Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening

被引:229
作者
Joseph, B
Przybilla, K
Stuhler, C
Schauer, K
Slaghuis, J
Fuchs, TM
Goebel, W
机构
[1] Univ Wurzburg, Theodor Boveri Inst, Biozentrum, Lehrstuhl Mikrobiol, D-97074 Wurzburg, Germany
[2] Tech Univ Munich, ZIEL, Abt Mikrobiol, D-85350 Freising Weihenstephan, Germany
关键词
D O I
10.1128/JB.188.2.556-568.2006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.
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页码:556 / 568
页数:13
相关论文
共 47 条
[1]   pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes [J].
Beauregard, KE ;
Lee, KD ;
Collier, RJ ;
Swanson, JA .
JOURNAL OF EXPERIMENTAL MEDICINE, 1997, 186 (07) :1159-1163
[2]   OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival [J].
Borezee, E ;
Pellegrini, E ;
Berche, P .
INFECTION AND IMMUNITY, 2000, 68 (12) :7069-7077
[3]  
Bubert A, 1999, MOL GEN GENET, V261, P323, DOI 10.1007/s004380050973
[4]   Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua:: clues for evolution and pathogenicity [J].
Buchrieser, C ;
Rusniok, C ;
Kunst, F ;
Cossart, P ;
Glaser, P .
FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, 2003, 35 (03) :207-213
[6]   COORDINATE REGULATION OF VIRULENCE GENES IN LISTERIA-MONOCYTOGENES REQUIRES THE PRODUCT OF THE PRFA GENE [J].
CHAKRABORTY, T ;
LEIMEISTERWACHTER, M ;
DOMANN, E ;
HARTL, M ;
GOEBEL, W ;
NICHTERLEIN, T ;
NOTERMANS, S .
JOURNAL OF BACTERIOLOGY, 1992, 174 (02) :568-574
[7]   clpB, a novel member of the Listeria monocytogenes CtsR regulon, is involved in virulence but not in general stress tolerance [J].
Chastanet, A ;
Derre, I ;
Nair, S ;
Msadek, T .
JOURNAL OF BACTERIOLOGY, 2004, 186 (04) :1165-1174
[8]   Hpt, a bacterial homolog of the microsomal glucose-6-phosphate translocase, mediates rapid intracellular proliferation in Listeria [J].
Chico-Calero, I ;
Suárez, M ;
González-Zorn, B ;
Scortti, M ;
Slaghuis, J ;
Goebel, W ;
Vázquez-Boland, JA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2002, 99 (01) :431-436
[9]   Molecular determinants of Listeria monocytogenes virulence [J].
Dussurget, O ;
Pizarro-Cerda, J ;
Cossart, P .
ANNUAL REVIEW OF MICROBIOLOGY, 2004, 58 :587-610
[10]   A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins [J].
Engelbrecht, F ;
Chun, SK ;
Ochs, C ;
Hess, J ;
Lottspeich, F ;
Goebel, W ;
Sokolovic, Z .
MOLECULAR MICROBIOLOGY, 1996, 21 (04) :823-837