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1
Second Symposium of the
Dublin Academy of Pathogenomics & Infection Biology
Wednesday 14th Sept 2011
09:00-‐17:00
Dublin Dental University Hospital
2
General Information
• The Dublin Dental University Hospital is located at the rear entrance to Trinity College at Lincoln Place (see map).
• All Talks will be in the Large Lecture Theatre on the third floor of the Dental Hospital. The third floor can be reached via the main stairs or the lift in the lobby
• Parking on TCD campus is for permit holders only; the nearest car park is at the Setanta Centre and can be accessed via Dawson St. The nearest DART station is Pearse Station (Westland Row) about five minutes walk away.
• Posters will be set up in the small lecture theatre and the Boardroom on the third floor. We request people with posters to arrive by 8:30am to allow posters to be in place before the first session starts at 09:00am.
• Posters, Teas/coffees/lunch will be in boardroom and the small lecture theatre
• There will be a prize for the best oral presentation and poster by a PhD student, to be presented at the wine reception, and funded by the Dublin Region Higher Education Alliance (DRHEA).
• The organisers are grateful to Roche, Alpha Technologies & Agilent Technologies for sponsorship of this DAPI Symposium
Trinity College Campus:
3
Programme
Large Lecture Theatre, Dublin Dental University Hospital
09:00 – 10.30 Chair: Gary Moran
09:00 -‐ 09:15 Welcome comments (Jay)
09:15 – 09:40 Shane Dillon, TCD. “Genome-‐wide Analysis of H-‐NS-‐like Proteins in Salmonella”
09:40 -‐ 10:05 Linda Holland, UCD. "Analysis of mating and biofilm formation by the pathogenic yeast Candida parapsilosis".
10:05 -‐ 10:30 Anna Shore, TCD, “Molecular Epidemiology of MRSA in Irish Hospital and Community Settings: Strain Replacement and Rapid Evolution”
10:30 – 11:00 Coffee and posters
11:00 – 12:50 Chair: Geraldine Butler
11:00 – 11:25 Belinda Maher, TCD. “Strain dependent activation of IL-‐1b regulates gammadeltaT-‐cell function and infection outcomes during Staphylococcus aureus surgical wound infection”
11:25 – 11:50 Nicole Tegtmeyer, UCD. "Novel role of the actin-‐binding protein cortactin in Helicobacter pylori infections".
11:50 – 12:50 Keynote lecture Prof. David Holden, Imperial College, London, UK. “Subversion of host cell functions by Salmonella”
12:50 – 13:45 Lunch and Poster viewing
13:45 – 15:25 Chair: Rachel McLoughlin
13:45 – 14:10 Nicolae Corcionivoschi, UCD. “Disruption of Campylobacter jejuni phosphotyrosine signaling by mucosal hydrogen peroxide” 14:10 – 14:35 Letal Salzberg, TCD. “WalRK (YycFG)-‐mediated control of cell wall
metabolism in Bacillus subtilis: linking autolysins and actin-‐like proteins”
14:35 – 15:00 Elaine Waters, UCD. “Relationship between methicillin resistance, biofilm and virulence in Staphylococcus aureus”
15:00 – 15:25 Pin Tong UCD, “RNA-‐seq analysis of the transcriptomes of the smooth and rough phenotypes of the human pathogen Mycobacterium abscessus.”
15:25 – 15:45 Coffee and posters
15:45 – 1700 Chair: Wim Meijer
15:45 – 16:10 Prof. Jay Hinton, TCD. “The virulence of Salmonella Typhimurium is regulated by sRNA”
16:10 – 16:35 Orla Condell, UCD. “Comparative proteomic response of isogenic susceptible and tolerant Salmonella to unrelated commercial biocides”
16:35 – 17:00 Joan Geoghegan, TCD. “The Mechanistic Basis of Fibronectin Binding Protein-‐Mediated Biofilm Formation in Staphylococcus aureus”
17:00 – 18.00 Closing remarks (Jay/Steve), Wine reception and prize giving.
4
Keynote Speaker
Prof David Holden Centre for Molecular Microbiology of Infection, Imperial College, London
Professor David Holden invented signature-‐tagged mutagenesis (STM), in which
molecular barcoding allows high-‐throughput screens by identifying mutants having
reduced or increased adaptation to certain environments. Using STM, his group
identified the Salmonella SPI-‐2 type III secretion system, which translocates effector
proteins from bacteria across their phagosomal membranes into host cells. This
results in a specialised compartment – the Salmonella-‐containing vacuole (SCV) -‐
which permits replication of Salmonella in epithelial cells and macrophages. The
group is now studying the mechanisms governing the secretion and translocation
process, as well as the functions of individual effector proteins.
This lecture is supported by the Dublin Region Higher Education Alliance (DRHEA) graduate strand, funded by Cycle II of the Strategic Innovation Fund
5
Speaker Abstracts 09:15 -‐ 09:40 1
Genome-wide analysis of the H-NS-like proteins in Salmonella Dillon SC1, Cameron AD1, Hokamp K2, Lucchini S3, Hinton JC1, Dorman CJ1.
1Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland.
2Department of Genetics, Trinity College Dublin, Dublin 2, Ireland.
3Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK.
Email: [email protected]
The conjugative IncHI1 plasmid pSfR27 from Shigella flexneri 2a strain 2457T
encodes the Sfh protein, a paralogue of the global transcriptional repressor H-NS.
Sfh allows pSfR27 to be transmitted to new bacterial hosts with minimal impact on
host fitness, providing a 'stealth' function whose molecular mechanism has yet to be
determined. The impact of the Sfh protein on the Salmonella enterica serovar
Typhimurium transcriptome was assessed and binding sites for Sfh in the Salmonella
Typhimurium genome were identified by chromatin immunoprecipitation. Sfh did not
bind uniquely to any sites. Instead, it bound to a subset of the larger H-NS regulatory
network. Analysis of Sfh binding in the absence of H-NS revealed a greatly expanded
population of Sfh binding sites that included the majority of H-NS target genes.
Furthermore, the presence of plasmid pSfR27 caused a decrease in H-NS
interactions with the S. Typhimurium chromosome, suggesting that the A + T-rich
DNA of this large plasmid acts to titrate H-NS, removing it from chromosomal
locations. It is proposed that Sfh acts as a molecular backup for H-NS and that it
provides its 'stealth' function by replacing H-NS on the chromosome, thus minimizing
disturbances to the H-NS-DNA binding pattern in cells that acquire pSfR27.
6
09:40 – 10:05 2
Analysis of mating in the Candida parapsilosis species group Linda Holland, Sixiang Sai, Conor Magee, Denise Lynch and Geraldine Butler. School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
Candida orthopsilosis and Candida metapsilosis are closely related to Candida
parapsilosis, a major cause of infection in premature neonates. Mating has not been
observed in these species. All C. parapsilosis isolates tested to date have an MTLa
idiomorph at the Mating-type like locus, containing an MTLa1 pseudogene. However,
many of the other genes required for mating in C. albicans are present and intact in
C. parapsilosis. Molecular fingerprinting of C. orthopsilosis isolates reveal that they
contain a mixture of MTLa and MTLalpha homozygotes, and a/alpha heterozygotes.
Isolates belong to two divergent groups characterized by restriction patterns at the
MTL, which probably represent subspecies. Idiomorphs from both groups were
sequenced and were shown to be 95% identical and that the regulatory genes are
intact. In contrast, isolates of C. metapsilosis contain only MTLalpha idiomorphs. Our
results suggest that the role of MTL in determining cell type is being eroded in the C.
parapsilosis species complex. The population structure of C. orthopsilosis indicates
that mating may occur. However, expression of genes in the mating signal
transduction pathway do not respond to exposure to alpha factor. C. parapsilosis is
also non-responsive even when the GTPase-activating protein gene SST2 is deleted.
In addition, splicing of introns in MTLa1 and MTLa2 is defective in C. orthopsilosis.
Mating is not detected. The alpha factor peptide, which is the same sequence in C.
parapsilosis, C. orthopsilosis and C. metapsilosis, can induce a mating response in
Candida albicans. It is therefore likely that either mating of C. orthopsilosis takes
place under certain unidentified conditions, or the mating pathway has been adapted
for other functions such as cross-species communication.
7
10:05 – 10:30 3
Molecular Epidemiology of MRSA in Irish Hospital and Community Settings: Strain Replacement and Rapid Evolution Anna C. Shore, Orla M. Brennan, Emily C. Deasy, Sarah Tecklenborg, David C. Coleman
1Microbiology Research Unit, Dublin Dental University Hospital, University of Dublin, Trinity College Dublin, Ireland. E-mail: [email protected]
Staphylococcus aureus is a versatile pathogen responsible for a wide range of infections reflecting the extensive array of virulence factors and antimicrobial resistance genes it can express, many encoded on mobile genetic elements (MGEs). Methicillin-resistant S. aureus (MRSA) have acquired a MGE termed the staphylococcal cassette chromosome (SCCmec) element encoding the methicillin resistance gene mecA. MRSA are a major nosocomial problem worldwide and have also emerged as significant cause of infections among otherwise healthy individuals in the community and among animals.
MRSA have been endemic in Irish hospitals for almost four decades and in-line with trends worldwide MRSA harbouring the Panton-Valentine leukocidin toxin have also emerged in the Irish community. In-depth molecular typing revealed that different MRSA strains predominated in Irish hospitals and in the community at different time periods [1, 2]. High-throughput DNA microarray and whole-genome sequence analysis has revealed that some of these MRSA strains have acquired MGEs encoding genes that enhance their virulence or antimicrobial resistance potential. For example, we have identified the multidrug-resistance gene cfr on a novel conjugative plasmid in the pandemic community-acquired MRSA USA300 (ST8-MRSA-IVa) [3] and a novel arginine catabolic mobile element (ACME), which may enhance the ability of S. aureus to spread and survive, in the ST22-MRSA-IV clone which is currently endemic in Irish hospitals [4].
We recently identified a new development in the evolution of MRSA with the detection of a novel mecA gene in MRSA isolates from humans that may be of animal origin [5]. This mecA gene cannot be detected by routine molecular screening methods and using whole-genome sequencing it was found to be located on a highly divergent SCCmec element designated SCCmec XI, indicating that it may have originated in another taxon.
The results of these studies demonstrate the ever-changing epidemiology of MRSA in Ireland and ability of S. aureus to change and adapt to different ecological niches through the acquisition of exogenous genes not just from S. aureus and other staphylococci but also from other bacteria. In addition, these studies demonstrate the potential of the DNA microarray and whole-genome sequencing for genotyping MRSA and for identifying and performing detailed analysis of new and emerging strains with increased virulence and extended antimicrobial resistance. This technology has and will continue to play a fundamental role in controlling, monitoring and reducing the spread of MRSA in Ireland.
[1] Rossney, A.S., et al. 2007. J. Clin. Microbiol. 45:2554-2563. [2] Shore, A.C., et al. 2005. Antimicrob. Agents Chemother. 49:2070-2083. [3] Shore, A. C., et al. 2010. Antimicrob. Agents Chemother. 54:4978-4984. [4] Shore, A. C., et al. 2011. Antimicrob. Agents Chemother. 55: 1896-1905. [5] Shore, A. C., et al. 2011. Antimicrob. Agents Chemother. 55:3765-3773.
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11:00 – 11:25 4
Strain dependent activation of IL-1b regulates gd+ T cell function and infection outcomes in a S.aureus surgical wound model. Belinda M Maher1, Alison G Murphy1, Rachel M McLoughlin.1 1Department of Biochemistry and Immunology, Trinity College Dublin. E-mail: [email protected] In this study we investigate a role for gd+T-cells during Staphylococcus aureus
infection. S.aureus surgical wound infection was established in wild-type (WT) and
gd+T-cell deficient (gd-/-) mice using two strains of S.aureus; SH1000, and PS80.
We demonstrate no difference in bacterial load between gd-/- and WT mice on day 3
post-infection with PS80 (102 CFU). However, gd-/- mice had significantly elevated
levels of infection compared to WT mice following infection with SH1000 (102 CFU).
To elucidate molecular events governing differential resolution of infection with each
strain, we assessed their effects on antigen presenting cell (APC) activation. APC’s
produced less IL-1b (41.9 vs. 96.3 pg/ml, p=0.004) and IL-23 (50.9 vs. 101.2 pg/ml)
following stimulation with SH1000 compared to PS80 respectively. IL-1b and IL-23
are proven regulators of IL-17 synthesis by gd+T-cells. To establish if differential
APC activation by the strains impacted IL-17 production by gd+T cells, culture
supernatants from APC’s stimulated with SH1000 or PS80 were added to purified
gd+T-cells. Supernatants from SH100 treated APCs induced little IL-17 from gd+T
cells in comparison to PS80 (26.9 vs. 262.2 pg/ml respectively). We then assessed
IL-17 production at the wound infection site on day 3 post-infection. IL-17 was
exclusively produced by gd+ T-cells during infection with both stains. However while
17.8% of gd+T cells produced IL-17 in response to PS80, significantly less gd+T
cells produced IL-17 in response to SH1000 (9.9%, p=0.05). This data indicates that
S.aureus strain-dependant effects on the IL-1b pathway regulate gd+T cell
production of IL-17 and subsequent bacterial clearance.
9
11:25 – 11:50 5 Diverse roles of the actin-binding protein cortactin in bacterial pathogenesis Nicole Tegtmeyer and Steffen Backert School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
Cell migration and invasion require the coordinated regulation of cytoskeletal
architectural changes by signaling factors, including the actin-binding protein
cortactin. Bacterial and viral pathogens subvert these signaling factors to promote
their uptake, spread and dissemination. We show that the gastric pathogen
Helicobacter pylori targets cortactin by two independent processes leading to its
tyrosine dephosphorylation and serine phosphorylation to regulate cell scattering
and elongation. The phosphorylation status of cortactin dictates its subcellular
localization and signaling partners. Upon infection, cortactin was found to interact
with and stimulate the kinase activity of focal adhesion kinase (FAK). This interaction
required the SH3 domain and phosphorylation of cortactin at serine 405 and a
proline-rich sequence in FAK. Using Helicobacter pylori as a model, this study
unravels a previously unrecognized FAK activation pathway. We propose that
Helicobacter pylori targets cortactin in order to trap active FAK, and to protect the
gastric epithelium from excessive cell lifting and ensure sustained infection in the
stomach.
10
13:45 – 14:10 6
Disruption of Campylobacter jejuni phosphotyrosine signaling by mucosal hydrogen peroxide Nicolae Corcionivoschi,2,6 Luis A. Alvarez,1,6 Thomas H. Sharp,3,4 Monika Strengert,1 Abofu Alemka,2 Judith Mantell,3,5 Paul Verkade,3,5 Ulla G. Knaus,1,7* and Billy Bourke 1,2,7*
1Conway Institute, School of Medicine and Medical Science, University College Dublin, and 2 National Children’s Research Centre, Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland 3School of Biochemistry, 4School of Chemistry, 5Wolfson Bioimaging Facility, University of Bristol, Bristol, England Homeostasis of intestinal mucosa requires maintenance of host-microbe equilibrium
while protecting from pathogenic challenges. Reactive oxygen species (ROS) play a
role in mucosal defense, yet how they are induced and the consequences for
pathogens are not clear. Here we report that infection with the diarrhoeal pathogen
Campylobacter jejuni stimulates ROS generation by epithelial NADPH oxidases
(Nox1, Duox2). Rather than having a direct bactericidal effect, ROS attenuate C.
jejuni pathogenicity by altering bacterial signal transduction. Specifically, ROS
released from the luminal surface of the mucosa switched off tyrosine
phosphorylation-controlled capsule production. Further, a novel, unconventional
bacterial tyrosine kinase in C. jejuni outer membranes was identified that regulates
capsule polysaccharide synthesis by phosphorylation of UDP-GlcNAc/Glc 4-
epimerase. Our results place epithelial Nox/Duox as an early antibacterial defense
system in the intestinal mucosa that may act as a general virulence modifier.
11
14:10 – 14:35 7
WalRK (YycFG)-mediated control of cell wall metabolism in Bacillus subtilis: linking autolysins and actin-like proteins. Letal Salzberg, Leagh Powell, Karsten Hokamp, Paola Bisicchia, David Noone and Kevin M Devine. Smurfit Institute of Genetics, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2. Ireland. E-mail: [email protected]
The Bacillus subtilis cell wall is a three-dimensional mesh composed of
peptidoglycan to which an approximately equal amount of teichoic acid is covalently
attached. It determines the shape of the bacterium, forms a protective barrier
between the cell and the external milieu and creates a buffer zone with a distinctive
environment in which synthetic activities can be executed. The cell wall is
extensively remodeled during cell growth and division requiring that intracellular
production of synthetic intermediates (eg. lipid II) and enzymes (eg. autolysins) is
coordinated with their utilization. The essential WalRK (YycFG) two-component
system plays an important role in this process, sensing cell growth through septum
formation and directing expression of cell division protein FtsZ, autolysins (YocH,
CwlO, LytE), the cell wall associated protein (YdjM) and inhibiting autolysin inhibitors
(IseA) and peptidoglycan modifying enzyme (YjeA). It has been difficult to identify
the complete WalRK regulon because of its essentiality. Here we will report on
studies that have established a comprehensive view of WalR~P activity in vivo using
ChIP on chip analysis. These newly identified members of the regulon appear to be
regulated by mechanisms additional to WalRK explaining why they were not
identified by transcriptome analyses. We will discuss the newly identified regulon
members, the linkage between WalRK controlled autolysins and the actin-like
proteins, and their role in cell wall metabolism during growth of B. subtilis.
12
14:35 – 15:00 8
Relationship between methicillin resistance, biofilm and virulence in Staphylococcus aureus Waters E.M., C. Pozzi, J. Rudkin, C. Schaeffer, G.B. Pier, P.D. Fey, R.C. Massey and J.P. O’Gara Conway Institute, School of Biomolecular and Biomedical Science, University College Dublin The acquisition of mecA, which encodes penicillin binding protein 2a (PBP2a) and
methicillin resistance, by Staphylococcus aureus has added to an already impressive
array of virulence mechanisms including enzyme and toxin production, biofilm
forming capacity and immune evasion. And yet clinical data does not indicate that
healthcare-associated methicillin resistant S. aureus (MRSA) strains are more
virulent than their methicillin-sensitive counterparts. Our findings suggest that MRSA
sacrifices virulence potential for antibiotic resistance and that expression of
methicillin resistance alters the biofilm phenotype but does not interfere with the
colonization of implanted medical devices in vivo. High level expression of PBP2a
resulted in these pleiotrophic effects by blocking icaADBC-dependent polysaccharide
type biofilm development and promoting an alternative PBP2a-mediated biofilm,
repressing the accessory gene regulator and extracellular protease production, and
attenuating virulence in a mouse device-infection model. Thus the adaptation of
MRSA to the hospital environment has apparently focused on the acquisition of
antibiotic resistance and retention of biofilm forming capacity, which are likely to be
more advantageous than metabolically-expensive enzyme and toxin production in
immunocompromised patients with implanted medical devices offering a route to
infection.
13
15:00 – 15:25 9
RNA-seq analysis of the transcriptomes of the smooth and rough phenotypes of the human pathogen Mycobacterium abscessus.
Pin Tong, Kevin Conlon, Amanda Lohan, Stephen Gordon and Brendan Loftus
Conway Institute, University College Dublin
Mycobacterium abscessus is a rapidly growing mycobacterium and causative agent
of chronic infections, particularly in those with immunodeficiency or cystic fibrosis. M.
abscessus infections represent a significant health threat because of the resistance
of this bacterium to the majority of antibiotics and biocides. M. abscessus can be
found in either smooth or rough colony morphology delineated by the presence of cell
wall associated glycopeptidolipids (GPLs). Smooth M. abscessus possesses surface
GPLs which are associated with transient infections, while rough morphology M.
abscessus lacks GPLs and is associated with persistent infections. Smooth to rough
transitions can occur during the course of infection however the rough phenotype
retains it morphology in culture.
In this study, RNA-seq was used to compare the log-phase transcriptomes of
colonies of smooth and rough phenotype M. abscessus derived from the same
inoculum. Previous genome sequencing could identify no genetic changes to explain
the phenotypic differences.
14
15:45– 16:10 10 The virulence of Salmonella Typhimurium is regulated by sRNA
Magali Hébrard1, Carsten Kröger1, Sathesh K. Sivasankaran1, Kristian Händler1, Samantha Paré1, Karsten Hokamp1, Alex Sittka, Yanjie Chao2, Kai Papenfort2, Cynthia Sharma2, Jörg Vogel2 & Jay C. D. Hinton1
1School of Genetics & Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin, Ireland 2Institute for Molecular Infection Biology, Research Centre of Infectious Diseases, University of Würzburg, Germany
E-mail: [email protected] Salmonella enterica serovar Typhimurium is an adaptable and robust micro-organism that thrives in a variety of environmental niches, including the GI tract of humans, farm animals, birds and reptiles, and inside mammalian cells. Since the first transcriptomic analysis of S. Typhimurium was published in 2002 [1], great progress has been made in the gene expression profiling of S. Typhimurium. A recent review describes the fifty transcriptomic datasets that are currently available [2] and how systems-level studies are beginning to unify the disparate datasets [3]. To elucidate new aspects of the regulation of Salmonella gene expression that are critical for survival, adaptation and disease, we used a deep sequencing-based strategy to identify and audit the sRNAs of S. Typhimurium expressed during the infection of mammalian cells. Our recent data suggest that the ability of S. Typhimurium to replicate within murine macrophages is controlled by novel small regulatory RNA molecules. [1] Clements MO, Eriksson S, Thompson A, Lucchini S, Hinton JC, et al. (2002)
Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica. Proc Natl Acad Sci U S A 99: 8784-8789.
[2] Hebrard M, Kröger C, Sivasankaran SK, Händler K, Hinton JC (2011) The challenge of relating gene expression to the virulence of Salmonella enterica serovar Typhimurium. Curr Opin Biotechnol.
[3] McDermott JE, Yoon H, Nakayasu ES, Metz TO, Hyduke DR, et al. (2011) Technologies and approaches to elucidate & model the virulence program of Salmonella. Front Microbiol 2: 121.
15
16:10 – 16:35 11
Comparative proteomic response of isogenic susceptible and tolerant Salmonella to unrelated commercial biocides O. Condell, A. Sheridan, C. Iversen, K. Power, R. Bonilla-Santiago, K. Burgess, J.E. Nally and S. Fanning School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents and their increasing use is a matter for concern. Specifically the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a cross-resistance phenotype to clinically important antimicrobial compounds, needs to be assessed. In this study we investigated the tolerance of a collection of susceptible and multi-drug resistant (MDR) Salmonella to a panel of eight food-grade biocide formulations. We explored their ability to adapt to these formulations and to individual biocidal agents including; triclosan, chlorhexidine and benzalkonium chloride. Susceptibility was evaluated in several ways; during planktonic growth, against surface-dried bacterial cells, and cells contained in a biofilm. Finally, cross-tolerance between different categories of biocidal formulations, their active agents and the potential for co-selection of resistance to clinically important antibiotics was investigated. Seven of the eight biocide formulations were bactericidal against planktonically grown cultures. MDR Salmonella exhibited the same pattern of susceptibility to the biocide formulations when compared to susceptible counterparts. However, all showed reduced activity against surface-dried and biofilm bacterial cultures. A stable tolerant phenotype to biocide formulations could not be selected for by in vitro means. Nevertheless, when exposed to single active biocidal compounds a high-level of tolerance was selected in a number of Salmonella serotypes. No cross-tolerance between the different biocidal agents or food-grade biocide formulations was observed in these mutant strains. Interestingly, all mutants displayed changes in their susceptibility patterns to a panel of antimicrobial compounds. The biocide tolerant Salmonella were studied at the proteomic level in order to elucidate the mechanisms involved in their tolerance phenotype. Our data pointed to a clear divergence in the response at the proteomic-level of wild type and susceptible strains and their isogenic, tolerant mutant counterparts. More than 400 proteins showed a differential expression in the chlorhexidine tolerant strain with over 50 proteins being differentially expressed in the triclosan tolerant strain. The number of alterations in the proteome may be indicative of the effects and corresponding tolerance mechanisms elicited by the biocidal agents; for example chlorhexidine is known to have a broad spectrum mechanism of action, whilst triclosan has a unique cell target. Two-hundred differentially expressed proteins were selected and subjected to mass spectrometry for identification. This work confirms that sub-lethal concentrations of food industry biocide active ingredients can induce discernible alterations in the proteome of exposed Salmonella. These changes are associated with a reduced susceptibility to clinically important antibiotics.
16
16:35 – 17:00 12
The Mechanistic Basis of Fibronectin Binding Protein-Mediated Biofilm Formation in Staphylococcus aureus. Joan A. Geoghegan1, Andrew Brentnall2, Jennifer R. Potts2, Timothy J. Foster1
1Microbiology Department, Moyne Institute of Preventive Medicine, Trinity College,
Dublin 2, Ireland; 2Department of Biology, University of York, York, UK.
E-mail: [email protected]
Certain strains of methicillin-resistant Staphylococcus aureus (MRSA) form biofilm in vitro that is dependent on expression of surface-located fibronectin binding proteins A and B (FnBPA, FnBPB). Primary attachment to hydrophobic surfaces is mediated by the major autolysin Atl and the initial development of biofilm requires autolytic activity and the release of some DNA. FnBP expression is required for biofilm accumulation which involves the N-terminal fibrinogen- and elastin-binding A domain, although the ability to bind to these ligand is not required for biofilm.
The mechanistic basis of FnBP-promoted biofilm formation has been investigated. In contrast to wild-type control strains, FnBPs of MRSA strain BH1CC were expressed at high levels and were detectable on cells from the late stationary phase of growth. This correlated with low level expression of the V8 serine protease (which was previously shown to degrade FnBPs on cells from stationary phase).
FnBP-mediated accumulation required a physiological concentration of Zn2+
and was inhibited by removal of Zn2+ with a chelator. In addition, biofilm accumulation was enhanced in mildly acidic conditions (pH = 5.5) triggered by growth in glucose-containing broth. These results were supported by data showing that the recombinant A domain of FnBPA formed dimers in vitro in a manner dependent on Zn2+ and low pH (5.5). The ability of recombinant FnBPs to dimerise suggests that a direct interaction between FnBP A domains on the surface of adjacent bacteria allows multicellular aggregates to form.
Studies are underway to localise regions of the FnBP A domain involved in mediating biofilm accumulation including crystallization of FnBP A domain dimers . Understanding how the A domains interact with each other could allow design of specific inhibitors.
17
Poster Presentations
Title Authors Addresses
1 Lactate Utilisation by the Pathogenic Actinomycete Rhodococcus equi
A. Miranda Caso Luengo, R. Miranda Caso Luengo, W. Meijer.
School of Biomolecular and Biomedical Science, UCD.
2 Characterization of the Interaction of Staphylococcus aureus with the Human Membrane Receptor CD36 on the Surface of Platelets
A. Murray and J. Mitchell School of Biomolecular and Biomedical Science, UCD.
3 Characterization of two proteases of Campylobacter jejuni and their potential role in disease development
A. Sheridan, M.Boehm, D.Baumann, B.Hoy, S. Wessler , U.Gross, G.Cagney
and S.Backert
School of Biomolecular and Biomedical Science, UCD.
4 S. aureus exposure expands a population of antigen specific T-‐cells that mediate protection against subsequent infection
Alison Murphy & Rachel M. McLoughlin
School of Biochemistry and Immunology , TCD
5 Transcriptional regulation of chemotaxis and motility through DNA topology in the enteric pathogen Campylobacter jejuni
C. Shortt, E. Scanlan, B. Bourke and T.Ó Cróinín
School of Biomolecular and Biomedical Science, UCD.
6 A novel secreted virulence factor of the
foodborne pathogen Campylobacter jejuni
D. Baumann , M.Boehm , B. Hoy , O. A. Oyarzabal , L. Bronsted , S.Wessler and Steffen Backert
School of Biomolecular and Biomedical Science, UCD.
7 Comparative Analysis of novel transcripts in Candida species
Denise B. Lynch, Kenneth H. Wolfe, Geraldine Butler
School of Biomolecular and Biomedical Science, UCD.
8 DNA supercoiling in C. jejuni: a potential global regulator of invasion
E. Scanlan, C. Shortt, B. Bourke and T. Ó Cróinín
School of Biomolecular and Biomedical Science, UCD.
9 Characterisation of Helicobacter pylori protease HtrA from worldwide patients revealed wide distribution of the gene, functional conservation and proteolytic activity on E-‐cadherin
E. De Poire, F.Rivas Traverso, B. Hoy, R. Rad, N. Tegtmeyer, S.Wessler
and S. Backert
School of Biomolecular and Biomedical Science, UCD.
10 Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid
Emma Jane Smith, Rebecca Corrigan, Angelika Grundling and Timothy J. Foster
Department of Microbiology, TCD
11 Antimitotic herbicides block development of intra-‐erythrocytic Plasmodium falciparum by binding to a novel site on tubulin
Enda Dempsey, Brian J. Fennell, Christine Mara, James W. Barlow and Angus Bell
Department of Microbiology, TCD
12 Cell envelope gene expression in phosphate limited Bacillus subtilis cells determined by live cell array (LCA) analysis.
Eric Botella, Sebastian Hübner, Karsten Hokamp, Annette Hansen, Paola Bisicchia, David Noone, Leagh Powell, Letal I Salzberg and Kevin M Devine
School of Genetics and Microbiology, Trinity College Dublin
13 Rhodococcus equi secretion VII is not related with proliferation in macrophage.
H. Luo and W. Meijer School of Biomolecular and Biomedical Science, UCD.
18
14 The ompR promoters of Salmonella enterica and Escherichia coli partially determine each species' differential response to pH.
Heather J. Quinn and Charles J. Dorman
Department of Microbiology, TCD
15 Response of extra-‐intestinal Escherichia coli to attack by human complement
Helen Miajlovic, Gary Moran, Thomas Rogers, Stephen Smith
Department of Clinical Microbiology, TCD
16 Improved toolbox for the genetic manipulation of Staphylococci
Ian R. Monk and Timothy J. Foster
Department of Microbiology, TCD
17 The telomere-‐associated (TLO) genes of Candida albicans and C. dubliniensis
J. Haran, T. Yeomans, D. Sullivan & G. Moran
TCD School of Dental Science, Dublin Dental University Hospital
18 Clostridium difficile ermB alleles associated with clinical outbreaks
K. Solomen, L. Kyne & S. Fanning
UCD Centre for Food Safety, School of Public Health, Physiotherapy & Population Science, UCD.
19 Cronobacter sakazakii SP291 –a persistent thermotolerant PIF factory-‐derived pathogen
K.A. Power, S. Cooney, C. Iversen & S. Fanning
UCD Centre for Food Safety, School of Public Health, Physiotherapy & Population Science, UCD.
20 Regulation of CwlO (YvcE) autolysin expression in Bacillus subtilis.
Leagh Powell, David Noone, Letal Salzberg and Kevin M Devine
School of Genetics and Microbiology, Trinity College Dublin
21 Analysis of the role of EFG1 in Candida parapsilosis
Leona Connolly, Geraldine Butler
School of Biomolecular and Biomedical Science, UCD.
22 Investigation of the endophytic colonization ability of Salmonella in mung bean sprouts
M. Chirico, S. Finn, M.P. Nuti & S. Fanning
UCD Centre for Food Safety, School of Public Health, Physiotherapy & Population Science, UCD.
23 Host cell invasion of Campylobacter jejuni: crucial role of fibronectin, integrins, tyrosine kinases and small Rho GTPases for the entry process
M.Boehm, M.Krause-‐Gruszcynska, N.Tegtmeyer,O.A.Oyarzabal, S.Takahashi & S. Backert
School of Biomolecular and Biomedical Science, UCD.
24 Genotypic and phenotypic characterization of Salmonella enterica serovar Agona SAGOXB.0066 –cause of the 2008 European outbreak
M.P. McCusker, M.S. Martins, K. Power & S. Fanning
UCD Centre for Food Safety, School of Public Health, Physiotherapy & Population Science, UCD.
25 Ex-‐vivo studies on Salmonella enterica serovar Agona SAGOXB.0066 –cause of the 2008 European outbreak
M.S. Martins, M.P. McCusker & S. Fanning
UCD Centre for Food Safety, School of Public Health, Physiotherapy & Population Science, UCD.
26 Identification of novel small non-‐coding RNAs and the transcriptional architecture of Salmonella Typhimurium
Magali Hébrard, Carsten Kröger, Sathesh K. Sivasankaran, Alex Sittka, Kai Papenfort, Sacha Lucchini, Cynthia Sharma, Jörg Vogel & Jay C.D. Hinton
Department of Microbiology, TCD Institute of Food Research, Norwich, UK University of Würzburg, Germany.