the application of biochar improves soils' available water capacity. Meta‐analysis clarifies the influence of biochar on soil hydraulic properties. Biochar addition at higher rates only improves the water holding capacity of sandy soils. Biochar types and pyrolysis temperatures do not influence soil hydraulic properties. The efficiency of biochar may depend on its pore size distribution and hydrophobicity.
Publisher: American Physiological Society
Date: 2008
DOI: 10.1152/AJPHEART.00829.2007
Abstract: Increased signaling by G i -coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted G i -coupled receptor (Ro1). Activation of G i signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist nor-binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum Ca 2+ -ATPase ( P 0.05) thereafter, there was progressive impairment of both Ca 2+ handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic Ca 2+ concentration was reduced to 0.61 ± 0.08 vs. 0.91 ± 0.07 μM for control ( n = 6–8 P 0.05), diastolic Ca 2+ concentration was elevated to 0.41 ± 0.07 vs. 0.23 ± 0.06 μM for control ( n = 6–8 P 0.01), and the decline phase of the Ca 2+ transient (time from peak to 50% decline) was slowed to 0.25 ± 0.02 s vs. 0.13 ± 0.02 s for control ( n = 6–8 P 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 ( P 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, Ca 2+ -saturated myofilament force in skinned trabeculae was reduced to 21 ± 2 vs. 38 ± 0.1 mN/mm 2 for controls ( n = 3 P 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of G i -coupled receptors leads to impaired Ca 2+ handling and myofilament injury, contributing to impaired ventricular pump function and heart failure.
Publisher: Springer Science and Business Media LLC
Date: 08-03-2017
DOI: 10.1038/NCOMMS14665
Abstract: DNA-binding proteins are central regulators of chromosome organization however, in genome-reduced bacteria their ersity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.
Publisher: Microbiology Society
Date: 09-2010
Abstract: Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various in vitro models have been developed to study P. aeruginosa pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of P. aeruginosa strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous in vitro models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of P. aeruginosa in CF.
Publisher: Springer Science and Business Media LLC
Date: 03-12-2019
DOI: 10.1038/S41598-019-54576-2
Abstract: Chronic wound treatment is becoming increasingly difficult and costly, further exacerbated when wounds become infected. Bacterial biofilms cause most chronic wound infections and are notoriously resistant to antibiotic treatments. The need for new approaches to combat polymicrobial biofilms in chronic wounds combined with the growing antimicrobial resistance crisis means that honey is being revisited as a treatment option due to its broad-spectrum antimicrobial activity and low propensity for bacterial resistance. We assessed four well-characterised New Zealand honeys, quantified for their key antibacterial components, methylglyoxal, hydrogen peroxide and sugar, for their capacity to prevent and eradicate biofilms produced by the common wound pathogen Pseudomonas aeruginosa . We demonstrate that: (1) honey used at substantially lower concentrations compared to those found in honey-based wound dressings inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms (2) the anti-biofilm effect of honey was largely driven by its sugar component (3) cells recovered from biofilms treated with sub-inhibitory honey concentrations had slightly increased tolerance to honey and (4) honey used at clinically obtainable concentrations completely eradicated established P. aeruginosa biofilms. These results, together with their broad antimicrobial spectrum, demonstrate that manuka honey-based wound dressings are a promising treatment for infected chronic wounds, including those with P. aeruginosa biofilms.
Publisher: American Society for Microbiology
Date: 06-2012
DOI: 10.1128/IAI.00239-12
Abstract: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous Gr1 + CD11b + population of immature cells containing granulocytic and monocytic progenitors, which expand under nearly all inflammatory conditions and are potent repressors of T-cell responses. Studies of MDSCs during inflammatory responses, including sepsis, suggest they can protect or injure. Here, we investigated MDSCs during early and late sepsis. To do this, we used our published murine model of cecal ligation and puncture (CLP)-induced polymicrobial sepsis, which transitions from an early proinflammatory phase to a late anti-inflammatory and immunosuppressive phase. We confirmed that Gr1 + CD11b + MDSCs gradually increase after CLP, reaching ∼88% of the bone marrow myeloid series in late sepsis. Adoptive transfer of early (day 3) MDSCs from septic mice into naive mice after CLP increased proinflammatory cytokine production, decreased peritoneal bacterial growth, and increased early mortality. Conversely, transfer of late (day 12) MDSCs from septic mice had the opposite effects. Early and late MDSCs studied ex vivo also differed in their inflammatory phenotypes. Early MDSCs expressed nitric oxide and proinflammatory cytokines, whereas late MDSCs expressed arginase activity and anti-inflammatory interleukin 10 (IL-10) and transforming growth factor β (TGF-β). Late MDSCs had more immature CD31 + myeloid progenitors and, when treated ex vivo with granulocyte-macrophage colony-stimulating factor (GM-CSF), generated fewer macrophages and dendritic cells than early MDSCs. We conclude that as the sepsis inflammatory process progresses, the heterogeneous MDSCs shift to a more immature state and from being proinflammatory to anti-inflammatory.
Publisher: Elsevier BV
Date: 10-2015
Publisher: IEEE
Date: 12-2010
Publisher: Frontiers Media SA
Date: 23-01-2016
Publisher: Springer Science and Business Media LLC
Date: 07-07-2013
DOI: 10.1007/S10096-013-1916-7
Abstract: Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF). This study examines the role of organism-specific factors in the pathogenesis of very early P. aeruginosa infection in the CF airway. A total of 168 longitudinally collected P. aeruginosa isolates from children diagnosed with CF following newborn screening were genotyped by pulsed-field gel electrophoresis (PFGE) and phenotyped for 13 virulence factors. Ninety-two strains were identified. Associations between virulence factors and gender, exacerbation, persistence, timing of infection and infection site were assessed using multivariate regression analysis. Persistent strains showed significantly lower pyoverdine, rhamnolipid, haemolysin, total protease, and swimming and twitching motility than strains eradicated by aggressive antibiotic treatments. Initial strains had higher levels of virulence factors, and significantly higher phospholipase C, than subsequent genotypically different strains at initial isolation. Strains from males had significantly lower pyoverdine and swimming motility than females. Colony size was significantly smaller in strains isolated during exacerbation than those isolated during non-exacerbation periods. All virulence factors were higher and swimming motility significantly higher in strains from bronchoalveolar lavage (BAL) and oropharyngeal sites than BAL alone. Using unadjusted regression modelling, age at initial infection and age at isolation of a strain showed U-shaped profiles for most virulence factors. Among subsequent strains, longer time since initial infection meant lower levels of most virulence factors. This study provides new insight into virulence factors underpinning impaired airway clearance seen in CF infants, despite aggressive antibiotic therapy. This information will be important in the development of new strategies to reduce the impact of P. aeruginosa in CF.
Publisher: Springer Science and Business Media LLC
Date: 06-12-2018
DOI: 10.1038/S41598-018-36054-3
Abstract: Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae , colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin β1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly (ii) integrin β1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly (iii) anti-integrin β1 antibodies block entry of M. hyopneumoniae into porcine cells and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.
Publisher: Public Library of Science (PLoS)
Date: 20-10-2015
Publisher: Hindawi Limited
Date: 2015
DOI: 10.1155/2015/387342
Abstract: The self-organisation of collective behaviours often manifests as dramatic patterns of emergent large-scale order. This is true for relatively “simple” entities such as microbial communities and robot “swarms,” through to more complex self-organised systems such as those displayed by social insects, migrating herds, and many human activities. The principle of stigmergy describes those self-organised phenomena that emerge as a consequence of indirect communication between in iduals of the group through the generation of persistent cues in the environment. Interestingly, despite numerous ex les of multicellular behaviours of bacteria, the principle of stigmergy has yet to become an accepted theoretical framework that describes how bacterial collectives self-organise. Here we review some ex les of multicellular bacterial behaviours in the context of stigmergy with the aim of bringing this powerful and elegant self-organisation principle to the attention of the microbial research community.
Publisher: American Physiological Society
Date: 05-2006
DOI: 10.1152/AJPHEART.01210.2005
Abstract: Postischemic reperfusion of rat or mouse hearts causes generation of inositol ( 1 , 4 , 5 )trisphosphate [Ins( 1 , 4 , 5 )P 3 ] and the initiation of arrhythmias. In the current study we investigated the possibility that the enhanced Ins( 1 , 4 , 5 )P 3 generation in postischemic reperfusion was associated with an increased availability of the precursor lipid phosphatidylinositol( 4 , 5 )bisphosphate (PIP 2 ) for α 1 -adrenergic receptor-activated phospholipase C (PLC). Isolated-perfused rat hearts were labeled with [ 3 H]inositol and subjected to ischemia-reperfusion or stimulation with norepinephrine under normoxic conditions. Caveolar fractions were prepared by buoyant density sucrose gradient centrifugation. [ 3 H]PIP 2 was concentrated in caveolae, along with Gαq and PLCβ1b. Caveolae contained only 27.3 ± 6.9% (means ± SE, n = 6) of the total α 1 -adrenergic receptor complement of the heart. These did not migrate to PIP 2 -containing caveolar fractions with norepinephrine stimulation under normoxic conditions, even though caveolar PIP 2 was depleted. In contrast, [ 3 H]PIP 2 in caveolae increased during 2 min of reperfusion, independently of norepinephrine release and thus of α 1 -adrenergic receptor activation. The increased PIP 2 in the caveolar fractions where signaling proteins are concentrated may be critical for the heightened generation of Ins( 1 , 4 , 5 )P 3 in early reperfusion.
Publisher: American Physiological Society
Date: 03-2006
DOI: 10.1152/AJPHEART.00732.2005
Abstract: Preconditioning protocols that protect the heart from ischemic injury may aid in the development of new therapies. However, the temporal window of cardioprotection is limited to a few days after the preconditioning stimulus. Here we report a sustained cardioprotected phenotype in mice expressing a tetracycline transactivator (tTA) transcription factor under the control of the α-myosin heavy chain (αMHC) promoter. αMHC-tTA mice were originally designed for tetracycline-regulated gene expression in the heart (Tet system). However, we found that after 45 min of global ischemia at 37°C, left ventricular developed pressure (LVDP) of Langendorff-perfused αMHC-tTA mouse hearts rapidly recovered in 5 min to 60% of initial levels, whereas LVDP of wild-type (WT) littermates recovered to only 10% of the initial level. Improved postischemic recovery of function for αMHC-tTA hearts was associated with a 50% decrease of infarct size and a significantly smaller release of lactate dehydrogenase to the coronary effluent. Improved postischemic recovery was not attributable to differences in coronary flow that was similar for WT- and αMHC-tTA hearts during recovery. Moreover, improved postischemic recovery of αMHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitochondrial ATP-sensitive K + channels, PKC, or adenosine receptors), suggesting a novel mechanism. Finally, the tetracycline analog doxycycline, which inhibits binding of tTA to DNA, did not abolish improved recovery for αMHC-tTA hearts. The sustained cardioprotected phenotype of αMHC-tTA hearts may have implications for developing new therapies to minimize cardiac ischemic injury. Furthermore, investigations of cardioprotection using the Tet system may be aberrantly influenced by sustained preconditioning induced by cardiac transgenesis with tTA.
Publisher: Elsevier BV
Date: 10-2003
DOI: 10.1016/S0022-2828(03)00227-X
Abstract: We used double-knockout mice (ABKO) lacking both predominant myocardial alpha(1)-adrenergic receptor (AR) subtypes (alpha(1A) and alpha(1B)) to determine if alpha(1)-ARs are required for normal myocardial contraction. Langendorff-perfused ABKO hearts had higher developed pressure than wild type (WT) hearts (123 +/- 3 mmHg n = 22 vs. 103 +/- 3 mmHg, n = 38, P < 0.001). Acutely inhibiting alpha(1)-ARs in WT hearts with prazosin did not increase pressure, suggesting that the increased pressure of ABKO hearts was mediated by long-term trophic effects on contraction rather than direct regulatory effects of alpha(1)-AR removal. Similar to perfused hearts, ABKO ventricular trabeculae had higher submaximal force at 2 mM extracellular [Ca(2+)] than WT (11.4 +/- 1.7 vs. 6.9 +/- 0.6 mN/mm(2), n = 8, P 0.05), suggesting ABKO myocardium had increased myofilament Ca(2+)-sensitivity. This conclusion was supported by measuring the Ca(2+)-force relationship using tetanization. Increased myofilament Ca(2+)-sensitivity was not explained by intracellular pH, which did not differ between ABKO and WT (7.41 +/- 0.01 vs. 7.39 +/- 0.02, n = 4-6, P > 0.05 from BCECF fluorescence). However, ABKO displayed impaired troponin I phosphorylation, which may have played a role. In contrast to increased submaximal force, ABKO trabeculae had lower maximal force than WT at high extracellular [Ca(2+)] (29.6 +/- 1.9 vs. 37.6 +/- 1.4 mN/mm(2), n = 7, P 0.05), suggesting ABKO myocardium had impaired myofilament function. Finally, ABKO myocardium had decreased responsiveness to beta-AR stimulation. We conclude: alpha(1)-ARs are required for normal myocardial contraction alpha(1)-ARs mediate long-term trophic effects on contraction loss of alpha(1)-AR function causes some of the functional abnormalities that are also found in heart failure.
Publisher: Wiley
Date: 06-07-2014
DOI: 10.1002/MBO3.187
Publisher: American Society for Clinical Investigation
Date: 23-03-2006
DOI: 10.1172/JCI22811
Publisher: Springer Science and Business Media LLC
Date: 06-02-2006
DOI: 10.1007/S00395-006-0585-4
Abstract: The goal of this study was to determine if an ischemic preconditioning (IPC) protocol improved post-ischemic functional recovery of female mouse hearts. A previous study found that IPC did not occur in hearts from 10-week-old females. We studied Langendorff-perfused hearts from both 10- and 18-week-old mice (males and females). Hearts were subjected to 45 min ischemia and 45 reperfusion (I/R) IPC involved pretreatment with 3 min ischemia. We measured hemodynamics, infarct size and levels of the phosphorylated prosurvival kinase Akt (p-Akt). Similar to a previous study, for 10- week-old mice we found that the IPC protocol appreciably improved recovery of LV developed pressure (LVDP) for hearts from males but not females. However, for 18-week-old mice we found that the IPC protocol doubled the recovery of LVDP for both males and females. For both ages, hearts from females had greater recovery of LVDP and higher levels of p-Akt compared to males. These findings are consistent with growing evidence that preconditioning induced by ischemia or other interventions can occur in hearts from females. However, for hearts from females, preconditioning depends on age. Moreover, consistent with previous studies, hearts from females have greater inherent resistance to ischemic injury, possibly involving increased signaling via p-Akt.
Publisher: Springer Science and Business Media LLC
Date: 16-07-2010
Abstract: Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I AES-I) has been found to be widespread in CF patients in eastern Australia. Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum s les. We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA ® Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.
Publisher: Frontiers Media SA
Date: 27-02-2018
Publisher: The Company of Biologists
Date: 2012
DOI: 10.1242/JCS.099002
Abstract: The most virulent of the human malaria parasites, Plasmodium falciparum, undergoes a remarkable morphological transformation as it prepares itself for sexual reproduction and transmission via mosquitoes. Indeed P. falciparum is named for the unique falciform or crescent shape of the mature sexual stages. Once the metamorphosis is completed the mature gametocyte releases from sequestration sites and enters the circulation making it accessible to feeding mosquitoes. Early ultrastructural studies showed that gametocyte elongation is driven by the assembly of a system of flattened cisternal membrane compartments underneath the parasite plasma membrane and a supporting network of microtubules. Here we describe the molecular composition and origin of the sub-pellicular membrane complex, and show that it is analogous to the inner membrane complex, an organelle with structural and motor functions that is well conserved across the apicomplexa. We identify novel cross-linking elements that may help stabilize the inner membrane complex during gametocyte development. We show that changes in gametocyte morphology are associated with an increase in cellular deformability and postulate that this enables the gametocytes to circulate in the blood stream without being detected and removed by the mechanical filtering mechanisms in the host's spleen.
Publisher: Springer Science and Business Media LLC
Date: 14-04-2016
DOI: 10.1038/NCOMMS11220
Abstract: Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.
Publisher: Springer Science and Business Media LLC
Date: 10-07-2018
DOI: 10.1038/S41598-018-28678-2
Abstract: Mycoplasma hyopneumoniae is an economically devastating, globally disseminated pathogen that can maintain a chronic infectious state within its host, swine. Here, we depict the events underpinning M . hyopneumoniae biofilm formation on an abiotic surface and demonstrate for the first time, biofilms forming on porcine epithelial cell monolayers and in the lungs of pigs, experimentally infected with M . hyopneumoniae . Nuclease treatment prevents biofilms forming on glass but not on porcine epithelial cells indicating that extracellular DNA (eDNA), which localises at the base of biofilms, is critical in the formation of these structures on abiotic surfaces. Subpopulations of M . hyopneumoniae cells, denoted by their ability to take up the dye TOTO-1 and release eDNA, were identified. A visually distinct sub-population of pleomorphic cells, that we refer to here as large cell variants (LCVs), rapidly transition from phase dark to translucent “ghost” cells. The translucent cells accumulate the membrane-impermeable dye TOTO-1, forming readily discernible membrane breaches immediately prior to lysis and the possible release of eDNA and other intracellular content (public goods) into the extracellular environment. Our novel observations expand knowledge of the lifestyles adopted by this wall-less, genome-reduced pathogen and provide further insights to its survival within farm environments and swine.
Publisher: Springer Science and Business Media LLC
Date: 22-12-2014
DOI: 10.1038/NATURE13983
Publisher: PeerJ
Date: 25-03-2014
DOI: 10.7717/PEERJ.326
Publisher: American Physiological Society
Date: 04-2003
DOI: 10.1152/AJPHEART.00441.2002
Abstract: Two functional α 1 -adrenergic receptor (AR) subtypes (α 1A and α 1B ) have been identified in the mouse heart. However, it is unclear whether the third known subtype, α 1D -AR, is also present. To investigate this, we determined whether there were α 1 -AR responses in hearts from a novel mouse model lacking α 1A - and α 1B -ARs (double knockout) (ABKO). In Langendorff-perfused hearts, α 1 -ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 ± 2% and 86 ± 4% of initial, respectively, n = 11, P 0.01). These effects were blocked by prazosin and 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspirol[4,5]decane-7,9-dione} dihydrochloride, suggesting that α 1D -AR-mediated responses were present. In contrast, right ventricular trabeculae from ABKO hearts did not respond to phenylephrine, suggesting that in ABKO perfused hearts, the effects of phenylephrine were not mediated by direct actions on cardiomyocytes. A novel finding was that α 1 -AR stimulation caused positive inotropy in the wild-type mouse heart, in contrast to negative inotropy observed in mouse cardiac muscle strips. We conclude that mouse hearts lacking α 1A - and α 1B -ARs retain functional α 1 -AR responses involving decreases of coronary flow and ventricular pressure that reflect α 1D -AR-mediated vasoconstriction. Furthermore, α 1 -AR inotropic responses depend critically on the experimental conditions.
Publisher: Frontiers Media SA
Date: 11-01-2018
Publisher: Elsevier BV
Date: 11-2012
Publisher: American Society for Microbiology
Date: 05-2008
DOI: 10.1128/JB.01807-07
Abstract: Type IV fimbriae are essential virulence factors of Dichelobacter nodosus , the principal causative agent of ovine foot rot. The fimA fimbrial subunit gene is required for virulence, but fimA mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the pilT or pilU gene eliminated the ability to carry out twitching motility. However, the pilT mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the pilU mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in D. nodosus . It was postulated that sufficient fimbrial retraction must occur in the pilU mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a pilU mutant of D. nodosus , aberrant motion was detected in an equivalent mutant of Pseudomonas aeruginosa . These observations explain how in D. nodosus protease secretion can occur in a pilU mutant but not in a pilT mutant. In addition, virulence studies with sheep showed that both the pilT and pilU mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.
Publisher: Life Science Alliance, LLC
Date: 09-06-2021
Abstract: Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-β. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.
Publisher: Elsevier BV
Date: 12-2012
DOI: 10.1016/J.JVIROMET.2012.07.003
Abstract: The study of host-pathogen interactions over past decades has benefited from advances in microscopy and fluorescent imaging techniques. A particularly powerful model in this field is vaccinia virus (VACV), which due to its amenability to genetic manipulation has been a productive model in advancing the understanding of the transport of subcellular cargoes. Conventional light microscopy imposes an upper limit of resolution of ~250nm, hence knowledge of events occurring at the sub-viral resolution is based predominantly on studies utilising electron microscopy. The development of super-resolution light microscopy presents the opportunity to bridge the gap between these two technologies. This report describes the analysis of VACV replication using fluorescent recombinant viruses, achieving sub-viral resolution with three-dimensional structured illumination microscopy. This is the first report of successfully resolving poxvirus particle morphologies at the scale of single virus particles using light microscopy.
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.BMCL.2013.06.082
Abstract: Described is the antibiotic activity of a marine natural product. Psammaplysin F (1) inhibited the growth of four Gram-positive strains by >80% at 50μM, and the amine at position C-20 is responsible for the observed antibacterial activity. When tested against two strains of methicillin resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentrations (MICs) for psammaplysin F (40-80μM) were similar to the structurally-related alkaloid psammaplysin H (2). Psammaplysin F (1) increased membrane permeability by two to four-fold compared to psammaplysin H (2) or control-treated bacteria, respectively. Unlike psammaplysin H (2), we show that psammaplysin F (1) inhibits equal partitioning of DNA into each daughter cell, suggesting that this natural product is a unique prokaryotic cell ision inhibitor.
Publisher: Proceedings of the National Academy of Sciences
Date: 24-06-2013
Abstract: Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa . We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze in idual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the “bulldozer” aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.
Publisher: Public Library of Science (PLoS)
Date: 11-09-2012
Publisher: Elsevier BV
Date: 03-2013
Publisher: American Physiological Society
Date: 16-06-2005
DOI: 10.1152/PHYSIOLGENOMICS.00016.2005
Abstract: The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac α-myosin heavy chain promoter (αMHC) drives lifetime expression of a tetracycline-inhibited transcription activator (tTA). Crossing αMHC-tTA mice with mice containing a tTA-responsive promoter linked to a target gene yields double-transgenic mice having tetracycline-repressed expression of the target gene in the heart. Using the tet-system, some studies use nontransgenic mice for the control group, whereas others use single-transgenic αMHC-tTA mice. However, previous studies found that high-level expression of a modified activator protein caused cardiomyopathy. Therefore, we tested whether cardiac expression of tTA was associated with altered function of αMHC-tTA mice compared with wild-type (WT) littermates. We monitored in vivo and in vitro function and gene expression profiles for myocardium from WT and αMHC-tTA mice. Compared with WT littermates, αMHC-tTA mice had a greater heart-to-body weight ratio (≈10%), ventricular dilation, and decreased ejection fraction, suggesting mild cardiomyopathy. In vitro, submaximal contractions were greater compared with WT and were associated with greater myofilament Ca 2+ sensitivity. Gene expression profiling revealed that the expression of 153 genes was significantly changed by % when comparing αMHC-tTA with WT myocardium. These findings demonstrate that introduction of the αMHC-tTA construct causes significant effects on myocardial gene expression and major functional abnormalities in vivo and in vitro. For studies using the tet-system, these results suggest caution in the use of controls, since αMHC-tTA myocardium differs appreciably from WT. Furthermore, the results raise the possibility that the phenotype conferred by a target gene may be influenced by the modified genetic background of αMHC-tTA myocardium.
Publisher: Portland Press Ltd.
Date: 11-12-2008
DOI: 10.1042/CS20070110
Abstract: Reperfusion of ischaemic rat or mouse hearts causes NE [noradrenaline (‘norepinephrine’)] release, stimulation of α1-ARs (α1-adrenergic receptors), PLC (phospholipase C) activation, Ins(1,4,5)P3 generation and the development of arrhythmias. In the present study, we examined the effect of increased α1A-AR drive on these responses. In hearts from non-transgenic mice (α1A-WT), Ins(1,4,5)P3 generation was observed after 2 min of reperfusion following 30 min of zero-flow ischaemia. No Ins(1,4,5)P3 response was observed in hearts from transgenic mice with 66-fold overexpression of α1A-AR (α1A-TG). This was despite the fact that α1A-TG hearts had 8–10-fold higher PLC responses to NE than α1A-WT under normoxic conditions. The immediate phospholipid precursor of Ins(1,4,5)P3, PtdIns(4,5)P2, responded to ischaemia and reperfusion similarly in α1A-WT and α1A-TG mice. Thus the lack of Ins(1,4,5)P3 generation in α1A-TG mice is not caused by limited availability of PtdIns(4,5)P2. Overall, α1-AR-mediated PLC activity was markedly enhanced in α1A-WT mice under reperfusion conditions, but responses in α1A-TG mice were not significantly different in normoxia and post-ischaemic reperfusion. Ischaemic preconditioning prevented Ins(1,4,5)P3 generation after 30 min of ischaemic insult in α1A-WT mice. However, the precursor lipid PtdIns(4,5)P2 was also reduced by preconditioning, whereas heightened α1A-AR activity did not influence PtdIns(4,5)P2 responses in reperfusion. Thus preconditioning and α1A-AR overexpression have different effects on early signalling responses, even though both prevented Ins(1,4,5)P3 generation. These studies demonstrate a selective inhibitory action of heightened α1A-AR activity on immediate post-receptor signalling responses in early post-ischaemic reperfusion.
Publisher: Society for Neuroscience
Date: 20-03-2013
DOI: 10.1523/JNEUROSCI.3560-12.2013
Abstract: Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlin C72 . Mini-dysferlin C72 -rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlin C72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.
Publisher: The Company of Biologists
Date: 2013
DOI: 10.1242/JCS.122119
Abstract: In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a sub-nuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex. However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging we show that the dynamic localization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochomatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and ChIP analyses revealed that in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used Peptide Nucleic Acids (PNA) to down-regulate PfSec13 and show that is essential for parasite proliferation in human erythrocytes.
Publisher: Wiley
Date: 07-08-2012
DOI: 10.1096/FJ.12-213876
Abstract: We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved hipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4(+) T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.
Publisher: The Royal Society
Date: 04-2013
DOI: 10.1098/RSOB.130017
Abstract: Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae . This unique interaction represents a new paradigm in microbial pathogenesis.
Publisher: Portland Press Ltd.
Date: 29-08-2013
DOI: 10.1042/BJ20130575
Abstract: We have described the presence of cell-membrane-associated κFLCs (free immunoglobulin light chains) on the surface of myeloma cells. Notably, the anti-κFLC mAb (monoclonal antibody) MDX-1097 is being assessed in clinical trials as a therapy for κ light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known whether λFLCs can associate with cell membranes of myeloma cells. In the present paper, we describe the presence of λFLCs on the surface of myeloma cells. We found that cell-surface-associated λFLCs are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that λFLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated λFLCs are aggregated. Given the present findings, we propose a model whereby in idual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.
Publisher: Elsevier BV
Date: 2010
Publisher: The Company of Biologists
Date: 2012
DOI: 10.1242/JCS.097790
Abstract: In vertebrates, two condensin complexes exist, condensin I and II, which have differing but unresolved roles in organizing mitotic chromosomes. To accurately dissect the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3/condensin II KO results in masses of chromatin forming anaphase bridges. CAP-H/condensin I KO anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals condensin I-depleted mitotic chromosomes as wider and shorter with a diffuse chromosome scaffold, while condensin II-depleted chromosomes retain a more defined scaffold with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.
Publisher: Springer Science and Business Media LLC
Date: 03-04-2020
DOI: 10.1186/S12860-020-00256-3
Abstract: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells. Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.
Publisher: Microbiology Society
Date: 07-2020
DOI: 10.1099/MIC.0.000911
Abstract: Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate twitching motility via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has an MCP PilJ and two CheW adapter proteins, PilI and ChpC, that likely interact with the histidine kinase ChpA to feed environmental signals into the system. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3′−5′-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Furthermore, we show that protease activity is required for the twitching motility response of P. aeruginosa to environmental signals. Based upon our data we propose a model whereby ChpC feeds these environmental signals into the Chp system, potentially via PilJ or another MCP, to control twitching motility. PilJ and PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa .
Publisher: IEEE
Date: 11-2009
Publisher: Springer Science and Business Media LLC
Date: 08-2017
DOI: 10.1038/S41598-017-07288-4
Abstract: Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.
Publisher: American Society for Microbiology
Date: 04-2011
DOI: 10.1128/EC.00244-10
Abstract: The most deadly of the human malaria parasites, Plasmodium falciparum , has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.
Publisher: Oxford University Press (OUP)
Date: 07-2010
DOI: 10.1086/653124
Publisher: Springer New York
Date: 03-12-2016
DOI: 10.1007/978-1-4939-6673-8_12
Abstract: Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good s le preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of s le preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.
Publisher: Frontiers Media SA
Date: 27-01-2015
Publisher: Wiley
Date: 23-02-2016
DOI: 10.1111/TRA.12370
Abstract: Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+)-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+)-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+)-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.
Publisher: Public Library of Science (PLoS)
Date: 15-09-2011
Publisher: American Society for Microbiology
Date: 25-10-2017
Abstract: Clostridium sordellii is a significant pathogen with mortality rates approaching 100%. It is the bacterial spore that is critical in initiating infection and disease. An understanding of spore structures as well as spore morphology across a range of strains may lead to a better understanding of C. sordellii infection and disease. However, the structural characteristics of the C. sordellii spores are limited. In this work, we have addressed this lack of detail and characterized the C. sordellii spore morphology. The use of traditional and advanced microscopy techniques has provided detailed new observations of C. sordellii spore structural features, which serve as a reference point for structural studies of spores from other bacterial species.
Publisher: Microbiology Society
Date: 02-2021
DOI: 10.1099/MIC.0.000990
Abstract: Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.
Publisher: Microbiology Society
Date: 03-2019
Abstract: Pandoraea species have been isolated from erse environmental s les and are emerging important respiratory pathogens, particularly in people with cystic fibrosis (CF). In the present study, two bacterial isolates initially recovered from consecutive sputum s les collected from a CF patient and identified as Pandoraea pnomenusa underwent a polyphasic taxonomic analysis. The isolates were found to be Gram-negative, facultative anaerobic motile bacilli and subsequently designated as strains 6399
Publisher: Wiley
Date: 28-11-2012
DOI: 10.1002/MBO3.49
Publisher: Cold Spring Harbor Laboratory
Date: 31-10-2019
DOI: 10.1101/825596
Abstract: Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate this expansion via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has two CheW adapter proteins, PilI and ChpC, and an MCP PilJ that likely interacts via PilI with the histidine kinase ChpA. It is thought that ChpC associates with other MCPs to feed environmental signals into the system, however no such signals have been identified. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3’-5’-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Based upon our data we propose a model whereby ChpC associates with an MCP other than PilJ to feed these environmental signals through the Chp system to control twitching motility. The MCP PilJ and the CheW adapter PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa .
Publisher: Cold Spring Harbor Laboratory
Date: 24-08-2019
DOI: 10.1101/737718
Abstract: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. Here, we demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/Akt activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/Akt activation. Mutation of Y180F strongly attenuated mouse xenograft tumor growth. An accompanying paper demonstrates altered metabolism, mutation incidence, and epigenetic status in these cells, indicating that PGRMC1 phosphorylation strongly influences cancer biology.
Publisher: Springer Science and Business Media LLC
Date: 26-05-2016
DOI: 10.1038/SREP26005
Abstract: Surface translocation by the soil bacterium Myxococcus xanthus is a complex multicellular phenomenon that entails two motility systems. However, the mechanisms by which the activities of in idual cells are coordinated to manifest this collective behaviour are currently unclear. Here we have developed a novel assay that enables detailed microscopic examination of M. xanthus motility at the interstitial interface between solidified nutrient medium and a glass coverslip. Under these conditions, M. xanthus motility is characterised by extensive micro-morphological patterning that is considerably more elaborate than occurs at an air-surface interface. We have found that during motility on solidified nutrient medium, M. xanthus forges an interconnected furrow network that is lined with an extracellular matrix comprised of exopolysaccharides, extracellular lipids, membrane vesicles and an unidentified slime. Our observations have revealed that M. xanthus motility on solidified nutrient medium is a stigmergic phenomenon in which multi-cellular collective behaviours are co-ordinated through trail-following that is guided by physical furrows and extracellular matrix materials.
Publisher: Oxford University Press (OUP)
Date: 17-05-2015
Publisher: Public Library of Science (PLoS)
Date: 21-03-2013
Publisher: Microbiology Society
Date: 10-2020
DOI: 10.1099/MIC.0.000956
Abstract: Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and ersity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa .
Location: United States of America
Start Date: 2016
End Date: 12-2016
Amount: $355,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 07-2010
End Date: 12-2013
Amount: $315,000.00
Funder: Australian Research Council
View Funded Activity