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Significance Statement
Staphylococcus aureus, a highly versatile Gram-positive bacterium, can cause a multitude of diseases ranging from mild superficial skin infections to life-threatening disseminated infections. In order to establish persistent colonization pathogens use specialized strategies to disrupt key cell functions and invade the epithelial barrier. Some of those strategies rely on cell cycle alteration. We recently found that S. aureus MW2 induces a G2/M phase transition delay in epithelial cells. The delay was associated with the accumulation of inactive Cyclin-dependent kinase 1 and unphosphorylated histone H3. Additionally, we showed that the G2 phase was preferential for bacterial internalization and intracellular replication. The aim of the current study was an identification of the bacterial factor that delays cell cycle phase transition and a deciphering of a biological significance of this phenomenon. We show here that the cell cycle is altered by S. aureus compounds that were secreted into the culture supernatant. Using size exclusion chromatography of MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin alpha (PSMα) peptides as the candidates for this effect. We have shown that N-formylated synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus LAC wt with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1 to 4. We also tested clinical human S. aureus isolates: a delayed G2/M phase transition was shown in only one clinical isolate and correlated with the detection of PSMα1 in its supernatant. Analysis of the proliferation of LAC wt and its isogenic mutant LAC∆psmα showed higher proliferation rates of internalized LAC wt. Moreover, we have shown, that PSMα-induced G2/M-transition delay was related to the decrease of the antibacterial functions of the epithelial cells. The expression level of antibacterial peptides (human β-defensins) was lower in G2/M-phase cells as compared to cells in the G1 phase. In conclusion, our study shows that particularly PSMα1 interferes in various ways with host cell cycle and host cell response. PSMα1induces G2/M phase transition delay, which prolongs the period of low antibacterial peptides expression. Moreover, PSMα1 promotes intracellular proliferation of S. aureus in HeLa cells. This is a novel mechanistic strategy of how S. aureus is able to subvert cell cycle progression accompanied by decreased immune response.
Figure. PSMα1 and PSMα3 induces G2/M phase transition delay.
Synchronized HeLa cells were exposed to the different concentrations of synthetic PSMα1 or PSMα3. Cell cycle was analyzed by flow cytometry. Time duration of the cell cycle phases in asynchronous cells is shown inside the diagram. Fulfilment of the cell cycle is indicated by the arrow. The exposure of HeLa cells to PSMα1 or PSMα3 slowed HeLa cell cycle progression.
Journal Reference
FASEB J. 2015 May;29(5):1950-9. doi: 10.1096/fj.14-260513. PMID: 25648996.
*Martine Deplanche1,2, *Rachid Aref El-Aouar Filho1,2,3, Ludmila Alekseeva1,2,4, Emilie Ladier1,2, Julien Jardin1,2, Gwénaële Henry1,2, Vasco Azevedo3, Anderson Miyoshi3, Laetitia Beraud5, Frederic Laurent5,6, Gerard Lina5,6, François Vandenesch5,6, Jean-Paul Steghens6, Yves Le Loir1,2, Michael Otto7, Friedrich Götz8, Nadia Berkova1,2
1Institut National de la Recherche Agronomique, UMR1253 STLO, F-35042 Rennes, France
2Agrocampus Ouest, UMR1253 STLO, F-35042 Rennes, France
3ICB–UFMG, CEP 31.270-901. Belo Horizonte-MG, Brazil
4Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RF
5Centre International de Recherche en Infectiologie, INSERM U1111, CNRS UMR5308, Université Lyon 1, Lyon, France
6 Hospices Civil de Lyon, Lyon, France
7Laboratory of Human Bacterial Pathogenesis, US National Institutes of Health, Bethesda, Maryland 20892, USA.
8Microbial Genetics, University of Tübingen, Tübingen, Germany
*These authors made an equal contribution to this work.
Correspondence: UMR1253 STLO INRA Agrocampus Ouest, 65 rue de Saint-Brieuc, 35042 Rennes, France. E-mail:[email protected]
[/expand]ABSTRACT
Staphylococcus aureus is a gram-positive bacterium responsible for a wide range of infections. Host cell cycle alteration is a sophisticated mechanism used by pathogens to hijack the defense functions of host cells. We previously demonstrated that S. aureus MW2 (USA400) bacteria induced a G2/M phase transition delay in HeLa cells. We demonstrate here that this activity is triggered by culture supernatant compounds. Using size exclusion chromatography of the MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin α (PSMα) peptides as the likely candidates for this effect. Indeed, synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus Los Angeles County clone (LAC) wild-type with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1-4. PSMα-induced G2/M transition delay correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. By testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production. We show that PSMs secreted by S. aureus alter the host cell cycle, revealing a newly identified mechanism for fostering an infection.
About the Corresponding Author:
Dr. Nadia Berkova
Current Position Principal Investigator,
INRA, UMR1253, Bacterial Biodiversity and In Situ Interactions, Rennes
65, rue de Saint Brieuc, Rennes, 35042, France
Email Address [email protected]
Education
2007 Habilitated as a Thesis Director (HDR), Marie and Pierre Curie University, Paris, France.
1986-1990 PhD in Immunology-Molecular Biology, Institute of Bioorganic Chemistry,
Moscow, RF and Central Institute of Molecular Biology, Berlin-Buch, Germany.
Scientific career
Since 2009 Principal Investigator, INRA, UMR1253, Bacterial Biodiversity and In Situ Interactions, Rennes, France.
2002-2009 Principal Investigator, Assistant-Director of Mycology Laboratory, UMR 956, Maisons Alfort, France.
1997-2002 Invited Scientist, CNRS UMR 6066, Genetic and Development, Rennes, France.
1994-1997 Associate Professor, Laval University, Faculty of Medicine, Québec, Canada.
From 1994 Accredited Clinical Researcher, FRSQ, Québec.
1991-1994 Clinical Researcher, Wyeth Ayerst Inc research fellow.
1990-1991 Postdoctoral Researcher, Laval University, Faculty of Medicine, Québec, Canada.
Research interests.
Main project is aimed at a multidisciplinary analysis of the mechanisms involved in the host-pathogen interaction.
The principal areas of scientific interest are:
-examination of bacteria-induced deregulation of host cellular signaling leading to cell growth perturbation and inflammation induction.
– analysis of host gene expression during SA long-term infection.