Angelo Bolchi

Differential Expression of a Metallothionein Gene during the Presymbiotic versus the Symbiotic Phase of an Arbuscular Mycorrhizal Fungus

A full-length cDNA encoding a metallothionein (MT)-like polypeptide, designated GmarMT1, was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita(BEG34). The GmarMT1 gene is composed of two exons separated by an 81-bp intron. It codes for a 65-amino acid polypeptide comprising a plant type 1 MT-like N-terminal domain and a C-terminal domain that is most closely related to an as-yet-uncharacterized fungal MT. As revealed by heterologous complementation assays in yeast,GmarMT1 encodes a functional polypeptide capable of conferring increased tolerance against Cd and Cu. TheGmarMT1 RNA is expressed in both presymbiotic spores and symbiotic mycelia, even in the absence of metal exposure, but is significantly less abundant in the latter stage. An opposite pattern was observed upon Cu exposure, which up-regulatedGmarMT1 expression in symbiotic mycelia but not in germinated spores. Together, these data provide the first evidence, to our knowledge, for the occurrence in an arbuscular mycorrhizal fungus of a structurally novel MT that is modulated in a metal and life cycle stage-dependent manner and may afford protection against heavy metals (and other types of stress) to both partners of the endomycorrhizal symbiosis.

Conformation-sensitive Antibodies against Alzheimer Amyloid-β by Immunization with a Thioredoxin-constrained B-cell Epitope Peptide

Immunotherapy against the amyloid-β (Aβ) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Aβ, and yet be capable of eliciting antibodies that recognize Aβ fibrils and neurotoxic Aβ oligomers but not the physiological monomeric species of Aβ. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Aβ1-15 (Aβ15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Aβ15)n polypeptides bearing one, four, or eight copies of Aβ15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Aβ15)4 antibody, in particular, recognized Aβ42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Aβ. We have also demonstrated that anti-Trx(Aβ15)4, which binds to human AD plaques, markedly reduces Aβ pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Aβ fragment repeat and identify Trx(Aβ15)4 as a promising new tool for AD immunotherapy.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium–host interactions

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.

Coordinate modulation of maize sulfate permease and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by L-cysteine.

To gain insight into the regulatory mechanisms and the signals responsible for the adaptation of higher plants to conditions of varying sulfate availability, we have isolated from a sulfate- deprived root library maize cDNAs encoding sulfate permease (ZmST1) and ATP sulfurylase (ZmAS1), the two earliest components of the sulfur assimilation pathway. The levels of ZmST1 and ZmAS1 transcripts concomitantly increased in both roots and shoots of seedlings grown under sulfate-deprived conditions, and rapidly decreased when the external sulfate supply was restored. This coordinate response, which was not observed under conditions of limiting nitrate or phosphate, correlated with the depletion of glutathione, rather than sulfate stores. However, drastically reducing glutathione levels through treatment with buthionine sulfoximine, a specific inhibitor of γ-glutamyl cysteine synthetase, did not provide an adequate stimulus for the up- regulation of either sulfate permease or ATP sulfurylase messengers. Indeed, L-cysteine, but not D-cysteine, effectively down-regulated both transcripts when supplied to sulfur-deficient seedlings under conditions of blocked glutathione synthesis. Altogether, these data provide evidence for the coordinate regulation of sulfur assimilation mRNAs in higher plants and for the glutathione-independent involvement of cysteine as a stereospecific pretranslational modulator of the expression of sulfur status-responsive genes.

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

A nutrient‐regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Important morphogenetic transitions in fungi are triggered by starvation‐induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up‐regulation of TbSP1, a surface‐associated, Ca2+‐dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A2 to be described in fungi and identifies a novel class of phospholipid‐hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre‐protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free‐living and symbiosis‐engaged hyphae. It thus appears that a dual localization phospholipase A2 is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1‐related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non‐filamentous microorganisms, points to a general role for TbSP1 phospholipases A2 in the organization of multicellular filamentous structures in bacteria and fungi.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein.

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified ι-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282–3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (Kd ≈ 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-Å x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

Potent anti-HPV immune responses induced by tandem repeats of the HPV16 L2 (20-38) peptide displayed on bacterial thioredoxin

The minor capsid protein L2 is a promising candidate for the construction of an anti-human papillomavirus (HPV) broadly protective vaccine for the prophylaxis of cervical cancer. However, L2-derived peptides are usually poorly immunogenic and extensive knowledge on the most relevant (cross)neutralizing epitope(s) is still needed. We systematically examined the immunogenicity and virus neutralization potential of six peptides encompassing the N-terminal (amino acids 1 -- 120) region of HPV16 L2 (20 -- 38; 28 -- 42; 56 -- 75; 64 -- 81; 96 -- 115; 108 -- 120) using bacterial thioredoxin (Trx) as a novel peptide scaffold. Mice antisera generated by 19 different Trx-L2 peptide fusions bearing one or multiple copies of each peptide were analyzed. Internal fusion to thioredoxin conferred strong immunogenicity to all the tested peptides, with a trend toward an increased immunogenicity for the multipeptide vs. the monopeptide forms of the various antigens. All Trx-L2 peptides induced HPV16 neutralizing antibodies in some of the immunized mice, but neutralization titers differed by more than two orders of magnitude. Trx-L2(20 -- 38) antisera were by far the most effective in HPV16 neutralization and did not differ significantly from those induced by a reference polypeptide covering the entire L2 (1 -- 120) region. The same antisera were also the most effective when challenged against the non-cognate HPV 18, 58, 45 and 31 pseudovirions. The data identify L2(20 -- 38) as the best (cross)neutralizing epitope among the six that were examined, and point to thioredoxin fusion derivatives of this peptide as excellent candidates for the formulation of a low-cost, broadly protective HPV vaccine.

The N-terminal region of the human papillomavirus L2 protein contains overlapping binding sites for neutralizing, cross-neutralizing and non-neutralizing antibodies.

The N-terminal region of the human papillomavirus (HPV) L2 protein has been shown to contain immune epitopes able to induce the production of neutralizing and cross-neutralizing antibodies (Gambhira et al., 2007; Kawana et al., 1999). Using bacterial thioredoxin as a scaffold, we managed to enhance the immunogenicity of putative L2 neutralizing epitopes, but only a minor fraction of the resulting immune responses was found to be neutralizing (Rubio et al., 2009). To determine the recognition patterns for non-neutralizing, neutralizing and cross-neutralizing antibodies, we isolated and characterized a panel of 46 monoclonal antibodies directed against different HPV16 L2 epitopes. Four of such antibodies proved to be neutralizing, and two of them, both targeting the amino acid (aa) 20-38 region of L2, were found to cross-neutralize a broad range of papillomaviruses. The epitopes recognized by neutralizing and cross-neutralizing antibodies were mapped at high resolution and were found to be characterized by distinct recognition patterns. Even in the case of the L2 20-38 epitope, cross-neutralization of HPV31 pseudovirions proved to be extremely inefficient, and this was found to be primarily due to the lack of a proline residue at position 30. HPV16 specific amino acids in this region also appear to be responsible for the lack of cross-neutralizing activity, thus suggesting a potential immune escape mechanism. For the aa 71-80 region, instead, the data indicate that restriction of neutralization to HPV16 is due to sequence (or structural) differences laying outside of the epitope. Besides providing new insights on the molecular bases of L2-mediated immune reactivity, the present data may pave the way to novel vaccination approaches specifically evoking cross-neutralizing antibody responses.

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells

sPLA(2)s (secretory phospholipases A(2)) belong to a broad and structurally diverse family of enzymes that hydrolyse the sn -2 ester bond of glycerophospholipids. We previously showed that a secreted fungal 15 kDa protein, named p15, as well as its orthologue from Streptomyces coelicolor (named Scp15) induce neurite outgrowth in PC12 cells at nanomolar concentrations. We report here that both p15 and Scp15 are members of a newly identified group of fungal/bacterial sPLA(2)s. The phospholipid-hydrolysing activity of p15 is absolutely required for neurite outgrowth induction. Mutants with a reduced PLA(2) activity exhibited a comparable reduction in neurite-inducing activity, and the ability to induce neurites closely matched the capacity of various p15 forms to promote fatty acid release from live PC12 cells. A structurally divergent member of the sPLA(2) family, bee venom sPLA(2), also induced neurites in a phospholipase activity-dependent manner, and the same effect was elicited by mouse group V and X sPLA(2)s, but not by group IB and IIA sPLA(2)s. Lysophosphatidylcholine, but not other lysophospholipids, nor arachidonic acid, elicited neurite outgrowth in an L-type Ca(2+) channel activity-dependent manner. In addition, p15-induced neuritogenesis was unaffected by various inhibitors that block arachidonic acid conversion into bioactive eicosanoids. Altogether, these results delineate a novel, Ca(2+)- and lysophosphatidylcholine-dependent neurotrophin-like role of sPLA(2)s in the nervous system.