Overexpression of Enterococcus faecalis elr operon protects from phagocytosis

BMC Microbiology

Pascale Serror¹  Lionel Rigottier-Gois¹  Brunella Posteraro⁴  Maurizio Sanguinetti³  Philippe Langella¹  Thierry Meylheuc¹  Kevin Piquand¹  Sophie Chat¹  Rebeca Martin¹  Francesca Bugli³  Caroline Lacoux¹  Stéphane Gaubert¹  Romain Dumoulin¹  Naima G. Cortes-Perez² 

[1] AgroParisTech, UMR1319 Micalis, Jouy-en-Josas, France;Current address: INRA, Unité d’Immuno-Allergie Alimentaire, iBiTecS/SPI, Gif-sur-Yvette, France;Institute of Microbiology, Università Cattolica del Sacro Cuore, Rome, Italy;Institute of Public Health (Section of Hygiene), Università Cattolica del Sacro Cuore, Rome, Italy

关键词: elr operon;    elrA;    Macrophage;    Enterococcus faecalis;   

Others  :  1212031
DOI  :  10.1186/s12866-015-0448-y

 received in 2015-01-14, accepted in 2015-05-14,  发布年份 2015

【 摘 要 】

Background

Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function.

Results

In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model.

Conclusions

Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.

【 授权许可】

   
2015 Cortes-Perez et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Brinster S, Furlan S, Serror P. C-terminal WxL domain mediates cell wall binding in Enterococcus faecalis and other gram-positive bacteria. J Bacteriol. 2007; 189(4):1244-1253.
• [2]Siezen R, Boekhorst J, Muscariello L, Molenaar D, Renckens B, Kleerebezem M. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria. BMC Genomics. 2006; 7:126. BioMed Central Full Text
• [3]Galloway-Pena JR, Liang X, Singh KV, Yadav P, Chang C, La Rosa SL et al.. The identification and functional characterization of WxL proteins from Enterococcus faecium reveal surface proteins involved in extracellular matrix interactions. J Bacteriol. 2015; 197(5):882-892.
• [4]Brinster S, Posteraro B, Bierne H, Alberti A, Makhzami S, Sanguinetti M et al.. Enterococcal Leucine-Rich Repeat-Containing Protein Involved in Virulence and Host Inflammatory Response. Infect Immun. 2007; 75(9):4463-4471.
• [5]Dumoulin R, Cortes-Perez N, Gaubert S, Duhutrel P, Brinster S, Torelli R et al.. Enterococcal Rgg-Like Regulator ElrR Activates Expression of the elrA Operon. J Bacteriol. 2013; 195(13):3073-3083.
• [6]Shepard BD, Gilmore MS. Differential expression of virulence-related genes in Enterococcus faecalis in response to biological cues in serum and urine. Infect Immun. 2002; 70(8):4344-4352.
• [7]Knodler LA, Celli J, Finlay BB. Pathogenic trickery: deception of host cell processes. Nat Rev Mol Cell Biol. 2001; 2(8):578-588.
• [8]Gentry-Weeks CR, Karkhoff-Schweizer R, Pikis A, Estay M, Keith JM. Survival of Enterococcus faecalis in Mouse Peritoneal Macrophages. Infect Immun. 1999; 67(5):2160-2165.
• [9]Verneuil N, Sanguinetti M, Le Breton Y, Posteraro B, Fadda G, Auffray Y et al.. Effects of the Enterococcus faecalis hypR gene encoding a new transcriptional regulator on oxidative stress response and intracellular survival within macrophages. Infect Immun. 2004; 72(8):4424-4431.
• [10]Baldassarri L, Bertuccini L, Creti R, Filippini P, Ammendolia MG, Koch S et al.. Glycosaminoglycans mediate invasion and survival of Enterococcus faecalis into macrophages. J Infect Dis. 2005; 191(8):1253-1262.
• [11]Coburn PS, Baghdayan AS, Dolan GT, Shankar N. An AraC-type transcriptional regulator encoded on the Enterococcus faecalis pathogenicity island contributes to pathogenesis and intracellular macrophage survival. Infect Immun. 2008; 76(12):5668-5676.
• [12]Thurlow LR, Thomas VC, Fleming SD, Hancock LE. Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion. Infect Immun. 2009; 77(12):5551-5557.
• [13]Hancock LE, Gilmore MS. The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall. Proc Natl Acad Sci U S A. 2002; 99(3):1574-1579.
• [14]Teng F, Jacques-Palaz KD, Weinstock GM, Murray BE. Evidence that the enterococcal polysaccharide antigen gene (epa) cluster is widespread in Enterococcus faecalis and influences resistance to phagocytic killing of E. faecalis. Infect Immun. 2002; 70(4):2010-2015.
• [15]Prajsnar TK, Renshaw SA, Ogryzko NV, Foster SJ, Serror P, Mesnage S. Zebrafish as a Novel Vertebrate Model To Dissect Enterococcal Pathogenesis. Infect Immun. 2013; 81(11):4271-4279.
• [16]Poyart C, Trieu-Cuot P. A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to β-galactosidase in Gram-positive bacteria. FEMS Microbiol Lett. 1997; 156(2):193-198.
• [17]Elliott JA, Winn WC. Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. Infect Immun. 1986; 51(1):31-36.
• [18]Gaillard JL, Berche P, Mounier J, Richard S, Sansonetti P. In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect Immun. 1987; 55(11):2822-2829.
• [19]Travier L, Guadagnini S, Gouin E, Dufour A, Chenal-Francisque V, Cossart P et al.. ActA promotes Listeria monocytogenes aggregation, intestinal colonization and carriage. PLoS Pathog. 2013; 9(1):e1003131.
• [20]Courtney HS, Ofek I, Penfound T, Nizet V, Pence MA, Kreikemeyer B et al.. Relationship between expression of the family of M proteins and lipoteichoic acid to hydrophobicity and biofilm formation in Streptococcus pyogenes. PLoS One. 2009; 4(1):e4166.
• [21]Bellon-Fontaine M-N, Rault J, van Oss CJ. Microbial adhesion to solvents: a novel method to determine the electron-donor/electron-acceptor or Lewis acid–base properties of microbial cells. Colloids Surf B: Biointerfaces. 1996; 7(1–2):47-53.
• [22]Garsin DA, Frank KL, Silanpaa J, Ausubel FM, Hartke A, Shankar N, Murray BE. Pathogenesis and Models of Enterococcal Infection. In: Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Edited by Gilmore MS, Clewell DB, Ike Y, Shankar N. Boston: Massachusetts Eye and Ear Infirmary; 2014.
• [23]Reid SD, Montgomery AG, Voyich JM, DeLeo FR, Lei B, Ireland RM et al.. Characterization of an extracellular virulence factor made by Group A Streptococcus with homology to the Listeria monocytogenes internalin family of proteins. Infect Immun. 2003; 71(12):7043-7052.
• [24]Nieto C, Espinosa M. Construction of the mobilizable plasmid pMV158GFP, a derivative of pMV158 that carries the gene encoding the green fluorescent protein. Plasmid. 2003; 49(3):281-285.
• [25]Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y; 1989.
• [26]Orman KL, Shenep JL, English BK. Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages. J Infect Dis. 1998; 178(6):1649-1657.
• [27]Maguin E, Duwat P, Hege T, Ehrlich D, Gruss A. New thermosensitive plasmid for gram-positive bacteria. J Bacteriol. 1992; 174(17):5633-5638.
• [28]Fouquier d'Herouel A, Wessner F, Halpern D, Ly-Vu J, Kennedy SP, Serror P, Aurell E, Repoila F: A simple and efficient method to search for selected primary transcripts: non-coding and antisense RNAs in the human pathogen Enterococcus faecalis. Nucleic acids research 2011;39(7):e46.
• [29]Mandin P, Repoila F, Vergassola M, Geissmann T, Cossart P. Identification of new noncoding RNAs in Listeria monocytogenes and prediction of mRNA targets. Nucleic Acids Res. 2007; 35(3):962-974.
• [30]Pils S, Schmitter T, Neske F, Hauck CR. Quantification of bacterial invasion into adherent cells by flow cytometry. J Microbiol Methods. 2006; 65(2):301-310.
• [31]Van Amersfoort ES, Van Strijp JA. Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes. Cytometry. 1994; 17(4):294-301.
• [32]Oxaran V, Ledue-Clier F, Dieye Y, Herry JM, Pechoux C, Meylheuc T et al.. Pilus biogenesis in Lactococcus lactis: molecular characterization and role in aggregation and biofilm formation. PLoS One. 2012; 7(12):e50989.
• [33]Rigottier-Gois L, Madec C, Navickas A, Matos RC, Akary-Lepage E, Mistou MY et al.. The Surface Rhamnopolysaccharide Epa of Enterococcus faecalis Is a Key Determinant of Intestinal Colonization. J Infect Dis. 2015; 211(1):62-71.
• [34]Pai SR, Singh KV, Murray BE. In vivo efficacy of the ketolide ABT-773 (cethromycin) against enterococci in a mouse peritonitis model. Antimicrob Agents Chemother. 2003; 47(8):2706-2709.
• [35]Dunny GM, Brown BL, Clewell DB. Induced cell aggregation and mating in Streptococcus faecalis: evidence for a bacterial sex pheromone. Proc Natl Acad Sci U S A. 1978; 75(7):3479-3483.
• [36]Gibson TJ. Studies on the Epstein-Barr virus genome. University of Cambridge, Cambridge, UK; 1984.
• [37]Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986; 189(1):113-130.
• [38]Rose RE. The nucleotide sequence of pACYC177. Nucleic Acids Res. 1988; 16(1):356.
• [39]Chastanet A, Fert J, Msadek T. Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria. Mol Microbiol. 2003; 47(4):1061-1073.