Teasing out the mechanisms of biofilm formation for the treatment of S. aureus infections on indwelling devices: the role of the surface protein SdrC

Antibiotic resistance has become a great challenge in the healthcare setting. In particular antibiotic resistant strains of Staphylococcus aureus pose further challenges. Methicillin resistant S. aureus (MRSA) is widespread in healthcare facilities and in the wider community and multi-drug resistant strains have been identified. S. aureus is normally present on the surface of the skin where it causes no harm. However, it can easily colonize open wounds causing infection. Systemic infections can result from these wound infections leading to severe problems such as sepsis and infective endocarditis. Infection after surgical implantation of devices, such as joint replacements, can result in the formation of biofilms coating the devices that are difficult to treat. Biofilms are an accumulation of bacteria on a surface which often persist as most antibiotics do not easily penetrate them. In biofilms, bacteria interact directly with the foreign surface, with host proteins coating the surface and can also accumulate through interactions directly with each other.

Thus, there are multiple mechanisms involved in biofilm formation. It is important to fully understand all the mechanisms of biofilm formation in order to be able to disrupt their formation and persistence. In our recent paper, we have characterized the direct binding interaction (cell-cell adhesion) through the S. aureus surface protein, serine-aspartate repeat protein C (SdrC). Our study also reveals the mechanism of interaction between SdrC and inert surfaces. Furthermore, we have demonstrated how a small peptide can be used to block these interactions preventing biofilm formation suggesting a possible approach that could be used to treat SdrC dependent S. aureus biofilms. This study is the result of a multi-disciplinary collaboration across research institutes in Ireland and Belgium with Dr. Brennan (RCSI) contributing to the molecular modelling, Prof. Joan Geoghegan, Prof. Timothy Foster and Leanne Hayes (Trinity College Dublin) leading the molecular biology and microbiological functional studies and researchers from University Catholique de Louvain characterizing the interactions quantitatively using atomic force microscopy.

Marian Brennan

More details can be found in “Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC”.

 

Decoding neuroblastoma microenvironment

MCT Research Talks – 24th April 2017

The main challenge in treating high-risk neuroblastoma is to combat tumour metastasis and development of resistance to multiple chemotherapeutic drugs. In the native tissue, cancer cells are surrounded by a three-dimensional (3D) microenvironment which provides biological and physical support and determines disease initiation, progression, patient prognosis and response to treatment. The conventional two-dimensional (2D) cell culture lacks this feature resulting in discrepancies between in vitro and in vivo results. Current neuroblastoma studies employ either 2D cell culture systems or murine models or alternatively a mix of both.

In collaboration with Dr Caroline Curtin and Prof Fergal O’Biren (TERG), we decided to bridge the gap between 2D culture and in vivo tumours in neuroblastoma research by developing a tissue-engineered cell culture model of neuroblastoma. This project is supported by a pilot grant from Neuroblastoma UK.

To understand what signalling pathways are activated in 2D, 3D and in vivo neuroblastoma models, we decided to look closer at the differences between conventional 2D neuroblastoma cells and their xenografts. This way we hope to find those targets that are activated in both tumour microenvironment and the 3D tissue engineered models. Ciara and Larissa have begun this search by profiling xenograft samples with a panel of antibodies. Ciara became particularly fascinated by the elevated levels of c-jun, TCF1 and LEF1 in cisplatin-resistant neuroblastoma xenografts suggesting that the development of cisplatin resistance in neuroblastoma may be accompanied by activation of the wnt/b-catenin pathway in vivo. Larissa identified that cisplatin-resistant neuroblastoma cells secrete chromogranin A (CgA) at levels higher that cisplatin-sensitive cells. CgA levels also correlated with increased vascularisation and volume of murine orthotopic neuroblastoma xenografts. Altogether it suggests that CgA can be used as a marker of neuroblastoma cell growth both in vitro and in vivo.

Olga Piskareva

Characterisation of Novel FCγRIIa Inhibitors

MCT Research Talks – 10th, April 2017

Research talks were presented by Sheila Zarros, Tatyana Devine, Afnan Ali and Padraig Norton. Tatyana and Sheila were talking about challenges in the characterisation of novel FCγRIIa inhibitors.

Fc receptors are a widely distributed family of receptors that mediate cellular responses to antibodies or immunoglobulins (Ig). The Fc gamma receptor II, FcgRII (also known as CD32) is a low-affinity receptor for Fc portion of immunoglobulin G (IgG) and has two isoforms FcgRIIa and b. Fcg RIIa is widely expressed by human innate immune cells and is the only Fc gamma receptor found on human platelets.

Our group and others have demonstrated the significance of this receptor in the activation of platelets by bacteria, suggesting that it could be an important target in the treatment of sepsis. Its implications in rheumatoid arthritis, cancer pathogenesis, allergic reactions and flu virus-induced thrombocytopenia were also demonstrated.

Our project is focused on characterisation of novel small molecule compounds designed for targeting FcgRIIa receptor’s IgG binding site to inhibit bacteria-induced platelet aggregation in primary human plasma and investigation of their interactions with the FcgRIIa using surface plasmon resonance technology.

Afnan Ali reported on the role of the Fc gamma Receptor IIa (FcγRIIa) in platelet activation. Platelets express the FcγRIIa and this receptor has been identified as a key receptor in bacterial activation of platelets leading to thrombocytopenia and platelet activation. The aim of this study was to identify drugs that could be re-purposed for the treatment of sepsis and immune-mediated thrombocytopenia. We identified 42 drugs predicted to inhibit binding of IgG1 to the FcγRIIa using virtual high throughput screening. This included 20 antibacterial agents, 3 anti-fungals, 3 antiviral agents, 7 antineoplastics and 3 immunosuppressives. A selection of drugs were tested for inhibition of platelet adhesion to IgG, S. aureus-induced platelet aggregation and assessed for platelet activation. This work has identified multiple drugs that have potential to be to be repositioned for thrombocytopenia, sepsis and autoimmune disorders, as well as providing a possible mechanism of action to explain the immunosuppressive effects of some anti-neoplastics and immunosuppressive drugs.

Japan Society for Promotion of Science – Short-term post-doctoral fellowship

Following a workshop conducted at Hoshi University, Tokyo, Japan organized through the ISCA-Japan initiative funded by SFI in October, 2015 a successful collaborative initiative was established between Dr. Sudipto Das (MCT, RCSI) and Prof. Hiroko Ikeda (Department of Neurophysiology, Hoshi University) to investigate the role of epigenetic modifications like DNA methylation in driving a neuronal dysfunction phenotype associated with Diabetes mellitus (DM). Moving this collaboration forward with support from his collaborators at Hoshi University Dr. Sudipto Das has recently received a prestigious short-term post-doctoral fellowship to further his work at Hoshi University from the Japan Society for Promotion of Science (JSPS), which would essentially cover travel, subsistence and a research consumable allowance of 562,000 Japanese Yen. As a part of this fellowship, Dr. Das will travel to Japan for a period of 1.5 months in January 2018. The successful completion of the proposed project as a part of this proposal will for the first time allow the scientific community to understand as to how epigenetic modifications like DNA methylation impact on neurological dysfunction in endocrine

The successful completion of the proposed project as a part of this proposal will for the first time allow the scientific community to understand as to how epigenetic modifications like DNA methylation impact on neurological dysfunction in endocrine related disorders such as DM, thus opening up avenues to utilize this modification to potentially predict such conditions in DM patients.

Sudipto Das

The circadian protein BMAL1 in myeloid cells is a negative regulator of allergic asthma

Asthma is of particular relevance to the area of circadian control of immunity, since it is a disease with very strong clinical evidence demonstrating regulation by circadian variation. Airway hypersensitivity and asthma attacks are more common at night in humans. The molecular basis for this is unknown and no model of asthma in animals with genetic distortion of the molecular clock exists.

Asthma is under strong circadian variation. Asthma symptoms worsen at night, particularly in the early hours of the morning. Lung function fluctuates in healthy individuals over 24 h period and these fluctuations are even more pronounced in asthmatics.

In this study, we showed that mice lacking the main clock transcription factor BMAL1 in myeloid cells have increased lung inflammation demonstrated by higher numbers of eosinophils and increased IL-5 (key pathogenic cytokine in asthma that recruits eosinophils).This suggests that Bmal1 is a potent negative regulator, in myeloid cells in the context of allergic asthma. Our findings might explain the increase in asthma incidents during the night in humans when BMAL1 expression is low.

Dr. Zbigniew Zaslona from TCD (pictured here) was the lead author on the study. Both Dr. Annie Curtis (MCT) and Prof. Luke O’Neill (TCD) were joint senior authors on the paper.

The circadian protein BMAL1 in myeloid cells is a negative regulator of allergic asthma.

Zaslona Z, Case S, Early JO, Lalor SJ, McLoughlin RM, Curtis AM*, O’Neill LA* – Both authors contributed equally to this study.

Am J Physiol Lung Cell Mol Physiol. 2017 Mar 23:ajplung.00072.2017. doi: 10.1152/ajplung.00072.2017. [Epub ahead of print]

Daffodil Day Bake sale – 24/03/2017

Daffodil day is marked on the annual calendar as one of the most significant days recognised for collecting donations from the Irish public to fund cancer research as well as various services provided by the Irish Cancer Society. Given the substantial amount of cancer researchers based in RCSI and in particular in MCT, a joint effort between the MCT and the Department of Physiology and Medical Physics was carried out to organise a “Bake sale” aimed to raise funds on this occasion. Dr. Sudipto Das (MCT) and Dr. Catriona Dowling (Physiology and Medical Physics) primarily organised the bake sale.

This year bake sale boasted a wide variety of baked goods prepared by various members of the staff including senior researchers and post-graduate students. One the main highlights of the bake sale was an auction for an exquisite chocolate biscuit cake with a daffodil theme baked by Ms. Ina Woods (Physiology and Medical Physics). The auction was successfully completed by selling the cake at the highest bid of 50 euro by Prof. Jochen Prehn. This year bake sale was a highly successful event, which effectively raised 800 euro with all proceeding going towards the Irish Cancer Society.

We thank everyone who made this fundraising event into an enjoyable and fruitful event.

 Reported by Sudipto Das

International Research and Education

Prof Tracy Robson (MCT), Prof Jochen Prehn (Physiology & Medical Physics) and Dr Darran O’Connor (MCT) have recently returned from 1 week at the College of Pharmaceutical Sciences, Soochow University, Suzhou, China where they participated in a workshop with faculty to explore research collaborations and future joint funding applications under the newly announced SFI-NSF Partnerships for International Research and Education. Supported by an Erasmus+ programme coordinated by Prof Marc Devocelle (Department of Pharmaceutical and Medicinal Chemistry), the workshop involved presentations from RCSI and Soochow investigators describing their work and discussion to identify areas of synergy. Afternoon lectures by RCSI faculty were opened to postgraduate and postdoctoral researchers from Soochow, leading to a vigorous and stimulating discussion and Prof Xinliang Mao from Soochow will visit RCSI next month to further strengthen future collaborative research opportunities. 

Left to Right: Prof Tracy Robson (MCT), Prof Jochen Prehn (Physiology & Medical Physics) and Dr Darran O’Connor (MCT)

At the invitation of the President of the British Pharmacological Society, Professor John Waddington (Emeritus, RCSI) has been elected to Fellowship of the Society; this is in recognition of his career contributions to research, education and service in the discipline of pharmacology, not just in Ireland but globally. He has recently returned from 3 weeks at the College of Pharmaceutical Sciences, Soochow University, China, under his joint appointment as a Professor of Pharmacology. While there, he continued collaborative research, gave undergraduate lectures and fostered further joint endeavours between RCSI and Soochow University, which is in the top 5% of Chinese research universities.   

Tracy Robson

The Time Evolution of Shear-Induced Particle Margination and Migration in Flowing Blood

MCT Research Talks – 24th March 2017

Prof. Eric S. G. Shaqfeh, Qin M. Qi, Departments of Chemical and of Mechanical Engineering, Stanford University

The inhomogeneous center-of-mass distribution of red blood cells and platelets normal to the flow direction in small vessels plays a significant role in hemostasis, drug delivery and microfluidics. Under pressure-driven flow in channels, the migration of deformable red blood cells at steady state is characterised by a concentration peak at the channel center and a cell-free layer or Fahraeus-Lindqvist layer near the vessel wall.

Eric Stefan G. Shaqfeh

Rigid particles such as platelets, however, “marginate” and thus develop a near-wall excess concentration. This margination controls the time it takes for the initial stages of platelet binding and clotting in response to trauma.
In this talk, we investigate the time-dependent concentration distribution of red blood cells and platelets in pressure-driven flow by developing and solving a Boltzmann model, advection-diffusion equation for both species. From a fluid mechanics point of view, deformability-induced hydrodynamic lift and shear-induced diffusion are essential mechanisms for the cross-flow particle migration and margination. The governing equation for the distribution of red blood cells includes both lift flux away from the wall and shear-induced diffusion due to cell-cell “collisions”. On the other hand, the governing transport equation for platelets includes shear-induced diffusion from cell-platelet “collisions” and platelet-platelet “collisions”. We demonstrate that these predictions are in good agreement with full boundary element simulations of the margination process and we also compare directly to experimental results. We then examine, within this model and our full boundary element simulations, the time evolution and “entrance length” for red blood cell migration and platelet margination. The resulting complete model can serve as a fast and computationally efficient alternative to large-scale simulation with the application, for example, as a real-time computational tool for microfluidic blood assay systems.

A novel platelet function test (The Platelet Monitoring Biochip)

MCT Research Talks –13th March 2017 

Jonathan Cowman reports

Cardiovascular disease (CVD) is the leading cause of death and disability in the world (approx. 1.9 million deaths per year within the EU). Platelet’s play a key role in this process and hence is why antiplatelet therapy such as aspirin is effective in reducing its incidences. Platelet function testing has a role in identifying those that are at high risk of a CVD related event (example a heart attack) and also identifying those patients that do not respond to their medication. There are a number of platelet function tests on the market however these tests suffer from a number of disadvantages such as expense, high sample volume, requirement for trained lab personal and single drug test capability.

Jonathan Cowman

My current research under the supervision of Prof. Dermot Kenny (RCSI) and Dr. Niamh Gilmartin (DCU and DIT) is to work alongside our multi-disciplinary team to produce a cost effective, rapid, small sample volume platelet function assay which can detect the effect of multiple antiplatelet drugs in a single patient blood sample. The project is known as the Platelet Monitoring Biochip (PMB). The PMB device consists of 6 micron sized fibrinogen dots, which are micro contact printed to a Zeonor (plastic) surface, a bright-field imaging system and a custom designed platelet analysis software. Blood is added to the device and rocked for 30 minutes to allow platelets to adhere to the fibrinogen dots. The device has 3 channels, a control (no agonist well), an adenosine diphosphate (ADP) well and an Arachidonic acid (AA) well which can be used to detect P2Y12 platelet inhibition and aspirin effect simultaneously. The PMB device provides a fast, easy and low cost way to determine the effectively of antiplatelet therapy against multiple agonists in whole blood. The device is currently in operation in RCSI Beaumont.