Regulation of the Mycobacterium tuberculosis mce1 Operon

ABSTRACT In the murine model of infection, a Mycobacterium tuberculosis mce1 operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the mce1 genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the mce1 genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of mce1 expression, we created an M. tuberculosis mce1R deletion mutant. There was no difference in expression of mce1 operon genes in Δmce1R compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of mce1 genes was significantly higher in Δmce1R. In addition, overexpression of mce1R resulted in repression of the mce1 genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the mce1 operon. We propose that Mce1R facilitates balanced temporal expression of the mce1 products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of M. tuberculosis.

[1]  J. Betts,et al.  Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling , 2002, Molecular microbiology.

[2]  K. Struhl,et al.  Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience). Host-Range Shuttle System for Gene Insertion into the Chromosomes of Gram-negative Bacteria. , 1988 .

[3]  Rajesh S. Gokhale,et al.  Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria , 2004, Nature.

[4]  Rick Lyons,et al.  The temporal expression profile of Mycobacterium tuberculosis infection in mice. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[5]  D. Schnappinger,et al.  Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis. , 2001, Gene.

[6]  W. Jacobs,et al.  Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence , 1994, Infection and immunity.

[7]  I. Smith,et al.  Mycobacterium tuberculosis Genes Induced during Infection of Human Macrophages , 2002, Infection and Immunity.

[8]  M. Horwitz,et al.  High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism , 2001, Infection and Immunity.

[9]  S T Cole,et al.  Analysis of the proteome of Mycobacterium tuberculosis in silico. , 1999, Tubercle and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[10]  Sangwei Lu,et al.  Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry , 2001, Cellular microbiology.

[11]  V. Brahmachari,et al.  Analysis of Expression Profile of Mammalian Cell Entry (mce) Operons of Mycobacterium tuberculosis , 2003, Infection and Immunity.

[12]  A. Henkel,et al.  Quantification of proteins dissolved in an electrophoresis sample buffer. , 1994, Analytical biochemistry.

[13]  S. Rigali,et al.  Subdivision of the Helix-Turn-Helix GntR Family of Bacterial Regulators in the FadR, HutC, MocR, and YtrA Subfamilies* , 2002, The Journal of Biological Chemistry.

[14]  Yang Liu,et al.  Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages , 2003, The Journal of experimental medicine.

[15]  Dirk Schnappinger,et al.  Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding α-crystallin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  G. Schoolnik,et al.  Genome-wide expression profiling of intracellular bacteria: The interaction of Mycobacterium tuberculosis with macrophages , 2002 .

[17]  M. Horwitz,et al.  Expression and Efficient Export of Enzymatically ActiveMycobacterium tuberculosis Glutamine Synthetase in Mycobacterium smegmatis and Evidence That the Information for Export is Contained within the Protein* , 1997, The Journal of Biological Chemistry.

[18]  B. Gicquel,et al.  Life on the inside: Probing Mycobacterium tuberculosis gene expression during infection , 2000, Immunology and cell biology.

[19]  W. Degrave,et al.  Method for high yield preparation in large and small scale of nucleic acids from mycobacteria , 1992 .

[20]  J. Guest,et al.  A new family of bacterial regulatory proteins. , 1991, FEMS microbiology letters.

[21]  Mustafa,et al.  Cloning, Expression and Immunological Reactivity of Two Mammalian Cell Entry Proteins Encoded by the mce1 Operon of Mycobacterium tuberculosis , 1999, Scandinavian journal of immunology.

[22]  G. Schoolnik,et al.  The Mycobacterium tuberculosis ECF sigma factor σE: role in global gene expression and survival in macrophages † , 2001, Molecular microbiology.

[23]  Geoffrey J. Barton,et al.  JPred : a consensus secondary structure prediction server , 1999 .

[24]  A. Cataldi,et al.  Negative transcriptional regulation of the mce3 operon in Mycobacterium tuberculosis. , 2002, Microbiology.

[25]  N. Casali,et al.  Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[27]  F. Kramer,et al.  Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis , 1999, Molecular microbiology.

[28]  S. Kaufmann,et al.  Immunology of infection , 1999 .

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

[30]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[31]  I. Smith,et al.  Mycobacterium tuberculosis Pathogenesis and Molecular Determinants of Virulence , 2003, Clinical Microbiology Reviews.

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

[33]  P. Black,et al.  Bacterial Long Chain Fatty Acid Transport: Gateway to a Fatty Acid-responsive Signaling System* , 2004, Journal of Biological Chemistry.

[34]  P. Butcher,et al.  Extraction of RNA from Intracellular Mycobacterium tuberculosis : Methods, Considerations, and Applications. , 2001, Methods in molecular medicine.

[35]  B. Gicquel,et al.  Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells. , 1999, Microbiology.

[36]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[37]  J. Graham,et al.  Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  T. Parish,et al.  Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. , 2000, Microbiology.