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Acquisition and regulation of virulence factors in Staphylococcus aureus

English title Acquisition and regulation of virulence factors in Staphylococcus aureus
Applicant Linder Patrick
Number 132638
Funding scheme Project funding (Div. I-III)
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Medical Microbiology
Start/End 01.10.2010 - 30.09.2013
Approved amount 548'745.00
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All Disciplines (2)

Discipline
Medical Microbiology
Molecular Biology

Keywords (6)

RNA helicase; restriction endonuclease; Staphylococcus aureus; gene expression; biofilm; restriction enzyme

Lay Summary (English)

Lead
Lay summary
The bacterium Staphylococcus aureus is an opportunistic pathogen present in 30 to 50% of the population without causing disease. Nevertheless, this bacterium can cause diverse infections ranging from furuncles to life threatening endocarditis or osteomyelitis, especially in people with a diminished defense system. Moreover, in recent years highly virulent strains were shown to infect healthy young people. In addition to a multitude of virulence factors, S. aureus strains readily acquire antibiotic resistance genes, making these strains difficult to treat and eradicate. S. aureus is responsible in the US for more deaths than the human immunodeficiency virus HIV.Using molecular genetic methods we are studying the expression of virulence factors in this bacterium. Amongst the virulence traits appears the possibility to form biofilms in which the bacteria adhere to a surface and secrete an extracellular matrix that protects them against clearing by the immune system. Moreover, bacteria in biofilms are less sensitive to antibiotics, making treatment difficult and resulting in persistent infections. We study a mutant deficient in a RNA helicase gene and that shows reduced biofilm formation. Using genetic approaches we aim at identifying the process in which this helicase participates and thereby regulates virulence factor expression, and in particullar biofilm formation. This work should identify the genes that are subject to regulation by the RNA helicase and required for biofilm formation.In a parallel project we analyze a second RNA helicase for which no function is known. We have detected that the inactivation of the corresponding gene shows reduced growth at lower temperatures. Interestingly the mutant dies if a gene that is involved in DNA repair is also inactivated. This strongly suggests that the RNA helicase gene and the DNA repair gene are required for the same cellular process. This is the first time that a link a link between RNA metabolism and DNA repair has been identified in bacteria. To identify the function of the RNA helicase, we will perform further genetic screens to find interacting genes.Finally we have identified a new type of restriction enzyme that limits horizontal gene transfer in S. aureus. We will analyze the expression of this gene to understand better the conditions that permit horizontal gene transfer. The analysis of horizontal gene transfer is important to understand the epidemiology of antibiotic resistant and highly virulent Staphylococcus aureus strains.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Looking back on the birth of DEAD-box RNA helicases
Linder Patrick, Fuller-Pace Frances V. (2013), Looking back on the birth of DEAD-box RNA helicases, in BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 1829(8), 750-755.
Special Issue: The Biology of RNA helicases - Modulation for life Preface
Linder Patrick, Fuller-Pace Frances (2013), Special Issue: The Biology of RNA helicases - Modulation for life Preface, in BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 1829(8), 749-749.
The CshA DEAD-box RNA helicase is important for quorum sensing control in Staphylococcus aureus
Oun Stella, Redder Peter, Didier Jean-Philippe, Francois Patrice, Corvaglia Anna-Rita, Buttazzoni Elena, Giraud Caroline, Girard Myriam, Schrenzel Jacques, Linder Patrick (2013), The CshA DEAD-box RNA helicase is important for quorum sensing control in Staphylococcus aureus, in RNA BIOLOGY, 10(1), 157-165.
DEAD-box RNA helicases in gram-positive RNA decay.
Redder Peter, Linder Patrick (2012), DEAD-box RNA helicases in gram-positive RNA decay., in Eckhard Jankowsky (ed.), 369-3201283.
New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus
Redder Peter, Linder Patrick (2012), New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus, in Appl Environ Microbio, 78(11), 3846-3854.
A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300.
Xu Shuang-Yong, Corvaglia Anna R, Chan Siu-Hong, Zheng Yu, Linder Patrick (2011), A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300., in Nucleic acids research, 39(13), 5597-610.
Analyses of the Functional Regions of DEAD-Box RNA "Helicases" with Deletion and Chimera Constructs Tested In Vivo and In Vitro.
Banroques Josette, Cordin Olivier, Doère Monique, Linder Patrick, Tanner N Kyle (2011), Analyses of the Functional Regions of DEAD-Box RNA "Helicases" with Deletion and Chimera Constructs Tested In Vivo and In Vitro., in Journal of molecular biology, 413(2), 451-72.
From unwinding to clamping - the DEAD box RNA helicase family.
Linder Patrick, Jankowsky Eckhard (2011), From unwinding to clamping - the DEAD box RNA helicase family., in Nature reviews. Molecular cell biology, 12(8), 505-16.
The CodY pleiotropic repressor controls virulence in gram-positive pathogens.
Stenz Ludwig, Francois Patrice, Whiteson Katrine, Wolz Christiane, Linder Patrick, Schrenzel Jacques (2011), The CodY pleiotropic repressor controls virulence in gram-positive pathogens., in FEMS immunology and medical microbiology, 62(2), 123-39.

Associated projects

Number Title Start Funding scheme
118309 DEAD-box RNA helicases orchestrate translation initiation in bacteria and eukaryotes 01.10.2007 Project funding (Div. I-III)
149228 RNA helicases in virulence expression in Staphylococcus aureus 01.10.2013 Project funding (Div. I-III)

Abstract

In this project we propose to study two RNA helicases of the DEAD-box family and a helicase superfamily 2 protein functioning as putative restriction endonuclease in Staphylococcus aureus.S. aureus is an opportunistic pathogen that can cause a diverse range of infections, from furuncles to life threatening endocarditis or osteomyelitis. It has been shown that S. aureus is able to persist in its host and to produce biofilms, conditions that make the eradication of the pathogen in a patient extremely difficult. The adaption to such different environmental conditions requires a sophisticated regulation of gene expression. In bacteria this is in large part governed by RNA molecules, an ideal play ground for RNA helicases of the DEAD-box family, involved in RNA metabolism from transcription to decay. A large body of knowledge on eukaryotic DEAD-box proteins is available, but relatively little is known about bacterial DEAD-box proteins. Five proteins are present in E. coli and they were shown to be involved in translational regulation, ribosome biogenesis, and RNA degradation. Mutations in DEAD-box protein genes from a variety of other bacteria resulted in a diverse range of specific phenotypes, such as biofilm formation, oxidative stress, cold shock induced gene expression, or aggregation of bacteria, without influencing the growth of the bacteria under normal growth conditions. This suggests that these effects are specific, and are not solely due to a general perturbation of cellular gene expression.S. aureus genomes encode two DEAD-box proteins. A mutant DEAD-box protein Sa1885 reveals a phenotype with decreased ability to form biofilm, without reducing growth rate. We propose to identify target genes of Sa1885 that are involved in biofilm formation or other measurable readouts. We will use genetic and biochemical methods to identify the targets of this DEAD-box protein and analyze its regulation. Identified target genes will be tested for their implication in biofilm formation and the requirement of Sa1885 in gene expression will be analyzed.In parallel we are studying a second RNA helicase, Sa1387. At present little is known about this DEAD-box protein. As for Sa1885, its disruption leads to a cold sensitive phenotype. We have identified an intriguing genetic interaction with a type IV endonuclease that suggests some implication in DNA metabolism, such as repair. To our knowledge, no such interaction was reported in bacteria. We are pursuing our genetic analyses and will use biochemical methods to characterize this DEAD-box protein further. Our approaches aim at defining interacting partners and target RNA(s). This also will help to analyze in-depth the enzymatic function of this protein, which displays no enzymatic activity on its own.The analysis of clinical S. aureus has shown a clonal structure of the strains, with relatively little exchange of genetic material. One explanation for this is the presence of multiple restriction systems. The presence of these barriers also explains the difficulties to transform clinical S. aureus strains, which hampers greatly the genetic analysis of these strains. We have therefore inactivated the known type I restriction system in two clinical strains but our analysis showed that additional systems must be present. Using a mutational analysis we have identified a new barrier. The identified gene resembles strongly a type III restriction endonuclease with all the motifs typical for a helicase superfamily 2 protein. We will continue our analysis of this gene and its role as a barrier to horizontal gene transfer. In addition to the better knowledge of the restriction system, the mutant strain will be an excellent tool to further analyze and understand horizontal gene transfer.Our analysis of the two DEAD-box proteins and the type III restriction endonuclease will contribute to a better understanding of gene expression and the acquisition of foreign DNA in the opportunistic pathogen S. aureus.
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