Virulence Genes of Prfa, the Key Regulator of Listerial Species-specific Differences in the Activity

ABSTRACT PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. PrfA is also present in the apathogenic species Listeria seeligeri, and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria. PrfA proteins of L. monocytogenes (PrfALm and PrfA*Lm), L. ivanovii (PrfALi), and L. seeligeri (PrfALs) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (Phly Lm) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters Phly and PactA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfALi resembled the constitutively active mutant PrfA*Lm more than the wild-type PrfALm, whereas PrfALs showed a drastically reduced capacity to bind to the PrfA-dependent promoters Phly and PactA. In contrast, the efficiencies of PrfALm, PrfA*Lm, and PrfALi forming ternary complexes and initiating transcription at Phly and PactA were rather similar, while those of PrfALs were also much lower. The low binding and transcriptional activation capacities of PrfALs seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfALm and PrfALi). In contrast to the significant differences in the biochemical properties of PrfALm, PrfALi, and PrfALs, the PrfA-dependent promoters of hly (Phly Lm, Phly L i, and Phly L s) and actA (PactA Lm, PactA L i, and PactA L s) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA Lm with prfA Ls in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes, suggesting that the altered properties of PrfALs protein are a major cause for the low virulence of L. seeligeri.

[1]  P. Youngman,et al.  Deregulation of Listeria monocytogenes virulence gene expression by two distinct and semi-independent pathways. , 2004, Microbiology.

[2]  W. Goebel,et al.  Molecular studies on the virulence of Listeria monocytogenes. , 1995, Genetic engineering.

[3]  W. Goebel,et al.  Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene , 1992, Journal of bacteriology.

[4]  W. Goebel,et al.  Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron , 1996, Molecular microbiology.

[5]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[6]  W. Goebel,et al.  Functional Similarities between the Listeria monocytogenes Virulence Regulator PrfA and Cyclic AMP Receptor Protein: the PrfA* (Gly145Ser) Mutation Increases Binding Affinity for Target DNA , 1998, Journal of Bacteriology.

[7]  W. Goebel,et al.  Differential interaction of the transcription factor PrfA and the PrfA‐activating factor (Paf) of Listeria monocytogenes with target sequences , 1998, Molecular microbiology.

[8]  I. Karunasagar,et al.  Complementation of Listeria seeligeri with the plcA-prfA genes from L. monocytogenes activates transcription of seeligerolysin and leads to bacterial escape from the phagosome of infected mammalian cells. , 1997, FEMS microbiology letters.

[9]  W. Goebel,et al.  New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure–function of the virulence regulator PrfA , 2004, Molecular microbiology.

[10]  D. Portnoy,et al.  Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo , 1994, Molecular microbiology.

[11]  P. Berche,et al.  Production of thiol-dependent haemolysins by Listeria monocytogenes and related species. , 1989, Journal of general microbiology.

[12]  H. Bouwer,et al.  Isolation of Listeria monocytogenes mutants with high‐level in vitro expression of host cytosol‐induced gene products , 2003, Molecular microbiology.

[13]  E. Domann,et al.  Intracellular Gene Expression Profile of Listeria monocytogenes , 2006, Infection and Immunity.

[14]  C. Buchrieser,et al.  New Aspects Regarding Evolution and Virulence of Listeria monocytogenes Revealed by Comparative Genomics and DNA Arrays , 2004, Infection and Immunity.

[15]  J. Vázquez-Boland,et al.  Pathogenicity islands and other virulence elements in Listeria. , 2002, Current topics in microbiology and immunology.

[16]  J. Vázquez-Boland,et al.  Regulation of virulence genes in Listeria. , 2001, International journal of medical microbiology : IJMM.

[17]  J. Vázquez-Boland,et al.  Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor , 2004, Molecular microbiology.

[18]  W. Goebel,et al.  PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA‐dependent virulence gene promoters resulting in a transcriptionally active complex , 2000, Molecular microbiology.

[19]  W. Goebel,et al.  Supportive and inhibitory elements of a putative PrfA‐dependent promoter in Listeria monocytogenes , 2005, Molecular microbiology.

[20]  J. Vázquez-Boland,et al.  A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes , 1997, Journal of bacteriology.

[21]  P. Cossart,et al.  An RNA Thermosensor Controls Expression of Virulence Genes in Listeria monocytogenes , 2002, Cell.

[22]  W. Goebel,et al.  LaXp180, a mammalian ActA‐binding protein, identified with the yeast two‐hybrid system, co‐localizes with intracellular Listeria monocytogenes , 2000, Cellular microbiology.

[23]  P. Youngman,et al.  Carbon‐source regulation of virulence gene expression in Listeria monocytogenes , 1997, Molecular microbiology.

[24]  P. Cossart,et al.  Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes, can be present but inactive , 1997, Infection and immunity.

[25]  W. Goebel,et al.  The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp‐Fnr family of transcription regulators , 1994, Molecular microbiology.

[26]  K. Schleifer,et al.  Evolutionary history of the genus Listeria and its virulence genes. , 2005, Systematic and applied microbiology.

[27]  Wolf-Dieter Schubert,et al.  The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA‐binding affinity by stabilizing the HTH motif , 2005, Molecular microbiology.

[28]  W. Goebel,et al.  In vitro transcription of PrfA‐dependent and 
‐independent genes of Listeria monocytogenes , 2001, Molecular microbiology.

[29]  E. Domann,et al.  Genome organization and the evolution of the virulence gene locus in Listeria species. , 2000, International journal of medical microbiology : IJMM.

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

[31]  P. Cossart,et al.  Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation , 1989, Infection and immunity.

[32]  C. Buchrieser,et al.  How Seryl-Phosphorylated HPr Inhibits PrfA, a Transcription Activator of Listeria monocytogenes Virulence Genes , 2006, Journal of Molecular Microbiology and Biotechnology.

[33]  Vincent A. Fischetti,et al.  Gram-positive pathogens , 2006 .

[34]  C. Nadon,et al.  The Listeria monocytogenes prfAP2 promoter is regulated by sigma B in a growth phase dependent manner. , 2005, FEMS microbiology letters.

[35]  P. Youngman,et al.  Transcriptional activation of the Listeria monocytogenes hemolysin gene in Bacillus subtilis , 1992, Journal of bacteriology.

[36]  W. Goebel,et al.  Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[37]  P. Cossart,et al.  The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species , 1994, Infection and immunity.

[38]  C. Nadon,et al.  Sigma B Contributes to PrfA-Mediated Virulence in Listeria monocytogenes , 2002, Infection and Immunity.

[39]  P. Berche,et al.  Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. , 1996, Research in microbiology.

[40]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[41]  R. Ebright,et al.  A single substitution in the putative helix‐turn‐helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence , 1996, Molecular microbiology.

[42]  P. Cossart,et al.  Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated , 1991, Molecular microbiology.

[43]  P. Cossart,et al.  Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator , 1995, Journal of bacteriology.

[44]  W. Goebel,et al.  Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA , 1996, Molecular microbiology.

[45]  E. Johnson,et al.  Development of an improved chemically defined minimal medium for Listeria monocytogenes , 1991, Applied and environmental microbiology.

[46]  W. Goebel,et al.  Regulation of Virulence Genes in Pathogenic Listeria spp. , 2006 .

[47]  W. Goebel,et al.  Positive Selection of Mutations Leading to Loss or Reduction of Transcriptional Activity of PrfA, the Central Regulator ofListeria monocytogenes Virulence , 2001, Journal of bacteriology.

[48]  N. Freitag,et al.  Pleiotropic Enhancement of Bacterial Pathogenesis Resulting from the Constitutive Activation of the Listeria monocytogenes Regulatory Factor PrfA , 2005, Infection and Immunity.

[49]  W. Goebel,et al.  In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA‐dependent and ‐independent promoters that are differentially activated by GTP , 2004, Molecular microbiology.

[50]  W. Goebel,et al.  Enhanced Synthesis of Internalin A in aro Mutants of Listeria monocytogenes Indicates Posttranscriptional Control of the inlAB mRNA , 2005, Journal of bacteriology.

[51]  W. Goebel,et al.  SigB-Dependent In Vitro Transcription of prfA and Some Newly Identified Genes of Listeria monocytogenes Whose Expression Is Affected by PrfA In Vivo , 2005, Journal of bacteriology.

[52]  Kendy K. Y. Wong,et al.  A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products , 2004, Journal of bacteriology.

[53]  W. Goebel,et al.  Listeria Pathogenesis and Molecular Virulence Determinants , 2001, Clinical Microbiology Reviews.

[54]  D. Portnoy,et al.  Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread , 1993, Infection and immunity.

[55]  T. Chakraborty,et al.  Comparative and functional genomics of Listeria spp. , 2006, Journal of biotechnology.