Publications by authors named "Valentina Rippa"

12 Publications

  • Page 1 of 1

The quorum-sensing regulator ComA from Bacillus subtilis activates transcription using topologically distinct DNA motifs.

Nucleic Acids Res 2016 Mar 17;44(5):2160-72. Epub 2015 Nov 17.

Center for Molecular Biology (ZMBH) and Center for the Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant), University of Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

ComA-like transcription factors regulate the quorum response in numerous Gram-positive bacteria. ComA proteins belong to the tetrahelical helix-turn-helix superfamily of transcriptional activators, which bind as homodimers to inverted sequence repeats in the DNA. Here, we report that ComA from Bacillus subtilis recognizes a topologically distinct motif, in which the binding elements form a direct repeat. We provide in vitro and in vivo evidence that the canonical and non-canonical site play an important role in facilitating type I and type II promoter activation, respectively, by interacting with different subunits of RNA polymerase. We furthermore show that there is a variety of contexts in which the non-canonical site can occur and identify new direct target genes that are located within the integrative and conjugative element ICEBs1. We therefore suggest that ComA acts as a multifunctional transcriptional activator and provides a striking example for complexity in protein-DNA interactions that evolved in the context of quorum sensing.
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http://dx.doi.org/10.1093/nar/gkv1242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797271PMC
March 2016

Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein.

Clin Vaccine Immunol 2015 Jul 6;22(7):769-77. Epub 2015 May 6.

Novartis Vaccines Srl, a GSK Company, Siena, Italy

Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.
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http://dx.doi.org/10.1128/CVI.00794-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478525PMC
July 2015

SslE elicits functional antibodies that impair in vitro mucinase activity and in vivo colonization by both intestinal and extraintestinal Escherichia coli strains.

PLoS Pathog 2014 May 8;10(5):e1004124. Epub 2014 May 8.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

SslE, the Secreted and surface-associated lipoprotein from Escherichia coli, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of in vitro bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired E. coli mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslEIHE3034), are able to inhibit translocation of E. coli strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslEIHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in E. coli colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic E. coli species.
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http://dx.doi.org/10.1371/journal.ppat.1004124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014459PMC
May 2014

EsiB, a novel pathogenic Escherichia coli secretory immunoglobulin A-binding protein impairing neutrophil activation.

mBio 2013 Jul 23;4(4). Epub 2013 Jul 23.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Unlabelled: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site.

Importance: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.
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http://dx.doi.org/10.1128/mBio.00206-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3735183PMC
July 2013

Adaptive response of Group B streptococcus to high glucose conditions: new insights on the CovRS regulation network.

PLoS One 2013 9;8(4):e61294. Epub 2013 Apr 9.

Novartis Vaccines and Diagnostics, Siena, Italy.

Although the contribution of carbohydrate catabolism to bacterial colonization and infection is well recognized, the transcriptional changes during these processes are still unknown. In this study, we have performed comparative global gene expression analysis of GBS in sugar-free versus high glucose milieu. The analysis revealed a differential expression of genes involved in metabolism, transport and host-pathogen interaction. Many of them appeared to be among the genes previously reported to be controlled by the CovRS two-component system. Indeed, the transcription profile of a ΔcovRS strain grown in high-glucose conditions was profoundly affected. In particular, of the total genes described to be regulated by glucose, ∼27% were under CovRS control with a functional role in protein synthesis, transport, energy metabolism and regulation. Among the CovRS dependent genes, we found bibA, a recently characterized adhesin involved in bacterial serum resistance and here reported to be down-regulated by glucose. ChIP analysis revealed that in the presence of glucose, CovR binds bibA promoter in vivo, suggesting that CovR may act as a negative regulator or a repressor. We also demonstrated that, as for other target promoters, chemical phosphorylation of CovR in aspartic acid increases its affinity for the bibA promoter region. The data reported in this study contribute to the understanding of the molecular mechanisms modulating the adaptation of GBS to glucose.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061294PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621830PMC
October 2013

A novel strategy for the construction of genomic mutants of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Methods Mol Biol 2012 ;824:219-33

Department of Organic Chemistry and Biochemistry, Università degli studi di Napoli Federico II, Naples, Italy.

The sequencing and the annotation of the marine Antarctic Pseudoalteromonas haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of several genetic tools for studying it at the molecular level. To allow a deeper understanding of the PhTAC125 physiology a genetic system for the reverse genetics in this bacterium was developed. In the present work, we describe a practical technique for allelic exchange and/or gene inactivation by in-frame deletion and the use of a counterselectable marker in P. haloplanktis. The construction of suitable non-replicating plasmid and methods used to carry out a two-step integration-segregation strategy in this bacterium are reported in detail.Furthermore two examples, in which the developed methodology was applied to find out gene function or to construct genetically engineered bacterial strains, were described.
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http://dx.doi.org/10.1007/978-1-61779-433-9_11DOI Listing
April 2012

Regulated recombinant protein production in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

Methods Mol Biol 2012 ;824:203-18

Department of Organic Chemistry and Biochemistry, Università degli studi di Napoli Federico II, Naples, Italy.

This review reports results from our laboratory on the development of an effective inducible expression system for the homologous/heterologous protein production in cold-adapted bacteria. Recently, we isolated and characterized a regulative genomic region from Pseudoalteromonas haloplanktis TAC125; in particular, a two-component regulatory system was identified. It is involved in the transcriptional regulation of the gene coding for an outer membrane porin (PSHAb0363) that is strongly induced by the presence of L: -malate in the growth medium.We used the regulative region comprising the two-component system located upstream the PSHAb0363 gene to construct an inducible expression vector - named pUCRP - under the control of L: -malate. Performances of the inducible system were tested using the psychrophilic β-galactosidase from P. haloplanktis TAE79 as model enzyme to be produced. Our results demonstrate that the recombinant cold-adapted enzyme is produced in P. haloplanktis TAC125 in good yields and in a completely soluble and catalytically competent form. Moreover, an evaluation of optimal induction conditions for protein production was also carried out in two consecutive steps: (1) definition of the optimal cellular growth phase in which the gene expression has to be induced; (2) definition of the optimal inducer concentration that has to be added in the growth medium.
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http://dx.doi.org/10.1007/978-1-61779-433-9_10DOI Listing
April 2012

Preferential DNA damage prevention by the E. coli AidB gene: A new mechanism for the protection of specific genes.

DNA Repair (Amst) 2011 Sep 23;10(9):934-41. Epub 2011 Jul 23.

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA.

aidB is one of the four genes of E. coli that is induced by alkylating agents and regulated by Ada protein. Three genes (ada, alkA, and alkB) encode DNA repair proteins that remove or repair alkylated bases. However, the role of AidB remains unclear despite extensive efforts to determine its function in cells exposed to alkylating agents. The E. coli AidB protein was identified as a component of the protein complex that assembles at strong promoters. We demonstrate that AidB protein preferentially binds to UP elements, AT rich transcription enhancer sequences found upstream of many highly expressed genes, several DNA repair genes, and housekeeping genes. AidB allows efficient transcription from promoters containing an UP element upon exposure to a DNA methylating agent and protects downstream genes from DNA damage. The DNA binding domain is required to target AidB to specific genes preferentially protecting them from alkylation damage. However, deletion of AidB's DNA binding domain does not prevent its antimutagenic activity, instead this deletion appears to allow AidB to function as a cytoplasmic alkylation resistance protein. Our studies identify the role of AidB in alkylating agent exposed cells and suggest a new cellular strategy in which a subset of the genome is preferentially protected from damage by alkylating agents.
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http://dx.doi.org/10.1016/j.dnarep.2011.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162126PMC
September 2011

Specific DNA binding and regulation of its own expression by the AidB protein in Escherichia coli.

J Bacteriol 2010 Dec 1;192(23):6136-42. Epub 2010 Oct 1.

Department of Organic Chemistry and Biochemistry, University Federico II of Naples, Naples, Italy,

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB "I-III"), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB "IV"), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.
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http://dx.doi.org/10.1128/JB.00858-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981221PMC
December 2010

The ribosomal protein L2 interacts with the RNA polymerase alpha subunit and acts as a transcription modulator in Escherichia coli.

J Bacteriol 2010 Apr 22;192(7):1882-9. Epub 2010 Jan 22.

Department of Organic Chemistry and Biochemistry, School of Biotechnological Sciences, Federico II University of Naples, Naples, Italy.

Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP alpha subunit (RNAPalpha) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAPalpha protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPalpha. A tagged form of Escherichia coli RNAPalpha was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAPalpha and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a beta-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase beta-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.
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http://dx.doi.org/10.1128/JB.01503-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838061PMC
April 2010

Identification of the transcription factor responsible for L-malate-dependent regulation in the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

FEMS Microbiol Lett 2009 Jun;295(2):177-86

Department of Public Health Sciences, La Sapienza University, Rome, Italy.

Two-component systems are widespread in nature and constitute the most common mechanism of transmembrane signal transduction in bacteria. Recently, a functionally active two-component system consisting of malS and malR genes possibly involved in the expression of a C4-dicarboxylate transporter system (dctAB operon) was identified in the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. In this paper, we performed a functional analysis of the two-component system and demonstrated its involvement in the regulation of the expression of C4-dicarboxylate transporter genes. The expression of the C4-dicarboxylate transporter genes was induced by l-malate with the promoter element located upstream of the dctA gene being active only in the presence of the inducer. A sigma(54) promoter responsible for the l-malate dependent transcription regulation was identified and functionally characterized. The molecular mechanism involves an inverted repeat sequence located upstream the sigma(54) promoter that was shown to bind regulatory proteins only in the presence of l-malate. The protein factor responsible for the induction of the dctAB operon expression was eventually identified as the transcriptional regulatory protein MalR. MalR is the first transcriptional factor identified in P. haloplanktis TAC125 and one of the few transcriptional modulators reported so far in cold adapted bacteria.
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http://dx.doi.org/10.1111/j.1574-6968.2009.01589.xDOI Listing
June 2009

An effective cold inducible expression system developed in Pseudoalteromonas haloplanktis TAC125.

J Biotechnol 2007 Jan 21;127(2):199-210. Epub 2006 Jul 21.

Department of Organic Chemistry and Biochemistry, Federico II University of Naples, Napoli, Italy.

A regulative two-component system previously identified in Pseudoalteromonas haloplanktis TAC125 was used to construct a cold inducible expression system that is under the control of l-malate. Performances of the inducible system were tested for both psychrophilic and mesophilic protein production using two "difficult" proteins as control. The results obtained demonstrated that both psychrophilic beta-galactosidase and yeast alpha-glucosidase are produced in a fully soluble and catalytically competent form. Optimal conditions for protein production, including growth temperature, growth medium and l-malate concentration were also investigated. Under optimized conditions yields of 620 and 27 mg/l were obtained for beta-galactosidase and alpha-glucosidase, respectively.
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http://dx.doi.org/10.1016/j.jbiotec.2006.07.003DOI Listing
January 2007