Mara Giangrossi

Involvement of FIS in the H‐NS‐mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli

The mechanism of pathogenicity in Shigella and enteroinvasive Escherichia coli (EIEC) requires the co‐ordinated expression of several genes located on both the virulence plasmid and the chromosome. We found that cells lacking a functional FIS protein (factor for inversion stimulation) are partially impaired in expressing the virulence genes and that full expression is totally restored when Shigella wild‐type fis gene is offered in trans. We also identified virF, among the virulence genes, as a target of FIS‐mediated activation and showed that FIS binds to four specific sites in the promoter region of virF. Previous studies have demonstrated that the expression of VirF, the first positive activator of a multistep regulatory cascade, is subject to temperature‐dependent regulation by H‐NS, one of the main nucleoid‐associated proteins. We now demonstrate that two of the four FIS sites overlap one of the two H‐NS sites responsible for thermoregulation (H‐NS site I). FIS was found to exercise a direct positive transcriptional control at permissive temperature (37°C), when H‐NS fails to repress virF, as well as an indirect effect by partially counteracting H‐NS inhibition at the transition temperature (32°C). Our data indicate that FIS may be relevant for the rapid increase in virF expression after penetration of bacteria into the host.

Antagonistic Role of H-NS and GadX in the Regulation of the Glutamate Decarboxylase-dependent Acid Resistance System in Escherichia coli

One of the most efficient systems of acid resistance in Escherichia coli, the gad system, is based on the coordinated action of two isoforms of glutamate decarboxylase (GadA and GadB) and of a specific glutamate/γ-aminobutyrate antiporter (GadC). The gadA/BC genes, activated in response to acid stress and in stationary phase cells, are subjected to complex circuits of regulation involving σ70, σS, cAMP receptor protein, H-NS, EvgAS, TorRS, GadE, GadX, GadW, and YdeO. Herein, we provide evidence that the nucleoid-associated protein H-NS directly functions as repressor of gadA, one of the structural genes, and gadX, a regulatory gene encoding one of the primary activators of the gad system. Band shift and DNase I footprints reveal that H-NS indeed binds to specific sites in the promoter regions of gadA and gadX and represses the transcription of these genes both in an in vitro system and in vivo. Moreover, we show that a maltose-binding protein MalE-GadX fusion is able to stimulate the promoter activity of gadA/BC, thus indicating that GadX is by itself able to up-regulate the gad genes and that a functional competition between H-NS and GadX takes place at the gadA promoter. Altogether, our results indicate that H-NS directly inhibits gadA and gadX transcription and, by controlling the intracellular level of the activator GadX, indirectly affects the expression of the whole gad system.

Selective expression of the β-subunit of nucleoid-associated protein HU during cold shock in Escherichia coli

Expression of Escherichia coli hupA and hupB, the structural genes encoding the most abundant nucleoid‐associated proteins HUα and HUβ has been studied during cold shock. This article demonstrates that: (i) transcriptional expression of hupA is blocked following a sudden temperature downshift (from 37°C to 10°C), whereas transcription of hupB from the P2 and P3 promoters is maintained at a constitutive level and is activated de novo from the P4 promoter; (ii) all three hupB mRNAs (transcribed from the three natural promoters P2, P3 and P4) become much more stable than the single hupA transcript; and (iii) the hupB transcripts, unlike that of hupA, are efficiently translated in vivo during cold acclimation and can be actively translated in vitro at low temperature. Taken together, the results indicate that during cold shock the expression of the HUβ subunit is preferentially stimulated and that of HUα repressed, suggesting that an altered HUα to HUβ expression ratio resulting in an increase of HUα/HUβ heterodimers and/or (HUβ)2 homodimers may play an important role during cold adaptation.

Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation

Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half‐life of its transcripts causes the steady‐state level of the P2 transcript to be substantially higher than that of P1 during growth at 37°C. After cold‐shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold‐shock mRNAs in the cold. Cold‐stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold‐shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.

Sanguinarine suppresses basal-like breast cancer growth through dihydrofolate reductase inhibition

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.

Interfering with the high-affinity interaction between wheat amylase trypsin inhibitor CM3 and toll-like receptor 4: in silico and biosensor-based studies

Wheat amylase/trypsin bi-functional inhibitors (ATIs) are protein stimulators of innate immune response, with a recently established role in promoting both gastrointestinal and extra-gastrointestinal inflammatory syndromes. These proteins have been reported to trigger downstream intestinal inflammation upon activation of TLR4, a member of the Toll-like family of proteins that activates signalling pathways and induces the expression of immune and pro-inflammatory genes. In this study, we demonstrated the ability of ATI to directly interact with TLR4 with nanomolar affinity, and we kinetically and structurally characterized the interaction between these macromolecules by means of a concerted approach based on surface plasmon resonance binding analyses and computational studies. On the strength of these results, we designed an oligopeptide capable of preventing the formation of the complex between ATI and the receptor.

VirF Relieves the Transcriptional Attenuation of the Virulence Gene icsA of Shigella flexneri Affecting the icsA mRNA–RnaG Complex Formation

VirF is the master activator of virulence genes of Shigella and its expression is required for the invasion of the human intestinal mucosa by pathogenic bacteria. VirF was shown to directly activate the transcription of virB and icsA, which encode two essential proteins involved in the pathogenicity process, by binding their promoter regions. In this study, we demonstrate by band shift, enzymatic probing and cross-linking experiments that VirF, in addition to DNA, can also bind the icsA transcript and RnaG, an antisense non-coding small RNA that promotes the premature termination of icsA mRNA through a transcriptional attenuation mechanism. Furthermore, we show that VirF binds in vitro also other species of RNAs, although with lower specificity. The existence of VirF–RnaG and VirF-icsA mRNA complexes is confirmed in a pulldown assay carried out under experimental conditions that very close reproduce the in vivo conditions and that allows immobilized VirF to “fish” out RnaG and icsA mRNA from a total RNA extract. The VirF binding sites identified on both icsA mRNA and RnaG contain a 13 nucleotides stretch (5′-UUUUaGYcUuUau-3′) that is the RNA-converted consensus sequence previously proposed for the VirF–DNA interaction. Band-shift assays with a synthetic RNA molecule whose sequence perfectly matches the consensus indicate that this signature plays a key role also in the VirF–RNA interaction, in particular when exposed in a stem–loop structure. To further explore the icsA-RnaG-VirF regulatory system, we developed an in vitro test (RNA–RNA Pairing Assay) in which pairing between icsA mRNA and synthetic RNAs that reproduce the individual stem–loop motifs of RnaG, was analyzed in the presence of VirF. This assay shows that this protein can prevent the formation of the kissing complex, defined as the initial nucleation points for RNA heteroduplex formation, between RnaG and icsA mRNA. Consistently, VirF alleviates the RnaG-mediated repression of icsA transcription in vitro. Therefore VirF, by hindering the icsA transcript-RnaG interaction, exhibits an activity opposed to that usually displayed by proteins, which generally assist the RNA–RNA interaction; this quite uncommon and new function and the regulatory implications of VirF as a potential RNA-binding protein are discussed.

An Interplay among FIS, H-NS, and Guanosine Tetraphosphate Modulates Transcription of the Escherichia coli cspA Gene under Physiological Growth Conditions

CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((p)ppGpp) and in vitro transcription assays, we show that the cspA promoter is sensitive to (p)ppGpp inhibition. The (p)ppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription.

De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli

During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°→10 °C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.

Corrigendum to “Different in vivo localization of the Escherichia coli proteins CspD and CspA”: [FEMS Microbiol. Lett. 202 (2002) 171–176]

0378-1097 / 02 /