Identification of MglA-Regulated Genes Reveals Novel Virulence Factors in Francisella tularensis

ABSTRACT The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.

[1]  M. Flickinger,et al.  Glutathione S-transferase-sspA fusion binds to E. coli RNA polymerase and complements delta sspA mutation allowing phage P1 replication. , 1994, Biochemical and biophysical research communications.

[2]  M. Horwitz,et al.  Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages , 2004, Infection and Immunity.

[3]  D. Kobayashi,et al.  Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. , 1988, Gene.

[4]  Na Zhang,et al.  A Francisella tularensis Pathogenicity Island Required for Intramacrophage Growth , 2004, Journal of bacteriology.

[5]  S. Salzberg,et al.  Improved microbial gene identification with GLIMMER. , 1999, Nucleic acids research.

[6]  D. Lackman,et al.  COMPARATIVE STUDIES OF FRANCISELLA TULARENSIS AND FRANCISELLA NOVICIDA , 1964, Journal of bacteriology.

[7]  Anne-Marie Hansen,et al.  SspA is required for acid resistance in stationary phase by downregulation of H‐NS in Escherichia coli , 2005, Molecular microbiology.

[8]  T. Takehara,et al.  Sequencing, expression and biochemical characterization of the Porphyromonas gingivalis pepO gene encoding a protein homologous to human endothelin‐converting enzyme , 1999, FEBS letters.

[9]  I. Golovliov,et al.  Francisella tularensis Induces Cytopathogenicity and Apoptosis in Murine Macrophages via a Mechanism That Requires Intracellular Bacterial Multiplication , 2001, Infection and Immunity.

[10]  P. Brown,et al.  DNA arrays for analysis of gene expression. , 1999, Methods in enzymology.

[11]  I. Golovliov,et al.  An Attenuated Strain of the Facultative Intracellular Bacterium Francisella tularensis Can Escape the Phagosome of Monocytic Cells , 2003, Infection and Immunity.

[12]  G. Baron,et al.  An erythromycin resistance cassette and mini-transposon for constructing transcriptional fusions to cat. , 1999, Gene.

[13]  D. Jin,et al.  Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes , 2003, Molecular microbiology.

[14]  V. Dixit,et al.  Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis , 2005, The Journal of experimental medicine.

[15]  T. Baker,et al.  A specificity-enhancing factor for the ClpXP degradation machine. , 2000, Science.

[16]  Minoru Kanehisa,et al.  The KEGG database. , 2002, Novartis Foundation symposium.

[17]  Tara L. Kieffer,et al.  Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS, and contributes to F. novicida murine pathogenesis. , 2003, Microbes and infection.

[18]  E. Rozengurt,et al.  Cytotoxic Necrotizing Factor 1 from Escherichia coli and Dermonecrotic Toxin from Bordetella bronchiseptica Induce p21rho-dependent Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin in Swiss 3T3 Cells* , 1997, The Journal of Biological Chemistry.

[19]  G. Baron,et al.  MglA and MglB are required for the intramacrophage growth of Francisella novicida , 1998, Molecular microbiology.

[20]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Barik,et al.  Construction of a pepO gene-deficient mutant of Porphyromonas gingivalis: potential role of endopeptidase O in the invasion of host cells. , 2003, Oral microbiology and immunology.

[22]  D. Maneval,et al.  Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains , 1995, Infection and immunity.

[23]  P. Berche,et al.  OppA of Listeria monocytogenes, an Oligopeptide-Binding Protein Required for Bacterial Growth at Low Temperature and Involved in Intracellular Survival , 2000, Infection and Immunity.

[24]  M. Flickinger,et al.  Glutathione S-transferase-sspA fusion binds to E. coli RNA polymerase and complements delta sspA mutation allowing phage P1 replication. , 1994, Biochemical and biophysical research communications.

[25]  P. Tsai,et al.  Effects of Oligopeptide Permease in Group A Streptococcal Infection , 2005, Infection and Immunity.

[26]  Ulrich Dobrindt,et al.  Genomic islands in pathogenic and environmental microorganisms , 2004, Nature Reviews Microbiology.

[27]  M. Telepnev,et al.  Francisella tularensis inhibits Toll‐like receptor‐mediated activation of intracellular signalling and secretion of TNF‐α and IL‐1 from murine macrophages , 2003, Cellular microbiology.

[28]  M. Kanehisa,et al.  Reconstruction of amino acid biosynthesis pathways from the complete genome sequence. , 1998, Genome research.

[29]  R. Burke,et al.  Growth of Francisella spp. in rodent macrophages , 1991, Infection and immunity.

[30]  A. Podbielski,et al.  Molecular characterization of group A streptococcal (GAS) oligopeptide permease (Opp) and its effect on cysteine protease production , 1996, Molecular microbiology.

[31]  F. Cohen,et al.  Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray , 2003, Genome Biology.

[32]  M. Mayer,et al.  A new set of useful cloning and expression vectors derived from pBlueScript. , 1995, Gene.

[33]  R. Isberg,et al.  IcmF and DotU Are Required for Optimal Effector Translocation and Trafficking of the Legionella pneumophila Vacuole , 2004, Infection and Immunity.

[34]  Jeffrey R. Barker,et al.  Allelic exchange in Francisella tularensis using PCR products. , 2003, FEMS microbiology letters.

[35]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Rose,et al.  Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence , 2001, Molecular microbiology.

[37]  Stanley Falkow,et al.  Gene Expression Profiling of Helicobacter pylori Reveals a Growth-Phase-Dependent Switch in Virulence Gene Expression , 2003, Infection and Immunity.

[38]  J. Sexton,et al.  Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex , 2004, Infection and Immunity.

[39]  D. Frank,et al.  Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid , 2004, Applied and Environmental Microbiology.

[40]  Jeffrey R. Barker,et al.  MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[41]  David Botstein,et al.  The Stanford Microarray Database , 2001, Nucleic Acids Res..

[42]  Yoshiyuki Yamada,et al.  Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis , 2004, Molecular microbiology.

[43]  F. Nano,et al.  Transformation and allelic replacement in Francisella spp. , 1991, Journal of general microbiology.

[44]  Anders Sjöstedt,et al.  The complete genome sequence of Francisella tularensis, the causative agent of tularemia , 2005, Nature Genetics.

[45]  W. Goebel,et al.  Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening , 2006, Journal of bacteriology.

[46]  Beth A. Lazazzera,et al.  The intracellular function of extracellular signaling peptides , 2001, Peptides.

[47]  S. Kaufmann,et al.  Studying trafficking of intracellular pathogens in antigen- presenting cells , 2002 .

[48]  Philip K. Russell,et al.  Tularemia as a biological weapon: medical and public health management. , 2001, JAMA.

[49]  S. Salzberg,et al.  Microbial gene identification using interpolated Markov models. , 1998, Nucleic acids research.

[50]  M. Flickinger,et al.  Starvation‐induced expression of SspA and SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation , 1994, Molecular microbiology.