Publications by authors named "Marta Perego"

47 Publications

The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy.

J Leukoc Biol 2021 Jun 4. Epub 2021 Jun 4.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA.

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22 and p47 at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.
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http://dx.doi.org/10.1002/JLB.2HI0421-123RDOI Listing
June 2021

Treatment options for localised renal cell carcinoma of the transplanted kidney.

World J Transplant 2020 Jun;10(6):147-161

Renal Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan 20122, Italy.

Currently, there is no consensus among the transplant community about the treatment of renal cell carcinoma (RCC) of the transplanted kidney. Until recently, graftectomy was universally considered the golden standard, regardless of the characteristics of the neoplasm. Due to the encouraging results observed in native kidneys, conservative options such as nephron-sparing surgery (NSS) (enucleation and partial nephrectomy) and ablative therapy (radiofrequency ablation, cryoablation, microwave ablation, high-intensity focused ultrasound, and irreversible electroporation) have been progressively used in carefully selected recipients with early-stage allograft RCC. Available reports show excellent patient survival, optimal oncological outcome, and preserved renal function with acceptable complication rates. Nevertheless, the rarity and the heterogeneity of the disease, the number of options available, and the lack of long-term follow-up data do not allow to adequately define treatment-specific advantages and limitations. The role of active surveillance and immunosuppression management remain also debated. In order to offer a better insight into this difficult topic and to help clinicians choose the best therapy for their patients, we performed and extensive review of the literature. We focused on epidemiology, clinical presentation, diagnostic work up, staging strategies, tumour characteristics, treatment modalities, and follow-up protocols. Our research confirms that both NSS and focal ablation represent a valuable alternative to graftectomy for kidney transplant recipients with American Joint Committee on Cancer stage T1aN0M0 RCC. Data on T1bN0M0 lesions are scarce but suggest extra caution. Properly designed multi-centre prospective clinical trials are warranted.
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http://dx.doi.org/10.5500/wjt.v10.i6.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360528PMC
June 2020

Allograft artery mycotic aneurysm after kidney transplantation: A case report and review of literature.

World J Clin Cases 2020 Mar;8(5):912-921

Renal Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan 20122, Italy.

Background: Allograft artery mycotic aneurysm (MA) represents a rare but life-threatening complication of kidney transplantation. Graftectomy is widely considered the safest option. Due to the rarity of the disease and the substantial risk of fatal consequences, experience with conservative strategies is limited. To date, only a few reports on surgical repair have been published. We describe a case of true MA successfully managed by aneurysm resection and arterial re-anastomosis.

Case Summary: An 18-year-old gentleman, on post-operative day 70 after deceased donor kidney transplantation, presented with malaise, low urinary output, and worsening renal function. Screening organ preservation fluid cultures, collected at the time of surgery, were positive for C. Doppler ultrasound and contrast-enhanced computer tomography showed a 4-cm-sized, saccular aneurysm of the iuxta-anastomotic segment of the allograft artery, suspicious for MA. The lesion was wide-necked and extended to the distal bifurcation of the main arterial branch, thus preventing endovascular stenting and embolization. After multidisciplinary discussion, the patient underwent surgical exploration, aneurysm excision, and re-anastomosis between the stump of the allograft artery and the internal iliac artery. The procedure was uneventful. Histology and microbiology evaluation of the surgical specimen confirmed the diagnosis of MA caused by infection. Three years after the operation, the patient is doing very well with excellent allograft function and no signs of recurrent disease.

Conclusion: Surgical repair represents a feasible option in carefully selected patients with allograft artery MA. Anti-fungal prophylaxis is advised when preservation fluid cultures are positive.
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http://dx.doi.org/10.12998/wjcc.v8.i5.912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062617PMC
March 2020

Systematic review of ablative therapy for the treatment of renal allograft neoplasms.

World J Clin Cases 2019 Sep;7(17):2487-2504

Renal Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan 20122, Italy.

Background: To date, there are no guidelines on the treatment of solid neoplasms in the transplanted kidney. Historically, allograft nephrectomy has been considered the only reasonable option. More recently, nephron-sparing surgery (NSS) and ablative therapy (AT) have been proposed as alternative procedures in selected cases.

Aim: To review outcomes of AT for the treatment of renal allograft tumours.

Methods: We conducted a systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2009 Checklist. PubMed was searched in March 2019 without time restrictions for all papers reporting on radiofrequency ablation (RFA), cryoablation (CA), microwave ablation (MWA), high-intensity focused ultrasound (HIFU), and irreversible electroporation (IRE) of solid tumours of the kidney allograft. Only original manuscripts describing actual cases and edited in English were considered. All relevant articles were accessed in full text. Additional searches included all pertinent references. Selected studies were also assessed for methodological quality using a tool based on a modification of the Newcastle Ottawa scale. Data on recipient characteristics, transplant characteristics, disease characteristics, treatment protocols, and treatment outcomes were extracted and analysed. Given the nature and the quality of the studies available (mostly retrospective case reports and small retrospective uncontrolled case series), a descriptive summary was provided.

Results: Twenty-eight relevant studies were selected describing a total of 100 AT procedures in 92 patients. Recipient age at diagnosis ranged from 21 to 71 years whereas time from transplant to diagnosis ranged from 0.1 to 312 mo. Most of the neoplasms were asymptomatic and diagnosed incidentally during imaging carried out for screening purposes or for other clinical reasons. Preferred diagnostic modality was Doppler-ultrasound scan followed by computed tomography scan, and magnetic resonance imaging. Main tumour types were: papillary renal cell carcinoma (RCC) and clear cell RCC. Maximal tumour diameter ranged from 5 to 55 mm. The vast majority of neoplasms were T1a N0 M0 with only 2 lesions staged T1b N0 M0. Neoplasms were managed by RFA ( = 78), CA ( = 15), MWA ( = 3), HIFU ( = 3), and IRE ( = 1). Overall, 3 episodes of primary treatment failure were reported. A single case of recurrence was identified. Follow-up ranged from 1 to 81 mo. No cancer-related deaths were observed. Complication rate was extremely low (mostly < 10%). Graft function remained stable in the majority of recipients. Due to the limited sample size, no clear benefit of a single procedure over the other ones could be demonstrated.

Conclusion: AT for renal allograft neoplasms represents a promising alternative to radical nephrectomy and NSS in carefully selected patients. Properly designed clinical trials are needed to validate this therapeutic approach.
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http://dx.doi.org/10.12998/wjcc.v7.i17.2487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745334PMC
September 2019

Forty years in the making: understanding the molecular mechanism of peptide regulation in bacterial development.

Authors:
Marta Perego

PLoS Biol 2013 19;11(3):e1001516. Epub 2013 Mar 19.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

Signal transduction systems are influenced by positive and negative forces resulting in an output reflecting the sum of the opposing forces. The Rap family of regulatory protein modules control the output of two-component signal transduction systems through protein∶protein and protein∶peptide interactions. These modules and their peptide regulators are found in complex signaling pathways, including the bacterial developmental pathway to sporulation, competence, and protease secretion. Two articles published in the current issue of PLOS Biology reveal by means of crystallographic analyses how the Rap proteins of bacilli are regulated by their inhibitor Phr peptide and provide a mechanistic explanation for a genetic phenotype isolated decades earlier. The Rap-Phr module of bacterial regulators was the prototype of a family that now extends to other bacterial signaling proteins that involve the use of the tetratricopeptide repeat structural fold. The results invite speculation regarding the potential exploitation of this module as a molecular tool for applications in therapeutic design and biotechnology.
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http://dx.doi.org/10.1371/journal.pbio.1001516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601992PMC
September 2013

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

J Bacteriol 2012 Mar 20;194(6):1378-88. Epub 2012 Jan 20.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, California, USA.

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.
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http://dx.doi.org/10.1128/JB.06747-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294843PMC
March 2012

Role of net charge on catalytic domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of phage lysins.

J Biol Chem 2011 Sep 4;286(39):34391-403. Epub 2011 Aug 4.

Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA.

The recombinant lysins of lytic phages, when applied externally to Gram-positive bacteria, can be efficient bactericidal agents, typically retaining high specificity. Their development as novel antibacterial agents offers many potential advantages over conventional antibiotics. Protein engineering could exploit this potential further by generating novel lysins fit for distinct target populations and environments. However, access to the peptidoglycan layer is controlled by a variety of secondary cell wall polymers, chemical modifications, and (in some cases) S-layers and capsules. Classical lysins require a cell wall-binding domain (CBD) that targets the catalytic domain to the peptidoglycan layer via binding to a secondary cell wall polymer component. The cell walls of Gram-positive bacteria generally have a negative charge, and we noticed a correlation between (positive) charge on the catalytic domain and bacteriolytic activity in the absence of the CBD (nonclassical behavior). We investigated a physical basis for this correlation by comparing the structures and activities of pairs of lysins where the lytic activity of one of each pair was CBD-independent. We found that by engineering a reversal of sign of the net charge of the catalytic domain, we could either eliminate or create CBD dependence. We also provide evidence that the S-layer of Bacillus anthracis acts as a molecular sieve that is chiefly size-dependent, favoring catalytic domains over full-length lysins. Our work suggests a number of facile approaches for fine-tuning lysin activity, either to enhance or reduce specificity/host range and/or bactericidal potential, as required.
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http://dx.doi.org/10.1074/jbc.M111.244160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190764PMC
September 2011

Transcription antitermination by a phosphorylated response regulator and cobalamin-dependent termination at a B₁₂ riboswitch contribute to ethanolamine utilization in Enterococcus faecalis.

J Bacteriol 2011 May 25;193(10):2575-86. Epub 2011 Mar 25.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, California 92037, USA.

The ability of bacteria to utilize ethanolamine (EA) as a carbon and nitrogen source may confer an advantage for survival, colonization, and pathogenicity in the human intestinal tract. Enterococcus faecalis, a Gram-positive human commensal organism, depends on a two-component signaling system (TCS-17) for sensing EA and regulating the expression of the ethanolamine utilization genes. Multiple promoters participate in eut gene expression in the presence of EA as the sole carbon source and cobalamin (CoB12), an essential cofactor in the enzymatic degradation process. By means of in vivo and in vitro approaches, this study characterized the transcriptional activity identified in the eutT-eutG intergenic region of the E. faecalis eut cluster. Two novel promoters in this region were shown to be active in vivo. The distal P2-1 promoter was associated with a B12 riboswitch that terminated transcription in the presence of CoB12. Transcription elongation from the proximal P2-2 promoter was regulated by antitermination mediated by the phosphorylated form of the response regulator of TCS-17 (RR17). 3'-Rapid amplification of cDNA ends (RACE) analyses of the terminated RNA products allowed precise identification of the hairpin loop structures involved in termination/antitermination. The results uncovered the role of the B12 riboswitch and RR17 in eut gene expression, adding to the complexity of this regulatory pathway and extending the knowledge of possible means of transcription regulation in Gram-positive organisms.
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http://dx.doi.org/10.1128/JB.00217-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133151PMC
May 2011

Enterococcus faecalis virulence regulator FsrA binding to target promoters.

J Bacteriol 2011 Apr 21;193(7):1527-32. Epub 2011 Jan 21.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, Mail Code MEM-116, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The FsrABDC signal transduction system is a major virulence regulator in Enterococcus faecalis. The FsrC sensor histidine kinase, upon activation by the gelatinase biosynthesis-activating pheromone (GBAP) peptide encoded by the fsrBD genes, phosphorylates the FsrA response regulator required for the transcription of the fsrBDC and the gelE-sprE genes from the fsrB promoter and the gelE promoter, respectively. FsrA belongs to the LytTR family of proteins, which includes other virulence regulators, such as AgrA of Staphylococcus aureus, AlgR of Pseudomonas aeruginosa, and VirR of Clostridium perfringens. The LytTR DNA-binding domain that characterizes these proteins generally binds to two imperfect direct repeats separated by a number of bases that place the repeats on the same face of the DNA helix. In this study, we demonstrated that FsrA also binds to two imperfect direct repeats separated by 13 bp, based on the consensus sequence of FsrA, T/AT/CAA/GGGAA/G, which is consistent with the binding characteristics of LytTR domains.
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http://dx.doi.org/10.1128/JB.01522-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067650PMC
April 2011

Structural insights into inhibition of Bacillus anthracis sporulation by a novel class of non-heme globin sensor domains.

J Biol Chem 2011 Mar 7;286(10):8448-8458. Epub 2011 Jan 7.

From the Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037,. Electronic address:

Pathogenesis by Bacillus anthracis requires coordination between two distinct activities: plasmid-encoded virulence factor expression (which protects vegetative cells from immune surveillance during outgrowth and replication) and chromosomally encoded sporulation (required only during the final stages of infection). Sporulation is regulated by at least five sensor histidine kinases that are activated in response to various environmental cues. One of these kinases, BA2291, harbors a sensor domain that has ∼35% sequence identity with two plasmid proteins, pXO1-118 and pXO2-61. Because overexpression of pXO2-61 (or pXO1-118) inhibits sporulation of B. anthracis in a BA2291-dependent manner, and pXO2-61 expression is strongly up-regulated by the major virulence gene regulator, AtxA, it was suggested that their function is to titrate out an environmental signal that would otherwise promote untimely sporulation. To explore this hypothesis, we determined crystal structures of both plasmid-encoded proteins. We found that they adopt a dimeric globin fold but, most unusually, do not bind heme. Instead, they house a hydrophobic tunnel and hydrophilic chamber that are occupied by fatty acid, which engages a conserved arginine and chloride ion via its carboxyl head group. In vivo, these domains may therefore recognize changes in fatty acid synthesis, chloride ion concentration, and/or pH. Structure-based comparisons with BA2291 suggest that it binds ligand and dimerizes in an analogous fashion, consistent with the titration hypothesis. Analysis of newly sequenced bacterial genomes points to the existence of a much broader family of non-heme, globin-based sensor domains, with related but distinct functionalities, that may have evolved from an ancestral heme-linked globin.
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http://dx.doi.org/10.1074/jbc.M110.207126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048729PMC
March 2011

Glucose-dependent activation of Bacillus anthracis toxin gene expression and virulence requires the carbon catabolite protein CcpA.

J Bacteriol 2011 Jan 22;193(1):52-62. Epub 2010 Oct 22.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-116, La Jolla, California 92037, USA.

Sensing environmental conditions is an essential aspect of bacterial physiology and virulence. In Bacillus anthracis, the causative agent of anthrax, transcription of the two major virulence factors, toxin and capsule, is triggered by bicarbonate, a major compound in the mammalian body. Here it is shown that glucose is an additional signaling molecule recognized by B. anthracis for toxin synthesis. The presence of glucose increased the expression of the protective antigen toxin component-encoding gene (pagA) by stimulating induction of transcription of the AtxA virulence transcription factor. Induction of atxA transcription by glucose required the carbon catabolite protein CcpA via an indirect mechanism. CcpA did not bind specifically to any region of the extended atxA promoter. The virulence of a B. anthracis strain from which the ccpA gene was deleted was significantly attenuated in a mouse model of infection. The data demonstrated that glucose is an important host environment-derived signaling molecule and that CcpA is a molecular link between environmental sensing and B. anthracis pathogenesis.
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http://dx.doi.org/10.1128/JB.01656-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019961PMC
January 2011

NMR solution structure and DNA-binding model of the DNA-binding domain of competence protein A.

J Mol Biol 2010 Apr 17;398(2):248-63. Epub 2010 Mar 17.

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.

Competence protein A (ComA) is a response regulator protein involved in the development of genetic competence in the Gram-positive spore-forming bacterium Bacillus subtilis, as well as the regulation of the production of degradative enzymes and antibiotic synthesis. ComA belongs to the NarL family of proteins, which are characterized by a C-terminal transcriptional activator domain that consists of a bundle of four helices, where the second and third helices (alpha 8 and alpha 9) form a helix-turn-helix DNA-binding domain. Using NMR spectroscopy, the high-resolution 3D solution structure of the C-terminal DNA-binding domain of ComA (ComAC) has been determined. In addition, surface plasmon resonance and NMR protein-DNA titration experiments allowed for the analysis of the interaction of ComAC with its target DNA sequences. Combining the solution structure and biochemical data, a model of ComAC bound to the ComA recognition sequences on the srfA promoter has been developed. The model shows that for DNA binding, ComA uses the conserved helix-turn-helix motif present in other NarL family members. However, the model reveals also that ComA might use a slightly different part of the helix-turn-helix motif and there appears to be some associated domain re-orientation. These observations suggest a basis for DNA binding specificity within the NarL family.
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http://dx.doi.org/10.1016/j.jmb.2010.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855743PMC
April 2010

Lung epithelial injury by B. anthracis lethal toxin is caused by MKK-dependent loss of cytoskeletal integrity.

PLoS One 2009 9;4(3):e4755. Epub 2009 Mar 9.

Department of Immunology & Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn(2+)-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649448PMC
May 2009

Two small c-type cytochromes affect virulence gene expression in Bacillus anthracis.

Mol Microbiol 2009 Apr 15;72(1):109-23. Epub 2009 Feb 15.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, Division of Cellular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis. Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c-type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c-type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis, but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.
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http://dx.doi.org/10.1111/j.1365-2958.2009.06627.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829433PMC
April 2009

The bicarbonate transporter is essential for Bacillus anthracis lethality.

PLoS Pathog 2008 Nov 21;4(11):e1000210. Epub 2008 Nov 21.

Department of Molecular and Experimental Medicine, Division of Cellular Biology, The Scripps Research Institute, La Jolla, California, United States of America.

In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO2/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO2 and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO2, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO2/bicarbonate, and suggests it may be a target for antibacterial intervention.
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http://dx.doi.org/10.1371/journal.ppat.1000210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582132PMC
November 2008

A unique GTP-dependent sporulation sensor histidine kinase in Bacillus anthracis.

J Bacteriol 2009 Feb 17;191(3):687-92. Epub 2008 Oct 17.

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The Bacillus anthracis BA2291 gene codes for a sensor histidine kinase involved in the induction of sporulation. Genes for orthologs of the sensor domain of the BA2291 kinase exist in virulence plasmids in this organism, and these proteins, when expressed, inhibit sporulation by converting BA2291 to an apparent phosphatase of the sporulation phosphorelay. Evidence suggests that the sensor domains inhibit BA2291 by titrating its activating signal ligand. Studies with purified BA2291 revealed that this kinase is uniquely specific for GTP in the forward reaction and GDP in the reverse reaction. The G1 motif of BA2291 is highly modified from ATP-specific histidine kinases, and modeling this motif in the structure of the kinase catalytic domain suggested how guanine binds to the region. A mutation in the putative coiled-coil linker between the sensor domain and the catalytic domains was found to decrease the rate of the forward autophosphorylation reaction and not affect the reverse reaction from phosphorylated Spo0F. The results suggest that the activating ligand for BA2291 is a critical signal for sporulation and in a limited concentration in the cell. Decreasing the response to it either by slowing the forward reaction through mutation or by titration of the ligand by expressing the plasmid-encoded sensor domains switches BA2291 from an inducer to an inhibitor of the phosphorelay and sporulation.
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http://dx.doi.org/10.1128/JB.01184-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632060PMC
February 2009

Ethanolamine activates a sensor histidine kinase regulating its utilization in Enterococcus faecalis.

J Bacteriol 2008 Nov 5;190(21):7147-56. Epub 2008 Sep 5.

Division of Cellular Biology, Mail Code MEM-116, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Enterococcus faecalis is a gram-positive commensal bacterium of the human intestinal tract. Its opportunistic pathogenicity has been enhanced by the acquisition of multiple antibiotic resistances, making the treatment of enterococcal infections an increasingly difficult problem. The extraordinary capacity of this organism to colonize and survive in a wide variety of ecological niches is attributable, at least in part, to signal transduction pathways mediated by two-component systems (TCS). Here, the ability of E. faecalis to utilize ethanolamine as the sole carbon source is shown to be dependent upon the RR-HK17 (EF1633-EF1632) TCS. Ethanolamine is an abundant compound in the human intestine, and thus, the ability of bacteria to utilize it as a source of carbon and nitrogen may provide an advantage for survival and colonization. Growth of E. faecalis in a synthetic medium with ethanolamine was abolished in the response regulator RR17 mutant strain. Transcription of the response regulator gene was induced by the presence of ethanolamine. Ethanolamine induced a 15-fold increase in the rate of autophosphorylation in vitro of the HK17 sensor histidine kinase, indicating that this is the ligand recognized by the sensor domain of the kinase. These results assign a role to the RR-HK17 TCS as coordinator of the enterococcal response to specific nutritional conditions existing at the site of bacterial invasion, the intestinal tract of an animal host.
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http://dx.doi.org/10.1128/JB.00952-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580688PMC
November 2008

Dual promoters control expression of the Bacillus anthracis virulence factor AtxA.

J Bacteriol 2008 Oct 1;190(19):6483-92. Epub 2008 Aug 1.

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have -10 and -35 consensus sequences compatible with recognition by sigma(A)-containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the level of AtxA in the cell.
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http://dx.doi.org/10.1128/JB.00766-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566008PMC
October 2008

A sensor histidine kinase co-ordinates cell wall architecture with cell division in Bacillus subtilis.

Mol Microbiol 2008 Aug 28;69(3):621-32. Epub 2008 Jun 28.

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.

The concerted interconnection between processes driving DNA synthesis, division septum formation and cell wall synthesis and remodelling in rapidly growing bacteria requires precise co-ordination by signalling mechanisms that are, for the most part, unknown. The YycG (sensor histidine kinase)-YycF (response regulator/transcription factor) two-component system of Bacillus subtilis controls the synthesis of enzymes and their inhibitors that function in cell wall remodelling and cell separation. Here it is shown that the YycG sensor histidine kinase is a component of the division septum in growing cells. RT-PCR quantification of YycF approximately PO(4)-regulated gene transcription, in wild type and FtsZ-depleted, septum-less cells, indicated that YycG kinase activity on YycF is dependent on YycG localization to a division septum. The data support a model in which the YycG sensor kinase perceives information at the division septum and regulates the reciprocal synthesis of autolysins and autolysin inhibitors to co-ordinate growth and division with cell wall restructuring.
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http://dx.doi.org/10.1111/j.1365-2958.2008.06308.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2574549PMC
August 2008

Virulence gene expression is independent of ResDE-regulated respiration control in Bacillus anthracis.

J Bacteriol 2008 Aug 6;190(15):5522-5. Epub 2008 Jun 6.

Department of Molecular and Experimental Medicine, Division of Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

The ResDE two-component system regulates the synthesis of several components of the aerobic and anaerobic respiratory pathways in bacilli. The ResD response regulator transcription factor has been implicated in the regulation of virulence factors in a number of gram-positive species, including Bacillus anthracis. The precise deletions of resD and resE in B. anthracis that retained the classical respiratory phenotypes did not affect the expression of the gene for the protective antigen of the anthrax toxin, pagA, or that of the toxin regulator, atxA. The results indicate that the loss of ResDE-controlled respiratory capacity does not affect the synthesis of anthrax toxin.
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http://dx.doi.org/10.1128/JB.00312-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493283PMC
August 2008

Commingling regulatory systems following acquisition of virulence plasmids by Bacillus anthracis.

Trends Microbiol 2008 May 18;16(5):215-21. Epub 2008 Apr 18.

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The conversion of a bacterium from a non-pathogenic to a pathogenic existence is usually associated with the acquisition of virulence factors, the genes of which gain entry through bacteriophage infection, transposable elements or plasmid transfer. Pathogenesis research is mostly focused on how these factors enable the bacterium to infect the host or evade the repertoire of host defenses. Less effort is expended on understanding how the invading genes are affected by the complex regulatory circuits of the bacterium and how virulence is the result of converting these regulatory circuits to make them complicit with pathogenesis. An example of such a conversion is seen in Bacillus anthracis, and how acquired plasmid regulatory functions affect the activity of the regulatory processes of the bacterium, and vice versa, is now being revealed.
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http://dx.doi.org/10.1016/j.tim.2008.01.010DOI Listing
May 2008

Functional role for a conserved aspartate in the Spo0E signature motif involved in the dephosphorylation of the Bacillus subtilis sporulation regulator Spo0A.

J Biol Chem 2008 Feb 28;283(5):2962-72. Epub 2007 Nov 28.

Department of Molecular and Experimental Medicine, Division of Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T(35)IXXSQ ELDCLI(46) residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0A approximately P was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr(35) and Cys(44) did not seem to have any critical functional or structural role. We propose that Asp(43) of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226 ).
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http://dx.doi.org/10.1074/jbc.M709032200DOI Listing
February 2008

New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis.

J Microbiol Methods 2007 Dec 21;71(3):332-5. Epub 2007 Sep 21.

The Scripps Research Institute, Department of Molecular and Experimental, Medicine, Division of Cellular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States.

Two new transposon delivery vector systems utilizing Mariner and mini-Tn10 transposons have been developed for in vivo insertional mutagenesis in Bacillus anthracis and other compatible Gram-positive species. The utility of both systems was directly demonstrated through the mutagenesis of a widely used B. anthracis strain.
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http://dx.doi.org/10.1016/j.mimet.2007.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175031PMC
December 2007

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage.

J Bacteriol 2007 Dec 5;189(24):8835-43. Epub 2007 Oct 5.

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.
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http://dx.doi.org/10.1128/JB.01311-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168621PMC
December 2007

Temporal separation of distinct differentiation pathways by a dual specificity Rap-Phr system in Bacillus subtilis.

Mol Microbiol 2007 Jul;65(1):103-20

Groningen Biomolecular Sciences and Biotechnology Institute, Department of Genetics, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.

In bacterial differentiation, mechanisms have evolved to limit cells to a single developmental pathway. The establishment of genetic competence in Bacillus subtilis is controlled by a complex regulatory circuit that is highly interconnected with the developmental pathway for spore formation, and the two pathways appear to be mutually exclusive. Here we show by in vitro and in vivo analyses that a member of the Rap family of proteins, RapH, is activated directly by the late competence transcription factor ComK, and is capable of inhibiting both competence and sporulation. Importantly, RapH is the first member of the Rap family that demonstrates dual specificity, by dephosphorylating the Spo0F-P response regulator and inhibiting the DNA-binding activity of ComA. The protein thus acts at the stage where competence is well initiated, and prevents initiation of sporulation in competent cells as well as contributing to the escape from the competent state. A deletion of rapH induces both differentiation pathways and interferes with their temporal separation. Together, these results indicate that RapH is an integral part of a multifactorial regulatory circuit affecting the cell's decision between distinct developmental pathways.
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http://dx.doi.org/10.1111/j.1365-2958.2007.05776.xDOI Listing
July 2007

Opposing effects of histidine phosphorylation regulate the AtxA virulence transcription factor in Bacillus anthracis.

Mol Microbiol 2007 Feb;63(3):644-55

The Scripps Research Institute, Department of Molecular and Experimental Medicine, Division of Cellular Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Expression of genes for Bacillus anthracis toxin and capsule virulence factors are dependent upon the AtxA transcription factor. The mechanism by which AtxA regulates the transcription of its target genes is unknown. Here we report that bioinformatic analyses suggested the presence in AtxA of two PTS (phosphenolpyruvate : sugar phosphotransferase system) regulation domains (PRD) generally regulated by phosphorylation/dephosphorylation at conserved histidine residues. By means of amino acid substitutions that mimic the phosphorylated (H to D) or the unphosphorylated (H to A) state of the protein, we showed that phosphorylation of H199 of PRD1 is likely to be necessary for AtxA activation while phosphorylation of H379 in PRD2 is inhibitory to toxin gene transcription. In vivo labelling experiments with radioactive phosphate allowed us to propose that H199 and H379 are AtxA residues subject to regulated phosphorylation. In support to these notions, we also show that deletion of ptsHI, encoding the HPr intermediate and the EI enzymes of PTS, or growth in the presence of glucose affect positively and negatively, respectively, the activity of AtxA. Our results link virulence factor production in B. anthracis to carbohydrate metabolism and, for the first time, provide a mechanistic explanation for AtxA transcriptional activity.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05543.xDOI Listing
February 2007

Negative regulation of Bacillus anthracis sporulation by the Spo0E family of phosphatases.

J Bacteriol 2007 Apr 26;189(7):2637-45. Epub 2007 Jan 26.

Division of Cellular Biology, Mail Code MEM-116, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The initiation of sporulation in Bacillus species is controlled by the phosphorelay signal transduction system. Multiple regulatory elements act on the phosphorelay to modulate the level of protein phosphorylation in response to cellular, environmental, and metabolic signals. In Bacillus anthracis nine possible histidine sensor kinases can positively activate the system, while two response regulator aspartyl phosphate phosphatases of the Rap family negatively impact the pathway by dephosphorylating the Spo0F intermediate response regulator. In this study, we have characterized the B. anthracis members of the Spo0E family of phosphatases that specifically dephosphorylate the Spo0A response regulator of the phosphorelay and master regulator of sporulation. The products of four genes were able to promote the dephosphorylation of Spo0A approximately P in vitro. The overexpression of two of these B. anthracis Spo0E-like proteins from a multicopy vector consistently resulted in a sporulation-deficient phenotype. A third gene was found to be not transcribed in vivo. A fourth gene encoded a prematurely truncated protein due to a base pair deletion that nevertheless was subject to translational frameshift repair in an Escherichia coli protein expression system. A fifth Spo0E-like protein has been structurally and functionally characterized as a phosphatase of Spo0A approximately P by R. N. Grenha et al. (J. Biol. Chem. 281:37993-38003, 2006). We propose that these proteins may contribute to maintain B. anthracis in the transition phase of growth during an active infection and therefore contribute to the virulence of this organism.
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http://dx.doi.org/10.1128/JB.01798-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855805PMC
April 2007

Structural characterization of Spo0E-like protein-aspartic acid phosphatases that regulate sporulation in bacilli.

J Biol Chem 2006 Dec 25;281(49):37993-8003. Epub 2006 Sep 25.

Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom.

Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.
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http://dx.doi.org/10.1074/jbc.M607617200DOI Listing
December 2006

Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase.

J Bacteriol 2006 Sep;188(17):6354-60

Division of Cellular Biology, Mail Code MEM-116, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.
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http://dx.doi.org/10.1128/JB.00656-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1595385PMC
September 2006
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