NanoViroStaph

Nanoscale analysis of bacteriophage-staphylococcus aureus interactions during host infection

Internal reference number : 23/28-131
Start date : October 1st, 2023
End date : September 30, 2028

Partners

PI (spokesperson) : Pr Yves Dufrêne, Université catholique de Louvain (UCLouvain), Louvain Institute of Biomolecular Science and Technology (LIBST)

Co-I 1: Pr David Alsteens, Université catholique de Louvain (UCLouvain), Louvain Institute of Biomolecular Science and Technology (LIBST)

Co-I 2: Pr Annika Gillis, Université catholique de Louvain (UCLouvain), Earth and Life Institute (ELI), Applied Microbiology (ELIM)

 

 

Aims of the Coordinated Research Project

Although antibiotics have saved countless lives, their widespread use has contributed to an increase in the incidence of antibiotic-resistant bacterial strains. More than 70% of hospital-acquired bacterial infections in the United States are now resistant to at least one of the main used antibiotics, and >50% of clinical strains of Staphylococcus aureus, a colonizer of the human skin, are now multidrug resistant (MDR), including methicillin resistant S. aureus (MRSA) strains. Bacterial viruses (bacteriophages) appear to have great therapeutic potential as an alternative to classical antimicrobials to fight MDR pathogens. Bacteriophages have been tested for clinical applications since the beginning of the twentieth century, and substantial progress has been made on phage genetics and physiology in the past 50 years. Yet, the phage-bacterial-host molecular interactions that lead to adhesion and invasion are still poorly understood. These are particularly critical when targeting bacterial intracellular pathogens, as they are protected from antibacterial agents and the host immune system, thus producing persistent infections.

At the crossroads of nanotechnology and microbiology, our multidisciplinary project aims at combining innovative state-of-the-art nanotechniques with biological methods to broaden our understanding of the molecular interactions and mechanisms taking place in the ternary system phage-bacterium-host cells, using S. aureus and the human skin as medically-important models. This will be possible owing to the integration of complementary expertises in nanophysics, microbiology, virology, microbial genetics/genomics, and molecular biology. The project addresses three objectives: deciphering S. aureus-phage interactions, unveiling S. aureus-skin cell interactions, and unravelling phage-skin cell interactions. Our findings will have broad impacts as i) there is currently a strong demand for single live-cell nanotechniques in microbiology and cell biology, and ii) MDR strains are becoming increasingly difficult to treat, meaning there is an urgent need for innovative virus-based antimicrobials.

The research team

Yves Dufrêne (PI - Spokesperson)

Yves Dufrêne holds a bioengineering degree with a major in physical chemistry. After his PhD (1996), he directed his research towards a rapidly emerging field of nanoscience, namely the study of the nanophysical properties of living cells using atomic force microscopy (AFM). At the crossroads between biophysics, cell biology and nanotechnology, this transdisciplinary research topic is highly original and very challenging. Yves Dufrêne’s research has resulted in significant innovations in AFM techniques for imaging and manipulating living cells at the molecular level. He has pioneered the use of advanced tools that are able to (bio)chemically recognize specific constituents of the cell surface, one molecule at a time. Using these tools and venturing into unexplored territories, he has contributed to a better understanding of cell surfaces through several major discoveries (1,2).

In a first breakthrough study he determined the binding mechanism and spatial arrangement of mycobacterial adhesion proteins involved in tuberculosis (3). He then discovered functional protein nanodomains on yeast cells and showed that these “nanoadhesomes” are able to grow in response to mechanical stimuli, a fascinating new mechanism for activating cell adhesion in microbial pathogens (4). He identified the role of the protein SasG of the pathogen Staphylococcus aureus in mediating the formation of biofilms, which are a common cause of infection (3). He then identified a peptide capable of preventing the formation of S. aureus biofilms, thus opening the way to the development of antiadhesion therapies (4). Recently, the team showed that staphylococcal adhesins bind to their host proteins with ultrastrong forces, enabling the bacteria to firmly attach to its host during colonization and infection (5-8).

Yves Dufrêne’s international impact is reflected by his h-index of 74 (Web of Science®; author = dufrene y*) and by several review/perspective articles for various Nature journals and Science. The impact of his research on the international scene is further illustrated by numerous research highlights in Nature, Science, Nature Rev. Microbiol., PNAS, Nature Methods. He has been invited to talk at >60 international conferences, including Gordon Research Conferences. Over the past few years, he has received several awards including the Quadrennial Life Sciences Award of the European Microscopy Society (2012) and the Léo Errera prize of the Royal Academy of Belgium (2013). He is Associate Editor for the high impact RSC journals Nanoscale Advances, Nanoscale (IF 7.4), and Nanoscale Horizons (IF 9.4), handling ~400 manuscripts per year, and Advisory Board member for Chem Soc Rev (IF 34). Since 2013 he was a member of the ERC Panel PE3 - Consolidator Grant - Condensed Matter Physics where he makes recommendations for funding. Yves Dufrêne has been the main PI of a number of competitive grant applications to a total value ~9 M €, including an ERC advanced grant.

In summary, using the tools of nanotechnology, Yves Dufrêne has made major contributions to the fields of biophysics and microbiology, by providing key insights into the structure-function relationships of cell surfaces. By the originality of his methodological approaches, he has cross-fertilized life sciences and nanotechnology. Continuous improvement of advanced AFM methods has enabled him to address pertinent questions such as how cell surface constituents are spatially organized, and how they interact with their environment. His current ambition is to further develop AFM nanoscopy to understand how staphylococcal pathogens attach to biomaterials and tissues, and to develop alternative strategies for treating staphylococcal infections.

1. Nat Rev Microbiol, (2020), 18:227-240.
2. Nat Rev Methods Primers, (2021), 1:63.
3. Nat. Methods, 2005, 2, 515-520.
4. Proc. Natl. Acad. Sci. USA, 2010, 107, 20744-20749.
5. Proc. Natl. Acad. Sci. USA, 2016, 113, 410-415.
6. Proc. Natl. Acad. Sci. USA, 2017, 114, 3738-3743.
7. Proc. Natl. Acad. Sci. USA, 2018, 115, 5564-5569.
8. Nat Commun, (2020), 11, 5431.

Research team: One postdoc, two PhD students, one technician

People funded by the project: Zhiyong Zheng, postdoc; Sylvie Derclaye, technicians
 

David Alsteens (Co-I)

David Alsteens graduated from the Faculty of Bioengineering of the UCLouvain in 2007, with a specialization in nanobiotechnology. Thanks to a FNRS fellowship, he completed a doctoral thesis in cellular nanobiophysics under the supervision of Prof. Yves Dufrêne. His thesis lead to the publication of height publications as first (first co-)authors in high-impact journals including Nat. Chem. Biol, PNAS, Nano Lett. and ACS Nano. The aim of his thesis was to gain insight in the nanoscale properties of the yeast cell wall, with an emphasis on the nanomechanics of a membrane sensor and a cell adhesion protein. Among others, this work revealed that the WSC1 sensors behave like nanosprings, capable of feeling mechanical force and activating intracellular signaling cascade (1). He also demonstrated that the modular and flexible nature of Als5p adhesins convey both strength and toughness to the protein and that the delivery of piconewton force on the yeast surface triggers the formation and propagation of adhesion domains. Such force-dependent adhesion domains could be a genral mechanism for activating cell adhesion (2). This thesis received two awards from the Royal Society for Microscopy and the DSM Science & Technology Award.

During a first post-doc thanks to a post-doc fellowship from the FNRS, he directed his research towards the world of virology by studying the extrusion mechanism of bacteriophages. Thanks to the use of a new technique based on the muliparametric imaging combined with biochemically sensitive tips, he reported that bacteriophages extrude from living bacteria at the septum, in the form of soft nanodomains surrounded by stiff cell wall material (3). Next, owing to a long-term EMBO post-doc fellowship, he moves to the ETH Zürich in Switzerland in the group of Prof. Daniel Müller, where he studied cell biology and the biological processes occuring at the plasma membrane of mammalian cells. In collaboration with Prof. Brian Kobilka (Nobel Prize in Chemsitry (2012)), he introduced an approach to simultaneously image single native G protein–coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands (4). The outstanding paper Award in life Science 2015 was attributed by the European Microscopy Society to this work. Next, he combined AFM and confocal microscopy to characterize the surface receptor landscape of cells and map the virus binding events sithin the first millisecond of contactat high-resolution (5).

Since 2015, he is leading an independent and very creative group of around 12-15 researcher on average thanks to significant funding acquired at the national (FNRS, Welbio, EoS) and international (ERC starting garnt, EMBO). His group is particularly interested in the molecular mechanisms of the interactions established by viruses with cell receptors, with a particular focus on the role played by glycans during these early stages. He has notably elucidated how sialic acid favors a strong multivalent binding to reovirus entry receptors and modulate the the conformation of the s1 viral glycoprotein dictating the further binding to integrin required for the endocytosis or reovirus virion following JAM-A binding (6).

David Alsteens’ international impact is reflected by his >110 articles in high-ranking journals. His h-index is 41 (mostly first/last authorship), with 6,409 citations (Scopus; author = Alsteens D*). David Alsteens has been invited to talk at >35 international conferences, including a Gordon Research Conference in Physical Virology. Over the past few years, he has received several awards including the BAEF Alumni Award (2019) in the field of Biomedical Science, the Heinrich Emanuel Merck Award (2019) for Analytical Science and the Prize of the Studie centrum Princess Joséphine Charlotte (2021) in virology. He is Associate Editor for the Journal of Structural Biology and Chief-editor for Frontiers in Biophysics. David Alsteens has been the main PI of a number of competitive grant applications to a total value ~4,5M €, including an ERC starting grant.

1. Nat. Chem. Biol. (2009) 5, 857- 862
2. PNAS (2010), 107, 20744-20749
3. Nat. Commun. (2013) 4, e2926
4. Nat. Methods. (2015) 12, 845-851
5. Nat. Nanotechnol. (2017) 12, 177-183
6. Nat. Commun. (2019) 10, 4460

Reserach team: Eight postdocs; six PhD students, one technician
 

Annika Gillis (Co-I)

Annika Gillis studied Biological Sciences (B.Sc.) at the University Simón Bolívar (Venezuela), where she also specialised in Molecular Biology (M.Sc.). She obtained her PhD (2014) from the Catholic University of Louvain (UCLouvain) in Belgium, under the supervision of Pr Jacques Mahillon. Her PhD thesis focused on studying the tectiviruses and their contribution to the adaptation and evolution of their Bacillus hosts. After her PhD, she directed her research towards the biology and genetics of other phages and Gram-positive bacterial pathogens. As summarised below, her research has mainly focused on the contribution of phages and other mobile genetic elements (MGEs), to the adaptation, evolution, and virulence of their Gram-positive hosts. Moreover, Annika has been involved in research associated with phage taxonomy and classification, phage therapy, biology of plant viruses and phytopathogenic bacteria, nanotechnologies, and biotechnological and biocontrol applications.

Annika has paid special attention to phages thriving in the Bacillus cereus group, mainly because of their potential role in the genetic diversity observed in this lineage of bacteria. In this context, she has focused her research on studying the Tectiviridae, a peculiar phage family that can establish a linear plasmidial prophage state upon infection of their hosts. Her Ph.D. work centred on characterizing the occurrence, distribution, and diversity of tectiviruses in the B. cereus group, allowing the discovery of some novel tectiviruses. Then, part of her postdoctoral work focused on answering the following question: can tectiviruses contribute to the ecological traits of their bacterial host? Another aspect of her research has addressed the impact of tectiviral resistance on the life traits of Bacillus thuringiensis. Then, during a post-doctoral training in the group of Pr Margarita Salas (CBMSO, Spain), Annika uncovered a tectivirus that encodes for a new family B DNA polymerase with efficient DNA polymerization activities, opening important avenues of exploration from a biotechnological point of view. Based on the data generated for the tectiviruses, Annika was awarded (2014-present) an official position as chair of the Tectiviridae Study Group of the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV).

In addition, her research has been also oriented on studying other extrachromosomal molecules and MGEs in the B. cereus group. She have been involved in i) the genomic characterization of the large conjugative plasmid pXO16, which uses a novel T4SS-mediated DNA for conjugation and it is able to transfer chromosomal loci, ii) the analysis of the plasmid content of B. cereus sensu lato strains using ultra-high throughput sequencing, and iii) the uncovering of the extrachromosomal element pBtic235, which is a molecule displaying plasmid- and phage- genetic modules. She has collaborated to develop nanoscale approaches to evaluate bacterial appendages and DNA transfer between single bacteria during plasmid conjugation using atomic force microscopy (AFM).

In 2019, Annika moved as a Research Associate to Imperial College London (UK), to the group of Pr Angelika Gründling, to study cyclic di-adenosine monophosphate (c-di-AMP) signalling in methicillin resistant Staphylococcus aureus. Annika has been recently appointed as Assistant Professor (September 2022) at UCLouvain where she is starting her research group on phage-bacteria interactions and phage-resistance/defence mechanisms, with a particular focus on Gram-positive pathogens such as S. aureus, Listeria monocytogenes and B. cereus.

Annika Gillis has published >45 articles mainly in Q1 (Microbiology) ranking journals. Her h-index is 22, with 2410 citations (Google Scholar; author = Gillis Annika). She has given more than >20 scientific talks at international conferences, research institutes and universities. She serves as an ad hoc reviewer of several scientific journals in microbiology, biotechnology and plant pathology, including Applied and Environmental Microbiology, European Plant Pathology, Genes, Viruses, Archives Virology, and Communications Biology. She is a founder member of the “Belgian Society for Viruses of Microbes”; and she has been elected as its Secretary since June 2022.

1. Nanoscale (2012) 4, 1585-1591.
2. Appl. Environ. Microbiol. (2014) 80, 4138-4152.
3. Appl. Environ. Microbiol. (2014) 80, 7620-7630.
4. FEMS Microbiol. Rev. (2018) 42, 829-856.
5. Syst. Biol. (2020). 69, 110–123.
6. Curr. Opin. Microbiol. (2021) 60, 24-33.

Reserach team: One postdoc, five PhD students, two technicians

People funded by the project: Félicie Chaumont, PhD Student; Alexia Nakoutsi, PhD Student
 

Theses defended in the context of the Coordinated Research Project

 

 

Activities organised as part of the Coordinated Research Project

 

 

Publications in connection with the Coordinated Research Project

 

 

Contact point in UCLouvain

Pr Yves Dufrêne, Principal Investigator (spokesperson/coordinator), Université catholique de Louvain (UCLouvain), Louvain Institute of Biomolecular Science and Technology (LIBST), E-mail: Yves Dufrêne