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Second Symposium of the

Dublin Academy of Pathogenomics & Infection Biology

Wednesday 14th Sept 2011

09:00-‐17:00

Dublin Dental University Hospital

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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:

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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.

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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

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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


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


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


7 Comparative Analysis of novel transcripts in Candida species

Denise B. Lynch, Kenneth H. Wolfe, Geraldine Butler


8 DNA supercoiling in C. jejuni: a potential global regulator of invasion

E. Scanlan, C. Shortt, B. Bourke and T. Ó Cróinín


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


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


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.

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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


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


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


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


22 Investigation of the endophytic colonization ability of Salmonella in mung bean sprouts

M. Chirico, S. Finn, M.P. Nuti & S. Fanning


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


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


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


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.

second’symposiumofthe’ dublinacademyofpathogenomics ... · 1 ! second’symposiumofthe’...

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