Search

The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.

Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins.

We have developed a system in which a foreign antigen replaces nearly all of the surface-exposed region of the fibrillar M protein from Streptococcus pyogenes and is fused to the C-terminal attachment motif of the M molecule. The fusion protein is thus expressed on the surface of Streptococcus gordonii, a commensal organism of the oral cavity. The antigen chosen to be expressed within the context of the M6 molecule was the E7 protein (98 amino acids) of human papillomavirus type 16. Stable recombinant streptococci were obtained by integrating genetic constructs into the chromosome, exploiting in vivo homologous recombination. The M6-E7 fusion protein expressed on the S. gordonii surface was shown to be immunogenic in mice. This is the first step in the construction of recombinant live vaccines in which nonpathogenic streptococci as well as other gram-positive bacteria may be used as vectors to deliver heterologous antigens to the immune system.

To understand how Mycobacterium tuberculosissurvives and grows in an infected host, we are studying the mycobacterial transcriptional machinery and its response to stresses encountered in vitro and in vivo. Much has been learned about σ factors and other transcriptional regulators concerning their roles in controlling mycobacterial gene expression. It has recently been shown that σAis the essential housekeeping σ factor and the alternative σ factor σB, not essential for growth in a laboratory setting, is required for a robust protective response to various environmental stresses. We are also studying the mechanism by which the R522H mutation in σAprevents the transcription of certain genes, including some that are believed necessary for virulence. Also under investigation is the mycobacterial iron acquisition apparatus and its regulation, as metabolism of this essential element plays a key role in microbial pathogenesis. We have identified and characterized the major mycobacterial iron regulator IdeR that blocks the synthesis of the iron uptake machinery and have identified target genes in M. smegmatisand M. tuberculosisthat are directly repressed by IdeR. Recent studies have examined the control of M. tuberculosisgene expression in vivo. Among these new approaches are an in vivo expression technology system to identify M. tuberculosisgenes that are induced in macrophages and mice and a novel RT-PCR method that allows an accurate comparison between the levels of specific mRNAs in M. tuberculosisgrown in vitro with those found in bacteria growing in human macrophages.

Preventive archaeology aims at assessing the archaeological interest of a given area, as well as discovering, interpreting, documenting, and protecting archaeological remains that might otherwise be destroyed by land development works. On request of private stakeholders or institutional bodies, archaeologists intervene on a given construction yard (of infrastructure or urbanisation works) in order to investigate the possible archaeological significance of zones subjected to excavation works. In many cases archaeological assays are carried out, which are associated with thorough stratigraphic documentations and definition of the final preservation conditions. The latter can involve suitable re-burying or musealization of some of the archaeological remains recovered. During these activities, the construction works are slowed down or completely interrupted up until the conclusion of the mentioned archaeological research, which can generate possible delays with respect to the planned time schedule and increase of the costs. Improvements are needed in order to minimise the duration of the archaeological assessment and, simultaneously, to guarantee quality standards even higher than tose stated by the competent superintendences. Within this framework, the project ARCHEO 3.0 "Integration of Key Enabling Technologies for rising the efficiency and quality of preventive archaeological excavations" (co-funded by Tuscany Region, POR-CReO 2014-2020) aims at speeding up the archaeological excavation and at improving its documentation. It foresees the development of a set of characterisation tools, their integration and validation in archaeological yards. Thanks to ICT solution such a multidisciplinary integration will be made possible by a dedicated web-based platform that will allow to manage 2D and 3D photogrammetry along with the data provided by electromagnetic, and acoustic sensors. The project aim at promoting a substantial step ahead of the preventive archaeology in order to overcome the traditional empiric approach. In this respect, data exchange and the ICT solutions for direct collection of information on archaeological evidences represent the major challenge. Here, we preliminarily focus on the general features of management protocols of the archaeometrical data, as well as on their suitable integration within the operative phases of the archaeological excavation.