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Dr Lynne Turnbull

Biography

Lynne Turnbull is a Senior Research Fellow at the ithree Institute. As well as Lynne's research focus on microbial biofilms, bacterial motility and pathogenicity, she also is the Manager of the Microbial Imaging Facility at UTS. Lynne has previously held a Chancellor's Postdoctoral Research Fellowship at UTS.

After completing her doctoral studies at Macquarie University in the area of cardiac biophysics, Lynne completed postdoctoral stints at the University of California San Francisco and the Baker Heart Research Institute (Melbourne).

Lynne changed her research focus to microbiology in 2005, taking a position at Monash University (Melbourne). Lynne moved to UTS in January 2008.

Watch Dr Lynne Turnbull's interview about DeltaVision OMX Blaze

Professional

Currently a member of the following professional societies:

  • Australian Society for Microbiology
  • American Society for Microbiology

Image of Lynne Turnbull
Associate of the Faculty, The ithree Institute
Core Member, ithree - Institute of Infection, Immunity and Innovation
B.Sc (Hons) (UNE), PhD (Macq), Ph. D
Associate, Australian Society for Microbiology
Member, American Society for Microbiology
 

Research Interests

Lynne has two areas of research focus. One area revolves around how bacteria create and sustain communities that provide protection from the environment. These communities are called biofilms and it is now acknowledged that many chronic infections and infections of medical implants are caused by bacterial biofilms. Biofilms are notoriously resistant to antibiotic treatment and are often too large to be attacked by the immune system. There are few good model systems to study these biofilms in medical settings. Lynne’s research interests are to design better techniques to study, treat and prevent biofilms that cause human disease. This research concentrates on the human pathogens Pseudomonas aeruginosa, non-typeable Haemophilus influenzae, enteropathogenic E. coli and Staphylococcus aureus.

Lynne’s second area of research focus involves the development of fluorescent microscopy techniques for microbial cell biology as part of her role within the Microbial Imaging Facility. This specifically involves using the super-resolution technique of 3D-structured illumination using the DeltaVision OMX imaging system. It also includes live cell imaging and wide-field fluorescent deconvolution microscopy of bacteria and bacterial-host interactions.

Chapters

Bottomley, A.L., Turnbull, L., Whitchurch, C.B. & Harry, E.J. 2017, 'Immobilization Techniques of Bacteria for Live Super-resolution Imaging Using Structured Illumination Microscopy.' in Pontus Nordenfelt and Mattias Collin (ed), Bacterial Pathogenesis, pp. 197-209.
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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 sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.
Turnbull, L. & Whitchurch, C.B. 2014, 'Motility Assay: Twitching Motility' in Filloux, A. & Ramos, J.L. (eds), Pseudomonas Methods and Protocols, Springer New York, New York, pp. 73-86.
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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 individual cells or small groups of bacteria during twitching motility mediated colony expansion.

Conferences

Lu, J., Whitchurch, C., Turnbull, L., Carter, D., Schlothauer, R. & Harry, L. 2012, 'THE THERAPEUTIC USE OF HONEY ON CHRONIC WOUND INFECTIONS', WOUND REPAIR AND REGENERATION, pp. A73-A73.
Mueller, P., Turnbull, L., Schlothauer, R., Whitchurch, C. & Harry, E. 2012, 'NEW ZEALAND MANUKA HONEY HELPS HUMAN KERATINOCYTES TO SURVIVE IN PRESENCE OF S-AUREUS', WOUND REPAIR AND REGENERATION, pp. A75-A75.
Vallotton, P., Mililli, L., Turnbull, L. & Whitchurch, C.B. 2010, 'Segmentation of Dense 2D Bacilli Populations', 2010 International Conference on Digital Image Computing: Techniques and Applications, International Conference on Digital Image Computing: Techniques and Applications, IEEE xplore, Sydney, pp. 82-86.
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Bacteria outnumber all other known organisms by far so there is considerable interest in characterizing them in detail and in measuring their diversity, evolution, and dynamics. Here, we present a system capable of identifying rod-like bacteria (bacilli) correctly in high resolution phase contrast images. We use a probabilistic model together with several purpose-designed image features in order to split bacteria at the septum consistently. Our method commits less than 1% error on test images. Our method should also be applicable to study dense 2D systems composed of elongated elements, such as some viruses, molecules, parasites (plasmodium, euglena), diatoms, and crystals.
Vallotton, P., Sun, C., Wang, D., Ranganathan, P., Turnbull, L. & Whitchurch, C.B. 2009, 'Segmentation and tracking of individual Pseudomonas aeruginosa bacteria in dense populations of motile cells', Proceedings of Image and Vision Computing New Zealand 2009, Image and Vision Computing Conference, IEEE, Wellington, NZ, pp. 221-225.
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The dynamics of individual bacteria underlies the manifestation of complex multicellular behaviours such as biofilm development and colony expansion. High resolution movies of expanding bacterial colonies reveal intriguing patterns of cell motions. A quantitative understanding of the observed behaviour in relation to the bacterias own motile apparatus and to hydrodynamic forces requires that bacteria be identified and tracked over time. This represents a demanding undertaking as their size is close to the diffraction limit; they are very close to each other; and a typical image may contain over a thousand cells. Here, we describe the approach that we have developed to segment individual bacteria and track them in high resolution phase contrast microscopy movies. We report that over 99% of nonoverlapping bacteria could be segmented correctly using mathematical morphology, and we present preliminary results that exploit this new capability.
Kaparakis, M., Turnbull, L., Carneiro, L., Firth, S., Coleman, H.A., Parkington, H.C., Le Bourhis, L., Karrar, A., Viala, J., Mak, J., Hutton, M., Davies, J.K., Philpott, D., Girardin, S., Whitchurch, C.B. & Ferrero, R.L. 2008, 'Bacterial outer membrane vesicles: A new mechanism for NOD1-dependent responses in epithelial cells', HELICOBACTER, pp. 394-394.
Turcato, S., Turnbull, L., Simpson, P.C. & Baker, A.J. 2005, 'Ischemic preconditioning in female mouse heart requires alpha 1-adrenergic receptors', BIOPHYSICAL JOURNAL, pp. 308A-308A.
Turnbull, L., McCloskey, D.T., Swigart, P., Simpson, P.C. & Baker, A.J. 2004, 'Novel cardioprotection in a tetracycline-regulated gene expression (tet) system', BIOPHYSICAL JOURNAL, pp. 48A-48A.
Turnbull, L., McCloskey, D.T., O'Connell, T.D., Simpson, P.C. & Baker, A.J. 2002, 'Evidence for functional alpha 1D-adrenoceptors in mouse heart', BIOPHYSICAL JOURNAL, pp. 601A-602A.
McCloskey, D.T., Turnbull, L., Simpson, G.L., Shalenberg, E.M., Simpson, P.C., Conklin, B.R. & Baker, A.J. 2002, 'Expression of a G(i)-coupled receptor causes decreased myocardial Ca2+ transients and contraction', BIOPHYSICAL JOURNAL, pp. 595A-595A.

Journal articles

Trussart, M., Yus, E., Martinez, S., Baù, D., Tahara, Y.O., Pengo, T., Widjaja, M., Kretschmer, S., Swoger, J., Djordjevic, S., Turnbull, L., Whitchurch, C., Miyata, M., Marti-Renom, M.A., Lluch-Senar, M. & Serrano, L. 2017, 'Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae.', Nat Commun, vol. 8, p. 14665.
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DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity 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 10kb 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 3kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33kb. 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.
Redpath, G.M., Sophocleous, R.A., Turnbull, L., Whitchurch, C.B. & Cooper, S.T. 2016, 'Ferlins show tissue-specific expression and segregate as plasma membrane/late endosomal or trans-Golgi/recycling ferlins.', Traffic, vol. 17, no. 3, pp. 245-266.
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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 (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. Dysferlin, myoferlin, and Fer1L6 are plasma membrane (PM) ferlins, whereas otoferlin and Fer1L5 localize predominantly to intracellular compartments. 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 plasma membrane 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.
Turnbull, L., Toyofuku, M., Hynen, A.L., Kurosawa, M., Pessi, G., Petty, N.K., Osvath, S.R., Carcamo-Oyarce, G., Gloag, E.S., Shimoni, R., Omasits, U., Ito, S., Yap, X., Monahan, L.G., Cavaliere, R., Ahrens, C.H., Charles, I.G., Nomura, N., Eberl, L. & Whitchurch, C.B. 2016, 'Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms', NATURE COMMUNICATIONS, vol. 7.
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Gloag, E.S., Turnbull, L., Javed, M.A., Wang, H., Gee, M.L., Wade, S.A. & Whitchurch, C.B. 2016, 'Stigmergy co-ordinates multicellular collective behaviours during Myxococcus xanthus surface migration.', Scientific reports, vol. 6, p. 26005.
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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 individual 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.
Gloag, E.S., Elbadawi, C., Zachreson, C.J., Aharonovich, I., Toth, M., Charles, I.G., Turnbull, L. & Whitchurch, C.B. 2016, 'Micro-Patterned Surfaces That Exploit Stigmergy to Inhibit Biofilm Expansion.', Front Microbiol, vol. 7, p. 2157.
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Twitching motility is a mode of surface translocation that is mediated by the extension and retraction of type IV pili and which, depending on the conditions, enables migration of individual cells or can manifest as a complex multicellular collective behavior that leads to biofilm expansion. When twitching motility occurs at the interface of an abiotic surface and solidified nutrient media, it can lead to the emergence of extensive self-organized patterns of interconnected trails that form as a consequence of the actively migrating bacteria forging a furrow network in the substratum beneath the expanding biofilm. These furrows appear to direct bacterial movements much in the same way that roads and footpaths coordinate motor vehicle and human pedestrian traffic. Self-organizing systems such as these can be accounted for by the concept of stigmergy which describes self-organization that emerges through indirect communication via persistent signals within the environment. Many bacterial communities are able to actively migrate across solid and semi-solid surfaces through complex multicellular collective behaviors such as twitching motility and flagella-mediated swarming motility. Here, we have examined the potential of exploiting the stigmergic behavior of furrow-mediated trail following as a means of controlling bacterial biofilm expansion along abiotic surfaces. We found that incorporation of a series of parallel micro-fabricated furrows significantly impeded active biofilm expansion by Pseudomonas aeruginosa and Proteus vulgaris. We observed that in both cases bacterial movements tended to be directed along the furrows. We also observed that narrow furrows were most effective at disrupting biofilm expansion as they impeded the ability of cells to self-organize into multicellular assemblies required for escape from the furrows and migration into new territory. Our results suggest that the implementation of micro-fabricated furrows that exploit stigmergy may be a ...
Raymond, B.B.A., Jenkins, C., Seymour, L.M., Tacchi, J.L., Widjaja, M., Jarocki, V.M., Deutscher, A.T., Turnbull, L., Whitchurch, C.B., Padula, M.P. & Djordjevic, S.P. 2015, 'Proteolytic processing of the cilium adhesin MHJ_0194 (P123(J)) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules', CELLULAR MICROBIOLOGY, vol. 17, no. 3, pp. 425-444.
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Duggin, I.G., Aylett, C.H.S., Walsh, J.C., Michie, K.A., Wang, Q., Turnbull, L., Dawson, E.M., Harry, E.J., Whitchurch, C.B., Amos, L.A. & Loewe, J. 2015, 'CetZ tubulin-like proteins control archaeal cell shape', NATURE, vol. 519, no. 7543, pp. 362-+.
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Liu, M., Lu, J., Muller, P., Turnbull, L., Burke, C.M., Schlothauer, R.C., Carter, D.A., Whitchurch, C.B. & Harry, E.J. 2015, 'Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey', Frontiers in Microbiology, vol. 6, no. JAN.
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Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey syner...
Gloag, E.S., Turnbull, L. & Whitchurch, C.B. 2015, 'Bacterial stigmergy: an organising principle of multicellular collective behaviours of bacteria.', Scientifica, vol. 2015, p. 387342.
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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 individuals of the group through the generation of persistent cues in the environment. Interestingly, despite numerous examples 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 examples 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.
Nolan, L.M., Cavaliere, R., Turnbull, L. & Whitchurch, C.B. 2015, 'Extracellular ATP inhibits twitching motility-mediated biofilm expansion by Pseudomonas aeruginosa.', BMC microbiology, vol. 15, no. 1, p. 392.
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BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that exploits damaged epithelia to cause infection. Type IV pili (tfp) are polarly located filamentous structures which are the major adhesins for attachment of P. aeruginosa to epithelial cells. The extension and retraction of tfp powers a mode of surface translocation termed twitching motility that is involved in biofilm development and also mediates the active expansion of biofilms across surfaces. Extracellular adenosine triphosphate (eATP) is a key "danger" signalling molecule that is released by damaged epithelial cells to alert the immune system to the potential presence of pathogens. As P. aeruginosa has a propensity for infecting damaged epithelial tissues we have explored the influence of eATP on tfp biogenesis and twitching motility-mediated biofilm expansion by P. aeruginosa. RESULTS: In this study we have found that eATP inhibits P. aeruginosa twitching motility-mediated expansion of interstitial biofilms at levels that are not inhibitory to growth. We have determined that eATP does not inhibit expression of the tfp major subunit, PilA, but reduces the levels of surface assembled tfp. We have also determined that the active twitching zone of expanding P. aeruginosa interstitial biofilms contain large quantities of eATP which may serve as a signalling molecule to co-ordinate cell movements in the expanding biofilm. The inhibition of twitching motility-mediated interstitial biofilm expansion requires eATP hydrolysis and does not appear to be mediated by the Chp chemosensory system. CONCLUSIONS: Endogenous eATP produced by P. aeruginosa serves as a signalling molecule to co-ordinate complex multicellular behaviours of this pathogen. Given the propensity for P. aeruginosa to infect damaged epithelial tissues, our observations suggest that eATP released by damaged cells may provide a cue to reduce twitching motility of P. aeruginosa in order to establish infection at the site of damage. Furth...
Nolan, L.M., Cavaliere, R., Turnbull, L. & Whitchurch, C.B. 2015, 'Extracellular ATP inhibits twitching motility-mediated biofilm expansion by Pseudomonas aeruginosa', BMC MICROBIOLOGY, vol. 15.
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Kennan, R.M., Lovitt, C.J., Han, X., Parker, D., Turnbull, L., Whitchurch, C.B. & Rood, J.I. 2015, 'A two-component regulatory system modulates twitching motility in Dichelobacter nodosus', Veterinary Microbiology, vol. 179, no. 1-2, pp. 34-41.
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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.
Liu, M., Lu, J., Muller, P., Turnbull, L., Burke, C.M., Schlothauer, R.C., Carter, D.A., Whitchurch, C.B. & Harry, E.J. 2015, 'Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey', Frontiers in Microbiology, vol. 6, no. JAN.
View/Download from: UTS OPUS or Publisher's site
Skin infections caused by antibiotic resistant Staphylococcus aureus are a significant health problem worldwide; often associated with high treatment cost and mortality rate. Complex natural products like New Zealand (NZ) manuka honey have been revisited and studied extensively as an alternative to antibiotics due to their potent broad-spectrum antimicrobial activity, and the inability to isolate honey-resistant S. aureus. Previous studies showing synergistic effects between manuka-type honeys and antibiotics have been demonstrated against the growth of one methicillin-resistant S. aureus (MRSA) strain. We have previously demonstrated strong synergistic activity between NZ manuka-type honey and rifampicin against growth and biofilm formation of multiple S. arueus strains. Here, we have expanded our investigation using multiple S. aureus strains and four different antibiotics commonly used to treat S. aureus-related skin infections: rifampicin, oxacillin, gentamicin, and clindamycin. Using checkerboard microdilution and agar diffusion assays with S. aureus strains including clinical isolates and MRSA we demonstrate that manuka-type honey combined with these four antibiotics frequently produces a synergistic effect. In some cases when synergism was not observed, there was a significant enhancement in antibiotic susceptibility. Some strains that were highly resistant to an antibiotic when present alone become sensitive to clinically achievable concentrations when combined with honey. However, not all of the S. aureus strains tested responded in the same way to these combinational treatments. Our findings support the use of NZ manuka-type honeys in clinical treatment against S. aureus-related infections and extend their potential use as an antibiotic adjuvant in combinational therapy. Our data also suggest that manuka-type honeys may not work as antibiotic adjuvants for all strains of S. aureus, and this may help determine the mechanistic processes behind honey syner...
Chaiyadet, S., Sotillo, J., Smout, M., Cantacessi, C., Jones, M.K., Johnson, M.S., Turnbull, L., Whitchurch, C.B., Potriquet, J., Laohaviroj, M., Mulvenna, J., Brindley, P.J., Bethony, J.M., Laha, T., Sripa, B. & Loukas, A. 2015, 'Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype.', The Journal of infectious diseases, vol. 212, no. 10, pp. 1636-1645.
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Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown.Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization.EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes.This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.
Chaiyadet, S., Smout, M., Johnson, M., Whitchurch, C., Turnbull, L., Kaewkes, S., Sotillo, J., Loukas, A. & Sripa, B. 2015, 'Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production.', International journal for parasitology, vol. 45, no. 12, pp. 773-781.
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Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-ea...
Smout, M.J., Sotillo, J., Laha, T., Papatpremsiri, A., Rinaldi, G., Pimenta, R.N., Chan, L.Y., Johnson, M.S., Turnbull, L., Whitchurch, C.B., Giacomin, P.R., Moran, C.S., Golledge, J., Sripa, B., Mulvenna, J.P., Brindley, P.J. & Loukas, A. 2015, 'Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia', PLoS Pathogens, vol. 11, no. 10.
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Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.
Liu, M., Lu, J., Mueller, P., Turnbull, L., Burke, C.M., Schlothauer, R.C., Carter, D.A., Whitchurch, C.B. & Harry, E.J. 2015, 'Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobiala combined with manuka honey', Frontiers in Microbiology, vol. 5.
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Turnbull, L. & Whitchurch, C.B. 2014, 'Motility assays: twitching motility', Methods in Molecular Biology, vol. 1149, no. 1.
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Lu, J., Turnbull, L., Burke, C.M., Liu, M.Y., Carter, D.A., Schlothauer, R.C., Whitchurch, C.B. & Harry, L. 2014, 'Manuka-type honeys can eradicate biofilms produced by Staphylococcus aureus strains with different biofilm-forming abilities', PeerJ, vol. 2.
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Chronic wounds are a major global health problem. Their management is difficult and costly, and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative wound treatments. Honey is now being revisited as an alternative treatment due to its broad-spectrum antibacterial activity and the inability of bacteria to develop resistance to it. Many previous antibacterial studies have used honeys that are not well characterized, even in terms of quantifying the levels of the major antibacterial components present, making it difficult to build an evidence base for the efficacy of honey as an antibiofilm agent in chronic wound treatment. Here we show that a range of well-characterized New Zealand manuka-type honeys, in which two principle antibacterial components, methylglyoxal and hydrogen peroxide, were quantified, can eradicate biofilms of a range of Staphylococcus aureus strains that differ widely in their biofilm-forming abilities. Using crystal violet and viability assays, along with confocal laser scanning imaging, we demonstrate that in all S. aureus strains, including methicillin-resistant strains, the manuka-type honeys showed significantly higher anti-biofilm activity than clover honey and an isotonic sugar solution. We observed higher anti-biofilm activity as the proportion of manuka-derived honey, and thus methylglyoxal, in a honey blend increased. However, methylglyoxal on its own, or with sugar, was not able to effectively eradicate S. aureus biofilms. We also demonstrate that honey was able to penetrate through the biofilm matrix and kill the embedded cells in some cases. As has been reported for antibiotics, sub-inhibitory concentrations of honey improved biofilm formation by some S. aureus strains, however, biofilm cell suspensions recovered after honey treatment did not develop resistance towards manuka-type honeys. New Zealand manuka-type honeys, at the concentrations they can be applie...
Monahan, L.G., Turnbull, L., Osvath, S.R., Birch, D., Charles, I.G. & Whitchurch, C.B. 2014, 'Rapid conversion of Pseudomonas aeruginosa to a spherical cell morphotype facilitates tolerance to carbapenems and penicillins but increases susceptibility to antimicrobial peptides', Antimicrobial Agents and Chemotherapy, vol. 58, no. 4, pp. 1956-1962.
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The Gram negative human pathogen Pseudomonas aeruginosa is able to tolerate high concentrations of -lactam antibiotics. Despite inhibiting the growth of the organism, these cell wall-targeting drugs exhibit remarkably little bactericidal activity. However, the mechanisms underlying -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 -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 therapeutics to combat bacterial infections.
Cavaliere, R., Ball, J.L., Turnbull, L. & Whitchurch, C.B. 2014, 'The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin', Microbiology Open, vol. 3, no. 4, pp. 557-567.
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Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethylenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix strength of biofilms of several bacterial species as well as to have bactericidal activity against various pathogens. EDTA exerts its antimicrobial activity by chelating divalent cations necessary for growth and membrane stability and by destabilizing the matrix thus enhancing the detachment of bacterial cells from the biofilm. In this study, we have explored the role of divalent cations in NTHi biofilm development and stability. We have utilized in vitro static and continuous flow models of biofilm development by NTHi to demonstrate that magnesium cations enhance biofilm formation by NTHi. We found that the divalent cation chelator EDTA is effective at both preventing NTHi biofilm formation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections.
Turnbull, L., Strauss, M.P., Liew, A.T.F., Monahan, L.G., Whitchurch, C.B. & Harry, E.J. 2014, 'Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)', JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, no. 91.
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Horsington, J., Lynn, H., Turnbull, L., Cheng, D., Braet, F., Diefenbach, R.J., Whitchurch, C.B., Karupiah, G. & Newsome, T.P. 2013, 'A36-dependent actin filament nucleation promotes release of vaccinia virus', Plos Pathogens, vol. 9, no. 3, pp. e1003239-e1003239.
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Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits.
Lek, A., Evesson, F.J., Lemckert, F.A., Redpath, G., Turnbull, L., Whitchurch, C.B., North, K.N. & Cooper, S.T. 2013, 'Calpains, Cleaved Mini-Dysferlin(C72), and L-Type Channels Underpin Calcium-Dependent Muscle Membrane Repair', Journal of Neuroscience, vol. 33, no. 12, pp. 5085-5094.
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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.
Lu, J., Carter, D.A., Turnbull, L., Rosendale, D., Hedderley, D., Stephens, J., Gannabathula, S., Steinhorn, G., Schlothauer, R.C., Whitchurch, C.B. & Harry, L. 2013, 'The effect of New Zealand kanuka, manuka and clover honeys on bacterial growth dynamics and cellular morphology varies according to the species', PLoS One, vol. 8, no. 2, pp. e55898-e55898.
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Treatment of chronic wounds is becoming increasingly difficult due to antibiotic resistance. Complex natural products with antimicrobial activity, such as honey, are now under the spotlight as alternative treatments to antibiotics. Several studies have shown honey to have broad-spectrum antibacterial activity at concentrations present in honey dressings, and resistance to honey has not been attainable in the laboratory. However not all honeys are the same and few studies have used honey that is well defined both in geographic and chemical terms. Here we have used a range of concentrations of clover honey and a suite of manuka and kanuka honeys from known geographical locations, and for which the floral source and concentration of methylglyoxal and hydrogen peroxide potential were defined, to determine their effect on growth and cellular morphology of four bacteria: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. While the general trend in effectiveness of growth inhibition was manuka.manuka-kanuka blend.kanuka.clover, the honeys had varying and diverse effects on the growth and cellular morphology of each bacterium, and each organism had a unique response profile to these honeys. P. aeruginosa showed a markedly different pattern of growth inhibition to the other three organisms when treated with sub-inhibitory concentrations of honey, being equally sensitive to all honeys, including clover, and the least sensitive to honey overall. While hydrogen peroxide potential contributed to the antibacterial activity of the manuka and kanuka honeys, it was never essential for complete growth inhibition. Cell morphology analysis also showed a varied and diverse set of responses to the honeys that included cell length changes, cell lysis, and alterations to DNA appearance. These changes are likely to reflect the different regulatory circuits of the organisms that are activated by the stress of honey treatment.
Gloag, E.S., Turnbull, L., Huang, A., Vallotton, P., Wang, H., Nolan, L.M., Mililli, L., Hunt, C., Lu, J., Osvath, S.R., Monahan, L.G., Cavaliere, R., Charles, I.G., Wand, M., Gee, M., Ranganathan, P. & Whitchurch, C.B. 2013, 'Self-organization of bacterial biofilms is facilitated by extracellular DNA', Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 28, pp. 11541-11546.
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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 individual 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
Hutchinson, A.T., Malik, A., Berkahn, M.B., Agostino, M., To, J., Tacchi, J.L., Djordjevic, S.P., Turnbull, L., Whitchurch, C.B., Edmundson, A.B., Jones, P.M., Raison, R.L. & Ramsland, P.A. 2013, 'Formation of Assemblies on Cell Membranes by Secreted Proteins: Molecular Studies of Free Lambda Light Chain Aggregates Found on the Surface of Myeloma Cells.', Biochemical Journal, vol. 454, no. 3, pp. 479-489.
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We have described the presence of cell membrane-associated ? free immunoglobulin light chains (FLC) on the surface of myeloma cells. Notably, the anti-?FLC mAb, MDX-1097, is being assessed in clinical trials as a therapy for ? 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 if ?FLCs can associate with cell membranes of myeloma cells. In this study, we describe the presence of ?FLCs on the surface of myeloma cells. We found that cell surface-associated ?FLC are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that ?FLCs 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 ?FLCs are aggregated. Given our findings, we propose a model whereby individual 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.
Ramsey, D., Islam, M., Turnbull, L., Davis, R., Whitchurch, C.B. & Mcalpine, S. 2013, 'Psammaplysin F: A Unique Inhibitor Of Bacterial Chromosomal Partitioning', Bioorganic & Medicinal Chemistry Letters, vol. 23, no. 17, pp. 4862-4866.
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Described is the antibiotic activity of a marine natural product. Psammaplysin F (1) inhibited the growth of four Gram-positive strains by >80% at 50 o(sic)M, and the amine at position C-20 is responsible for the observed antibacterial activity. When tes
Gloeckl, S., Ong, V., Patel, P., Tyndall, J., Timms, P., Beagley, K., Allan, J., Armitage, C., Turnbull, L., Whitchurch, C.B., Merdanovic, M., Ehrmann, M., Powers, J., Oleksyszyn, J., Verdoes, M., Bogyo, M. & Huston, W. 2013, 'Identification Of A Serine Protease Inhibitor Which Causes Inclusion Vacuole Reduction And Is Lethal To Chlamydia Trachomatis', Molecular Microbiology, vol. 89, no. 4, pp. 676-689.
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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 b
Dahan-pasternak, N., Nasereddin, A., Kolevzon, N., Pe'er, M., Wong, W., Shinder, V., Turnbull, L., Whitchurch, C.B., Elbaum, M., Gilberger, T., Yavin, E., Baum, J. & Dzikowski, R. 2013, 'Pfsec13 Is An Unusual Chromatin-associated Nucleoporin Of Plasmodium Falciparum That Is Essential For Parasite Proliferation In Human Erythrocytes', Journal of Cell Science, vol. 126, no. 14, pp. 3055-3069.
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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 envelo
Manos, J., Hu, H., Rose, B.R., Wainwright, C.E., Zablotska, I., Cheney, J., Turnbull, L., Whitchurch, C.B., Grimwood, K., Harmer, C., Anuj, S., Harbour, C. & ACFBAL study investigator, T. 2013, 'Virulence factor expression patterns in Pseudomonas aeruginosa strains from infants with cystic fibrosis', European Journal of Clinical Microbiology & Infectious Diseases, vol. 32, no. 12, pp. 1583-1592.
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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
Hu, H., Harmer, C., Anuj, S., Wainwright, C.E., Manos, J., Cheney, J., Harbour, C., Zablotska, I., Turnbull, L., Whitchurch, C.B., Grimwood, K., Rose, B. & ACFBAL study investigator, T. 2013, 'Type 3 secretion system effector genotype and secretion phenotype of longitudinally collected Pseudomonas aeruginosa isolates from young children diagnosed with cystic fibrosis following newborn screening', Clinical Microbiology and Infection, vol. 19, pp. 266-272.
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Studies of the type 3 secretion system (T3SS) in Pseudomonas aeruginosa isolates from chronically infected older children and adults with cystic fibrosis (CF) show a predominantly exoS+/exoU) (exoS+) genotype and loss of T3SS effector secretion over time. Relatively little is known about the role of the T3SS in the pathogenesis of early P. aeruginosa infection in the CF airway. In this longitudinal study, 168 P. aeruginosa isolates from 58 children diagnosed with CF following newborn screening and 47 isolates from homes of families with or without children with CF were genotyped by pulsed-field gel electrophoresis (PFGE) and T3SS genotype and phenotype determined using multiplex PCR and western blotting. Associations were sought between T3SS data and clinical variables and comparisons made between T3SS data of clinical and environmental PFGE genotypes. Seventy-seven of the 92 clinical strains were exoS+ (71% secretors (ExoS+)) and 15 were exoU+ (93% secretors (ExoU+)). Initial exoS+ strains were five times more likely to secrete ExoS than subsequent exoS+ strains at first isolation. The proportion of ExoS+ strains declined with increasing age at acquisition. No associations were found between T3SS characteristics and gender, site of isolation, exacerbation, a persistent strain or pulmonary outcomes. Fourteen of the 23 environmental strains were exoS+ (79% ExoS+) and nine were exoU+ (33% ExoU+). The exoU+ environmental strains were significantly less likely to secrete ExoU than clinical strains. This study provides new insight into the T3SS characteristics of P. aeruginosa isolated from the CF airway early in life.
Muller, P., Alber, D.G., Turnbull, L., Schlothauer, R.C., Carter, D.A., Whitchurch, C.B. & Harry, L. 2013, 'Synergism between medihoney and rifampicin against methicillin-resistant Staphylococcus aureus (MRSA)', PLoS One, vol. 8, no. 2, pp. e57679-e57679.
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Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.
Gloag, E.S., Javed, M.A., Wang, H., Gee, M.L., Wade, S.A., Turnbull, L. & Whitchurch, C.B. 2013, 'Stigmergy: A key driver of self-organization in bacterial biofilms.', Communicative & integrative biology, vol. 6, no. 6, p. e27331.
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Bacterial biofilms are complex multicellular communities that are often associated with the emergence of large-scale patterns across the biofilm. How bacteria self-organize to form these structured communities is an area of active research. We have recently determined that the emergence of an intricate network of trails that forms during the twitching motility mediated expansion of Pseudomonas aeruginosa biofilms is attributed to an interconnected furrow system that is forged in the solidified nutrient media by aggregates of cells as they migrate across the media surface. This network acts as a means for self-organization of collective behavior during biofilm expansion as the cells following these vanguard aggregates were preferentially confined within the furrow network resulting in the formation of an intricate network of trails of cells. Here we further explore the process by which the intricate network of trails emerges. We have determined that the formation of the intricate network of furrows is associated with significant remodeling of the sub-stratum underlying the biofilm. The concept of stigmergy has been used to describe a variety of self-organization processes observed in higher organisms and abiotic systems that involve indirect communication via persistent cues in the environment left by individuals that influence the behavior of other individuals of the group at a later point in time. We propose that the concept of stigmergy can also be applied to describe self-organization of bacterial biofilms and can be included in the repertoire of systems used by bacteria to coordinate complex multicellular behaviors.
Riglar, D.T., Rogers, K.L., Hanssen, E., Turnbull, L., Bullen, H.E., Charnaud, S.C., Przyborski, J., Gilson, P.R., Whitchurch, C.B., Crabb, B.S., Baum, J. & Cowman, A.F. 2013, 'Spatial association with PTEX complexes defines regions for effector export into Plasmodium falciparum-infected erythrocytes', NATURE COMMUNICATIONS, vol. 4.
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Robinson, M.W., Buchtmann, K.A., Jenkins, C., Tacchi, J.L., Raymond, B.B.A., To, J., Chowdhury, P.R., Woolley, L.K., Labbate, M., Turnbull, L., Whitchurch, C.B., Padula, M.P. & Djordjevic, S.P. 2013, 'MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 3.
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Dearnley, M.K., Yeoman, J.A., Hanssen, E., Kenny, S., Turnbull, L., Whitchurch, C.B., Tilley, L. & Dixon, M. 2012, 'Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes', Journal of Cell Science, vol. 125, no. 8, pp. 2053-2063.
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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 falci
Green, L.C., Kalitsis, P., Chang, T.M., Cipetic, M., Kim, J.K., Marshall, O., Turnbull, L., Whitchurch, C.B., Vagnarelli, P., Samejima, K., Earnshaw, W.C., Choo, K. & Hudson, D. 2012, 'Contrasting Roles Of Condensin I And Condensin Ii In Mitotic Chromosome Formation', Journal of Cell Science, vol. 125, no. 6, pp. 1591-1604.
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In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first verteb
Ivanov, I.E., Boyd, C.D., Newell, P.D., Schwartz, M.E., Turnbull, L., Johnson, M.S., Whitchurch, C.B., O'Toole, G.A. & Camesano, T.A. 2012, 'Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens', Research in Microbiology, vol. 163, no. 9-10, pp. 685-691.
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Pseudomonas fluorescence Pf0-1 requires the large repeat protein LapA for stable surface attachment. This study presents direct evidence that LapA is a cell-surface-localized adhesin. Atomic force microscopy (AFM) revealed a significant 2-fold reduction in adhesion force for mutants lacking the LapA protein on the cell surface compared to the wild-type strain. Deletion of lapG, a gene encoding a periplasmic cysteine protease that functions to release LapA from the cell surface, resulted in a 2-fold increase in the force of adhesion. Three-dimensional structured illumination microscopy (3D-SIM) revealed the presence of the LapA protein on the cell surface, consistent with its role as an adhesin. The protein is only visualized in the cytoplasm for a mutant of the ABC transporter responsible for translocating LapA to the cell surface. Together, these data highlight the power of combining the use of AFM and 3D-SIM with genetic studies to demonstrate that LapA, a member of a large group of RTX-like repeat proteins, is a cell-surface adhesin
Zuccala, E.S., Gout, A.M., Dekiwadia, C., Marapana, D.S., Angrisano, F., Turnbull, L., Rogers, K.L., Whitchurch, C.B., Ralph, S.A., Speed, T.P. & Baum, J. 2012, 'Subcompartmentalisation of proteins in the rhoptries correlates with ordered events of erythrocyte invasion by the blood stage malaria parasite', PLoS One, vol. 7, no. 9, p. e46160.
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Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction - the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion.
Strauss, M., Liew, A.T., Turnbull, L., Whitchurch, C.B., Monahan, L.G. & Harry, L. 2012, '3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis', Plos Biology, vol. 10, no. 9, p. e1001389.
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FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.
Robinson, M.W., Alvarado, R., To, J., Hutchinson, A.T., Dowdell, S.N., Lund, M.E., Turnbull, L., Whitchurch, C.B., O'Brien, B., Dalton, J.P. & Donnelly, S.M. 2012, 'A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase', Faseb Journal, vol. 26, no. 11, pp. 4614-4627.
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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 amphipathic 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.-Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase
Horsington, J., Turnbull, L., Whitchurch, C.B. & Newsome, T.P. 2012, 'Sub-viral imaging of vaccinia virus using super-resolution microscopy', Journal of Virological Methods, vol. 186, pp. 132-136.
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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 similar to 250 nm, 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.
Angrisano, F., Sturm, A., Volz, J.C., Delves, M.J., Zuccala, E.S., Turnbull, L., Dekiwadia, C., Olshina, M.A., Marapana, D.S., Wong, W., Mollard, V., Bradin, C.H., Tonkin, C.J., Gunning, P.W., Ralph, S.A., Whitchurch, C.B., Sinden, R.E., Cowman, A., McFadden, G.I. & Baum, J. 2012, 'Spatial localisation of actin filaments across developmental stages of the malaria parasite', PLoS One, vol. 7, no. 2, p. e32188.
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Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu.
Nolan, L.M., Croft, L., Jones, P.M., George, A.M., Turnbull, L. & Whitchurch, C.B. 2012, 'Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels', Microbiology Open, vol. 1, no. 4, pp. 490-501.
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Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.
Baldi, D.L., Higginson, E.E., Hocking, D.M., Praszkier, J., Cavaliere, R., James, C.E., Bennett-Wood, V., Azzopardi, K.I., Turnbull, L., Lithgow, T., Robins-Browne, R.M., Whitchurch, C.B. & Tauschek, M. 2012, 'The Type II Secretion System and Its Ubiquitous Lipoprotein Substrate, SslE, Are Required for Biofilm Formation and Virulence of Enteropathogenic Escherichia coli', INFECTION AND IMMUNITY, vol. 80, no. 6, pp. 2042-2052.
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Riglar, D.T., Richard, D., Wilson, D.W., Martin, B., Dekiwadia, C., Turnbull, L., Angrisano, F., Marapana, D.S., Rogers, K.L., Whitchurch, C.B., Beeson, J.G., Cowman, A., Ralph, S.A. & Baum, J. 2011, 'Super-Resolution Dissection of Coordinated Events during Malaria Parasite Invasion of the Human Erythrocyte', Cell Host and Microbe, vol. 9, no. 1, pp. 9-20.
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Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vi
Yeoman, J.A., Hanssen, E., Maier, A.G., Klonis, N., Maco, B., Baum, J., Turnbull, L., Whitchurch, C.B., Dixon, M. & Tilley, L. 2011, 'Tracking glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum', Eukaryotic Cell, vol. 10, no. 4, pp. 556-564.
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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 tha
Naughton, S., Parker, D., Seemann, T., Thomas, T., Turnbull, L., Bye, P., Cordwell, S.J., Whitchurch, C.B. & Manos, J. 2011, 'Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung', PLoS ONE, vol. 6, no. 9, p. e24526.
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Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.
Hollands, A., Pence, M.A., Timmer, A.M., Osvath, S.R., Turnbull, L., Whitchurch, C.B., Walker, M.J. & Nizet, V. 2010, 'Genetic switch to hypervirulence impairs colonization phenotypes of the globally disseminated Group A Streptococcus M1T1 clone', Journal of Infectious Diseases, vol. 202, no. 1, pp. 11-19.
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Background.The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation has been linked to mutations in the covR/S 2-component regulator. We investigated whether a fitness cost is associated with the covS mutation that counterbalances hypervirulence. Methods.Wild-type M1T1 group A Streptococcus and an isogenic covS-mutant strain derived from animal passage were compared for adherence to human laryngeal epithelial cells, human keratinocytes, or fibronectin; biofilm formation; and binding to intact mouse skin. Targeted mutagenesis of capsule expression of both strains was performed for analysis of its unique contribution to the observed phenotypes. Results.The covS-mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS-mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS-mutant strain was demonstrated in a murine model.
Williams, H.L., Turnbull, L., Thomas, S.J., Murphy, A., Stinear, T., Armstrong, D.S. & Whitchurch, C.B. 2010, 'A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa', Annals of Clinical Microbiology and Antimicrobials, vol. 9, no. 18, pp. 1-8.
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Background 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. Methods 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. Results 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 samples.
Kaparakis, M., Turnbull, L., Carneiro, L., Firth, S., Coleman, H.A., Parkington, H.C., Le Bourhis, L., Karrar, A., Viala, J., Mak, J., Hutton, M.L., Davies, J.K., Crack, P.J., Hertzog, P.J., Philpott, D.J., Girardin, S.E., Whitchurch, C.B. & Ferrero, R.L. 2010, 'Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells', Cellular Microbiology, vol. 12, no. 3, pp. 372-385.
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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.
Donnelly, S.M., O'Neill, S., Stack, C.M., Robinson, M.W., Turnbull, L., Whitchurch, C.B. & Dalton, J.P. 2010, 'Helminth cysteine proteases inhibit TRIF - dependent activation of macrophages via degradation of TLR3.', Journal Of Biological Chemistry, vol. 285, no. 5, pp. 3383-3392.
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Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCLl) and Schistosoma mansoni (SmCBI) protect mice from the lethal effects oflipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor a, and interleukin- 12, from macro phages. The proteases specifically blocl< the MyDSS-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation ofTLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development ofThl responses.
Fung, C., Naughton, S., Turnbull, L., Tingpej, P., Rose, B., Arthur, J., Hu, H., Harmer, C., Harbour, C., Hassett, D.J., Whitchurch, C.B. & Manos, J. 2010, 'Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum', Journal of Medical Microbiology, vol. 59, no. 9, pp. 1089-1100.
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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.
Han, X., Kennan, R.M., Davies, J.K., Reddacliff, L.A., Dhungyel, O.P., Whittington, R.J., Turnbull, L., Whitchurch, C.B. & Rood, J.I. 2008, 'Twitching Motility Is Essential For Virulence In Dichelobacter Nodosus', Journal Of Bacteriology, vol. 190, no. 9, pp. 3323-3335.
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Type W 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
McCloskey, D.T., Turcato, S., Wang, G.-.Y., Turnbull, L., Zhu, B.-.Q., Bambino, T., Nguyen, A.P., Lovett, D.H., Nissenson, R.A., Karliner, J.S. & Baker, A.J. 2008, 'Expression of a G(i)-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy', AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, vol. 294, no. 1, pp. H205-H212.
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Amirahmadi, F., Turnbull, L., Du, X.-.J., Graham, R.M. & Woodcock, E.A. 2008, 'Heightened alpha(1A)-adrenergic receptor activity suppresses ischaemia/reperfusion-induced Ins(1,4,5)P-3 generation in the mouse heart: a comparison with ischaemic preconditioning', CLINICAL SCIENCE, vol. 114, no. 1-2, pp. 157-164.
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O'connell, T.D., Swigart, P., Rodrigo, M., Ishizaka, S., Joho, S., Turnbull, L., Tecott, L., Baker, A.J., Foster, E., Grossman, W. & Simpson, P. 2006, 'Alpha(1)-Adrenergic Receptors Prevent A Maladaptive Cardiac Response To Pressure Overload', Journal Of Clinical Investigation, vol. 116, no. 4, pp. 1005-1015.
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An alpha(1)-adrenergic receptor (alpha(1)-AR) antagonist increased heart failure in the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), but it is unknown whether this adverse result was due to alpha(1)-AR inhibition
Turcato, S., Turnbull, L., Wang, G., Honbo, N., Simpson, P., Karliner, J. & Baker, A.J. 2006, 'Ischemic Preconditioning Depends On Age And Gender', Basic Research in Cardiology, vol. 101, no. 3, pp. 235-243.
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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 Langendo
Lanzafame, A.A., Turnbull, L., Amiramahdi, F., Arthur, J.F., Huynh, H. & Woodcock, E.A. 2006, 'Inositol phospholipids localized to caveolae in rat heart are regulated by alpha(1)-adrenergic receptors and by ischemia-reperfusion', AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, vol. 290, no. 5, pp. H2059-H2065.
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Turnbull, L., Zhou, H.Z., Swigart, P.M., Turcato, S., Karliner, J.S., Conklin, B.R., Simpson, P.C. & Baker, A.J. 2006, 'Sustained preconditioning induced by cardiac transgenesis with the tetracycline transactivator', AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, vol. 290, no. 3, pp. H1103-H1109.
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Mccloskey, D., Turnbull, L., Swigart, P., Zambon, A., Turcato, S., Joho, S., Grossman, W., Conklin, B., Simpson, P. & Baker, A.J. 2005, 'Cardiac Transgenesis With The Tetracycline Transactivator Changes Myocardial Function And Gene Expression', Physiological Genomics, vol. 22, no. 1, pp. 118-126.
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The cardiac-specific tetracycline-regulated gene expression system (tet-system) is a powerful tool using double-transgenic mice. The cardiac alpha-myosin heavy chain promoter (alpha MHC) drives lifetime expression of a tetracycline-inhibited transcriptio
Mccloskey, D., Turnbull, L., Swigart, P., O'connell, T.D., Simpson, P. & Baker, A.J. 2003, 'Abnormal Myocardial Contraction In Alpha(1A)- And Alpha(1B)-Adrenoceptor Double-Knockout Mice', Journal of Molecular Cell Cardiology, vol. 35, no. 10, pp. 1207-1216.
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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
Turnbull, L., McCloskey, D.T., O'Connell, T.D., Simpson, P.C. & Baker, A.J. 2003, 'alpha(1)-Adrenergic receptor responses in alpha(1AB)-AR knockout mouse hearts suggest the presence alpha(1D)-AR', AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, vol. 284, no. 4, pp. H1104-H1109.
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Turnbull, L., McCloskey, D.T., O'Connell, T.D., Simpson, P.C. & Baker, A.J. 2003, '1-adrenergic receptor responses in 1AB-AR knockout mouse hearts suggest the presence of 1D-AR', American Journal of Physiology - Heart and Circulatory Physiology, vol. 284, no. 4 53-4.
Two functional 1-adrenergic receptor (AR) subtypes (1A and 1B) have been identified in the mouse heart. However, it is unclear whether the third known subtype, 1D-AR, is also present. To investigate this, we determined whether there were 1-AR responses in hearts from a novel mouse model lacking 1A- and 1B-ARs (double knockout) (ABKO). In Langendorff-perfused hearts, 1-ARs were stimulated with phenylephrine. For ABKO hearts, phenylephrine reduced left ventricular pressure and coronary flow (to 87 &plusmn; 2% and 86 &plusmn; 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 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 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 1A- and 1B-ARs retain functional 1-AR responses involving decreases of coronary flow and ventricular pressure that reflect 1D-AR-mediated vasoconstriction. Furthermore, 1-AR inotropic responses depend critically on the experimental conditions.
Turnbull, L., Hoh, J., Ludowyke, R. & Rossmanith, G. 2002, 'Troponin I Phosphorylation Enhances Crossbridge Kinetics During Beta-Adrenergic Stimulation In Rat Cardiac Tissue', Journal Of Physiology-london, vol. 542, no. 3, pp. 911-920.
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Inotropic agents that increase the intracellular levels of cAMP have been shown to increase crossbridge turnover kinetics in intact rat ventricular muscle, as measured by the parameter f(min) (the frequency at which dynamic stiffness is minimum). These a
Rossmanith, G., Hoh, J., Turnbull, L. & Ludowyke, R. 1997, 'Mechanism Of Action Of Endothelin In Rat Cardiac Muscle: Cross-Bridge Kinetics And Myosin Light Chain Phosphorylation', Journal Of Physiology-london, vol. 505, no. 1, pp. 217-227.
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1. The molecular mechanism of inotropic action of endothelin was investigated in rat ventricular muscle by studying its effects on characteristics of isometric twitch, barium-induced steady contracture and the level of incorporation of P-32(i) into myosi