- Email: [email protected]
Altered gene expression in response to Bordetella pertussis infection
News & Comment
TRENDS in Microbiology Vol.9 No.2 February 2001
57
Journal Club
The ubiquitous nature of budding The final stage of retrovirus and lentivirus budding occurs when virion particles pinch off from the plasma membrane. The late (L)-domain region in the Gag polyprotein is responsible for promoting virus release, but the mechanism by which this event occurs is unknown. One clue came with the discovery of conserved sequences that confer L-domain function. In HIV-1 Gag and certain rhabdovirus and filovirus proteins, the L-domain core sequence is PT/SAP, whereas the core sequence PPXY is found in the L-domains of Rous sarcoma virus (RSV), Mason-Pfizer monkey virus and Moloney murine leukemia virus. PPXY motifs are known to interact with proteins containing WW domains, suggesting that L-domains mediate interactions with cellular proteins. A series of recent papers in PNAS have provided further insight into L-domain function by implicating ubiquitin and components of the ubiquitination machinery in the late stages of budding. Ubiquitin is a 76-residue protein, which, when covalently attached to proteins, targets the modified proteins for degradation by the proteasome. Treatment of cells with proteasome inhibitors, which causes a build up of polyubiquitinated proteins and decreases the pool of free
ubiquitin, has now been shown to inhibit the release of virus particles from RSV(Ref. 1), SIV- (Ref. 2) and HIV-1- (Ref. 3) infected cells. Electron microscopic analyses of the drug-treated cells revealed the accumulation of late-budding structures at the plasma membrane, which, in the case of RSV, appear as multilayered crystalline-like arrays of particles. Moreover, ubiquitination of a minimal HIV-1 Gag construct and the VP40 protein of Ebola virus has been shown to occur in a manner that is dependent upon the PPXY L-domain sequence4. ‘Components of the ubiquitination machinery...are recruited via the L-domain to the sites of assembly.’ What is the function of ubiquitin in the assembly process? It is possible that ubiquitination is necessary for removal of misfolded Gag proteins, which could interfere with particle assembly. However, this is unlikely, given that fusion of ubiquitin to the carboxyl terminus of RSV Gag overcomes the block to budding in the presence of proteasome inhibitors. Instead, the authors favor the notion that components of the ubiquitination machinery, specifically ubiquitin ligase, are
recruited via the L-domain to the sites of assembly. This is supported by the findings that Nedd4 and Rsp5, ubiquitin ligases that contain WW domains, bind to viral L-domains in vitro. Ubiquitination of cell-surface receptors has been shown to trigger endocytosis, and it is possible that ubiquitination serves to recruit endocytic components for assistance in the pinchingoff stage of budding. The exact role of ubiquitin in virus assembly still remains to be determined, but it is likely that it reflects the ubiquitous nature of budding for multiple virus types. 1 Patnaik, A. et al. (2000) Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. U. S. A. 97, 13069–13074 2 Strack, B. et al. (2000) A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. U. S. A. 97, 13063–13068 3 Schubert, U. et al. (2000) Proteasome inhibition interferes with Gag polyprotein processing, release and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. U. S. A. 97, 13057–13062 4 Harty, R.N. et al. (2000) A PPXY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Sci. U. S. A. 97, 13871–13876
Marilyn D. Resh [email protected]
Altered gene expression in response to Bordetella pertussis infection Whooping cough is a highly contagious disease caused by Bordetella pertussis, which has a highly selective tropism in humans for the ciliated epithelium of the respiratory tract. This pathogen produces an array of powerful toxins that penetrate tissues, kill cells, immobilize the ciliary elevator and cause the accumulation of thick mucus in the airway. Despite the well-known clinical manifestations of B. pertussis infection, relatively little is known about the host response to infection with this pathogen. To further delineate the role respiratory epithelial cells play in initiating and modulating the host response to B. pertussis infection, Belcher et al.1 recently published the results of their use of
high-density cDNA arrays to analyse genome-wide gene expression in a human bronchial epithelial cell line in response to B. pertussis infection. ‘These host cell responses...offer important insights into pertussis pathology.’ Following infection with B. pertussis, bronchial epithelial cells upregulated mRNA expression of a number of proinflammatory cytokines and chemokines, anti-apoptotic factors, and nuclear factor κB (NF-κB)regulated genes. At the same time, the levels of expression of numerous genes decreased, including several encoding DNA-binding proteins and cellular adhesin molecules.
Cytokine protein expression and neutrophil chemoattraction were also independently verified by the authors in a variety of functional analyses. The majority of these host cell responses have not been previously implicated in B. pertussis infection, and therefore offer important insights into pertussis pathology. Moreover, several of these results are consistent with the clinical manifestations of pertussis in humans. The authors also demonstrated that, in addition to inducing mucin gene expression in infected bronchial tissues, B. pertussis is also able to use mucin as a binding substrate. This novel finding has important implications for the pathology of pertussis, considering that the B. pertussis-induced
http://tim.trends.com 0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
58
News & Comment
damage to the mucociliary elevator results in a build-up of respiratory secretions and mucus. Accordingly, the increase in mucus production, coupled with the inability to clear these respiratory secretions effectively, might prove to be an ideal
TRENDS in Microbiology Vol.9 No.2 February 2001
scenario for Bordetella to colonize the host by binding to mucin and thereby subverting the host innate defenses. 1 Belcher, C.E. et al. (2000) The transcriptional responses of respiratory eptihelial cells to
Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies. Proc. Natl. Acad. Sci. U. S. A. 97, 13847–13852
Cheryl A. Nickerson [email protected]
Removing repression: novel roles for solute Microbial genomics transporters in regulating gene expression Genome sequence of an extreme Bacteria utilize a plethora of different mechanisms to regulate gene expression. One of the most common and well studied mechanisms is the binding of a repressor protein to a specific operator sequence in the promoter region of a regulated gene, which then prevents binding of RNA polymerase and subsequent transcription. The binding of a repressor to its operator sequence is classically modulated by the presence or absence of an inducer molecule that binds directly to the repressor. For example, the Lac repressor normally binds tightly to its operator sequence, but when the inducer – lactose – is present, the repressor undergoes a conformational change and can no longer bind to its operator sequence, thereby removing the repression of that particular gene. Recently, a new Escherichia coli repressor protein, Mlc, was discovered and shown to be involved in the regulation of a series of genes in response to the presence of glucose in the growth medium. Initially, Jacqueline Plumbridge and colleagues from the Institut de Biologie Physico-Chimique in Paris attempted to identify an inducer molecule that would bind to Mlc, but were unable to identify any such small molecule. Instead, they proposed an alternative mechanism for modulation of Mlc activity that involved the glucose transporter PtsG in a signal transduction pathway from external glucose to Mlt. Now, Plumbridge, collaborating with Winfried Boos and Jean-Pierre Bouché and, independently, the group of Hiroji Aiba from Nagoya University, have presented two papers that confirm and extend this hypothesis1,2. The PtsG glucose transporter is a component of the phosphotransferase system and phosphorylates glucose as it enters the cell. When glucose is absent from the extracellular medium, the
transporter is in a phosphorylated state waiting for substrate. Under these conditions, Mlc is free in the cytoplasm and represses transcription of its target genes. However, when glucose is present outside the cell, it is transported and phosphorylated by PtsG, leaving the transporter unphosphorylated. Both research teams found that unphosphorylated PtsG binds Mlc, which effectively removes the repressor from the cytoplasm and prevents binding to promoter regions to repress transcription1,2. As ptsG expression is regulated by Mlc, this in turn leads to increased synthesis of PtsG and more derepression in a positive-feedback loop. ‘No bacterial repressor protein has previously been demonstrated to operate in this manner.’ This work is significant as no bacterial repressor protein has previously been demonstrated to operate in this manner. It also raises the possibility that the role of transporters in regulating gene expression could be a more widespread phenomenon. For many bacterial regulatory systems that respond to the presence of an extracellular substrate for a metabolic process, it makes sense for the cell to consider how much substrate is actually getting into the cell as well as sensing if it is present in the environment. 1 Lee, S-J. et al. (2000) Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli. EMBO J. 19, 5353–5361 2 Tanaka, Y. et al. (2000) A novel regulatory role of glucose transporter of Escherichia coli: membrane sequestration of a global repressor Mlc. EMBO J. 19, 5344–5352
Gavin H. Thomas [email protected]
halophile A consortium of North American laboratories has recently reported the complete 2.5-Mb genome sequence of an extreme halophile, Halobacterium sp. NRC-1 (Ref. 1). The genome is organized into a large chromosome and 2 related minichromosomes. Salient features identified by sequence analysis include copious insertion sequences, pathways for uptake and utilization of amino acids, active sodium–proton antiporter and potassiumuptake systems, photosensory and signal transduction pathways, and complex DNA replication, transcription and translation systems. Ease of culture and genetic manipulation, added to the availability of the whole genome sequence, promise to make this organism as an excellent model system among the Archaea. 1 Ng, W.V. et al. (2000) Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 97, 12176–12181
Mark Pallen [email protected]
Commentaries Did you know that some of the Journal Club articles that appear in TIM can also be accessed online as BioMedNet Commentaries? Access to Commentaries is FREE. Simply go to http://www.bmn.com, click on the ‘News and Comment’ link and then press the Commentaries button (you might need to register as a BioMedNet member if you haven’t already). If you would be interested in contributing to this section, please contact the Editor: [email protected]
http://tim.trends.com 0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
TRENDS in Microbiology Vol.9 No.2 February 2001
57
Journal Club
The ubiquitous nature of budding The final stage of retrovirus and lentivirus budding occurs when virion particles pinch off from the plasma membrane. The late (L)-domain region in the Gag polyprotein is responsible for promoting virus release, but the mechanism by which this event occurs is unknown. One clue came with the discovery of conserved sequences that confer L-domain function. In HIV-1 Gag and certain rhabdovirus and filovirus proteins, the L-domain core sequence is PT/SAP, whereas the core sequence PPXY is found in the L-domains of Rous sarcoma virus (RSV), Mason-Pfizer monkey virus and Moloney murine leukemia virus. PPXY motifs are known to interact with proteins containing WW domains, suggesting that L-domains mediate interactions with cellular proteins. A series of recent papers in PNAS have provided further insight into L-domain function by implicating ubiquitin and components of the ubiquitination machinery in the late stages of budding. Ubiquitin is a 76-residue protein, which, when covalently attached to proteins, targets the modified proteins for degradation by the proteasome. Treatment of cells with proteasome inhibitors, which causes a build up of polyubiquitinated proteins and decreases the pool of free
ubiquitin, has now been shown to inhibit the release of virus particles from RSV(Ref. 1), SIV- (Ref. 2) and HIV-1- (Ref. 3) infected cells. Electron microscopic analyses of the drug-treated cells revealed the accumulation of late-budding structures at the plasma membrane, which, in the case of RSV, appear as multilayered crystalline-like arrays of particles. Moreover, ubiquitination of a minimal HIV-1 Gag construct and the VP40 protein of Ebola virus has been shown to occur in a manner that is dependent upon the PPXY L-domain sequence4. ‘Components of the ubiquitination machinery...are recruited via the L-domain to the sites of assembly.’ What is the function of ubiquitin in the assembly process? It is possible that ubiquitination is necessary for removal of misfolded Gag proteins, which could interfere with particle assembly. However, this is unlikely, given that fusion of ubiquitin to the carboxyl terminus of RSV Gag overcomes the block to budding in the presence of proteasome inhibitors. Instead, the authors favor the notion that components of the ubiquitination machinery, specifically ubiquitin ligase, are
recruited via the L-domain to the sites of assembly. This is supported by the findings that Nedd4 and Rsp5, ubiquitin ligases that contain WW domains, bind to viral L-domains in vitro. Ubiquitination of cell-surface receptors has been shown to trigger endocytosis, and it is possible that ubiquitination serves to recruit endocytic components for assistance in the pinchingoff stage of budding. The exact role of ubiquitin in virus assembly still remains to be determined, but it is likely that it reflects the ubiquitous nature of budding for multiple virus types. 1 Patnaik, A. et al. (2000) Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. U. S. A. 97, 13069–13074 2 Strack, B. et al. (2000) A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. U. S. A. 97, 13063–13068 3 Schubert, U. et al. (2000) Proteasome inhibition interferes with Gag polyprotein processing, release and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. U. S. A. 97, 13057–13062 4 Harty, R.N. et al. (2000) A PPXY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Sci. U. S. A. 97, 13871–13876
Marilyn D. Resh [email protected]
Altered gene expression in response to Bordetella pertussis infection Whooping cough is a highly contagious disease caused by Bordetella pertussis, which has a highly selective tropism in humans for the ciliated epithelium of the respiratory tract. This pathogen produces an array of powerful toxins that penetrate tissues, kill cells, immobilize the ciliary elevator and cause the accumulation of thick mucus in the airway. Despite the well-known clinical manifestations of B. pertussis infection, relatively little is known about the host response to infection with this pathogen. To further delineate the role respiratory epithelial cells play in initiating and modulating the host response to B. pertussis infection, Belcher et al.1 recently published the results of their use of
high-density cDNA arrays to analyse genome-wide gene expression in a human bronchial epithelial cell line in response to B. pertussis infection. ‘These host cell responses...offer important insights into pertussis pathology.’ Following infection with B. pertussis, bronchial epithelial cells upregulated mRNA expression of a number of proinflammatory cytokines and chemokines, anti-apoptotic factors, and nuclear factor κB (NF-κB)regulated genes. At the same time, the levels of expression of numerous genes decreased, including several encoding DNA-binding proteins and cellular adhesin molecules.
Cytokine protein expression and neutrophil chemoattraction were also independently verified by the authors in a variety of functional analyses. The majority of these host cell responses have not been previously implicated in B. pertussis infection, and therefore offer important insights into pertussis pathology. Moreover, several of these results are consistent with the clinical manifestations of pertussis in humans. The authors also demonstrated that, in addition to inducing mucin gene expression in infected bronchial tissues, B. pertussis is also able to use mucin as a binding substrate. This novel finding has important implications for the pathology of pertussis, considering that the B. pertussis-induced
http://tim.trends.com 0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
58
News & Comment
damage to the mucociliary elevator results in a build-up of respiratory secretions and mucus. Accordingly, the increase in mucus production, coupled with the inability to clear these respiratory secretions effectively, might prove to be an ideal
TRENDS in Microbiology Vol.9 No.2 February 2001
scenario for Bordetella to colonize the host by binding to mucin and thereby subverting the host innate defenses. 1 Belcher, C.E. et al. (2000) The transcriptional responses of respiratory eptihelial cells to
Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies. Proc. Natl. Acad. Sci. U. S. A. 97, 13847–13852
Cheryl A. Nickerson [email protected]
Removing repression: novel roles for solute Microbial genomics transporters in regulating gene expression Genome sequence of an extreme Bacteria utilize a plethora of different mechanisms to regulate gene expression. One of the most common and well studied mechanisms is the binding of a repressor protein to a specific operator sequence in the promoter region of a regulated gene, which then prevents binding of RNA polymerase and subsequent transcription. The binding of a repressor to its operator sequence is classically modulated by the presence or absence of an inducer molecule that binds directly to the repressor. For example, the Lac repressor normally binds tightly to its operator sequence, but when the inducer – lactose – is present, the repressor undergoes a conformational change and can no longer bind to its operator sequence, thereby removing the repression of that particular gene. Recently, a new Escherichia coli repressor protein, Mlc, was discovered and shown to be involved in the regulation of a series of genes in response to the presence of glucose in the growth medium. Initially, Jacqueline Plumbridge and colleagues from the Institut de Biologie Physico-Chimique in Paris attempted to identify an inducer molecule that would bind to Mlc, but were unable to identify any such small molecule. Instead, they proposed an alternative mechanism for modulation of Mlc activity that involved the glucose transporter PtsG in a signal transduction pathway from external glucose to Mlt. Now, Plumbridge, collaborating with Winfried Boos and Jean-Pierre Bouché and, independently, the group of Hiroji Aiba from Nagoya University, have presented two papers that confirm and extend this hypothesis1,2. The PtsG glucose transporter is a component of the phosphotransferase system and phosphorylates glucose as it enters the cell. When glucose is absent from the extracellular medium, the
transporter is in a phosphorylated state waiting for substrate. Under these conditions, Mlc is free in the cytoplasm and represses transcription of its target genes. However, when glucose is present outside the cell, it is transported and phosphorylated by PtsG, leaving the transporter unphosphorylated. Both research teams found that unphosphorylated PtsG binds Mlc, which effectively removes the repressor from the cytoplasm and prevents binding to promoter regions to repress transcription1,2. As ptsG expression is regulated by Mlc, this in turn leads to increased synthesis of PtsG and more derepression in a positive-feedback loop. ‘No bacterial repressor protein has previously been demonstrated to operate in this manner.’ This work is significant as no bacterial repressor protein has previously been demonstrated to operate in this manner. It also raises the possibility that the role of transporters in regulating gene expression could be a more widespread phenomenon. For many bacterial regulatory systems that respond to the presence of an extracellular substrate for a metabolic process, it makes sense for the cell to consider how much substrate is actually getting into the cell as well as sensing if it is present in the environment. 1 Lee, S-J. et al. (2000) Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli. EMBO J. 19, 5353–5361 2 Tanaka, Y. et al. (2000) A novel regulatory role of glucose transporter of Escherichia coli: membrane sequestration of a global repressor Mlc. EMBO J. 19, 5344–5352
Gavin H. Thomas [email protected]
halophile A consortium of North American laboratories has recently reported the complete 2.5-Mb genome sequence of an extreme halophile, Halobacterium sp. NRC-1 (Ref. 1). The genome is organized into a large chromosome and 2 related minichromosomes. Salient features identified by sequence analysis include copious insertion sequences, pathways for uptake and utilization of amino acids, active sodium–proton antiporter and potassiumuptake systems, photosensory and signal transduction pathways, and complex DNA replication, transcription and translation systems. Ease of culture and genetic manipulation, added to the availability of the whole genome sequence, promise to make this organism as an excellent model system among the Archaea. 1 Ng, W.V. et al. (2000) Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. U. S. A. 97, 12176–12181
Mark Pallen [email protected]
Commentaries Did you know that some of the Journal Club articles that appear in TIM can also be accessed online as BioMedNet Commentaries? Access to Commentaries is FREE. Simply go to http://www.bmn.com, click on the ‘News and Comment’ link and then press the Commentaries button (you might need to register as a BioMedNet member if you haven’t already). If you would be interested in contributing to this section, please contact the Editor: [email protected]
http://tim.trends.com 0966-842X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.