Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

Cell death mechanisms triggered by monoclonal antibodies against CD99 in Ewing sarcoma: Cross-talk between MDM2, IGF-1R and Ras/MAPK

CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Involvement of microRNAs in Androgen Receptor-dependent Breast Cancers

Triple negative breast cancer (TNBC) is a very aggressive tumor subtype characterized by the lack of expression of estrogen receptor 1 (ESR1), due in the most of cases to an increased expression of DNA methyltransferases (DNMTs) and hypermethylation in CpG islands, resulting in gene silencing. Furthermore, in ESR1- negative breast cancers, androgen receptor (AR) is highly expressed and some studies suggest that it can drive tumor progression and might represent a therapeutic target. A correlation between microRNAs, small non-coding RNAs that regulate gene expression, and DNMTs was investigated in a TNBC cell line to restore a normal methylation pattern of ESR1, leading to its re-expression and conferring again sensitivity to selective estrogen receptor modulators (SERMs). miR-148A and miR-29B were found to be involved in the reduction of the expression of DNMT1 and DNMT3A and in a slight increase of ESR1 expression, but not at protein level. Then, we found a down-regulation of AR by miRs-7, -9, -27a, -27b, -29a, -29b, -29c, -127-3p, -127-5p and -376 at 48h post transfection and an up-regulation by miR-15a and miR-16 at every time considered. We concomitantly investigated a possible increase of Tamoxifen, Herceptin and Metformin sensitivity after AR silencing in MDA-MB 453 and T-47D cell lines. Cells seemed more sensitive when silenced for AR only in MDA-MB-453 at 24h post Tamoxifen treatment. Studies on Metformin have basically confirmed an increase of drug sensitivity due to AR silencing in both cell lines. Analysis of Herceptin showed how MDA-MB 453 samples silenced for AR have a slight decrease in the percentage of proliferating cells, demonstrating a possible increase in the response to treatment. These preliminary data provide the basis for further study of the modulation of the expression of AR by microRNAs and it will be interesting to understand the molecular mechanisms underlying these interactions.

Role of Reactive Oxygen Species in signalling and oxidative stress

Results reported in this Thesis contribute to the comprehension of the complicated world of “redox biology”. ROS regulate signalling pathways both in physiological responses and in pathogenesis and progression of diseases. In cancer cells, the increase in ROS generation from metabolic abnormalities and oncogenic signalling may trigger a redox adaptation response, leading to an up-regulation of antioxidant capacity in order to maintain the ROS level below the toxic threshold. Thus, cancer cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS-generating agents or compounds that inhibit the antioxidant system. Results here reported indicate that the development of new drugs targeting specific Nox isoforms, responsible for intracellular ROS generation, or AQP isoforms, involved in the transport of extracellular H2O2 toward intracellular targets, might be an interesting novel anti-leukaemia strategy. Furthermore, also the use of CSD peptide, which simulate the VEGFR-2 segregation into caveolae in the inactive form, might be a strategy to stop the cellular response to VEGF signalling. As above stated, in the understanding of the redox biology, it is also important to identify and distinguish the molecular effectors that maintain normal biological and physiological responses, such as agents that stimulate our adaptation systems and elevate our endogenous antioxidant defences or other protective systems. Data here reported indicate that the nutraceutical compound sulforaphane and the Klotho protein are able to stimulate the HO-1 and Prx-1 expression, as well as the GSH levels, confirming their antioxidant and protective role. Finally, results here reported demonstrated that Stevia extracts are involved in insulin regulated glucose metabolism, suggesting that the use of these compounds goes beyond their sweetening power and may also offer therapeutic benefits hence improving the quality of life.

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management Bull’s eye rot (BER) caused by Neofabraea alba is one of the most frequent and damaging latent infection occurring in stored pome fruits worldwide. Fruit infection occurs in the orchard, but disease symptoms appear only 3 months after harvest, during refrigerated storage. In Italy BER is particularly serious for late harvest apple cultivar as ‘Pink Lady™’. The purposes of this thesis were: i) Evaluate the influence of ‘Pink Lady™’ apple primary metabolites in N. alba quiescence ii) Evaluate the influence of pH in five different apple cultivars on BER susceptibility iii) To find out not chemical method to control N. alba infection iv) Identify some fungal volatile compounds in order to use them as N. alba infections markers. Results regarding the role of primary metabolites showed that chlorogenic, quinic and malic acid inhibit N. alba development. The study based on the evaluation of cultivar susceptibility, showed that Granny Smith was the most resistant apple cultivar among the varieties analyzed. Moreover, Granny Smith showed the lowest pH value from harvest until the end of storage, supporting the thesis that ambient pH could be involved in the interaction between N. alba and apple. In order to find out new technologies able to improve lenticel rot management, the application of a non-destructive device for the determination of chlorophyll content was applied. Results showed that fruit with higher chlorophyll content are less susceptible to BER, and molecular analyses comforted this result. Fruits with higher chlorophyll content showed up-regulation of PGIP and HCT, genes involved in plant defence. Through the application of PTR-MS and SPME GC-MS, 25 volatile organic compounds emitted by N. alba were identified. Among them, 16 molecules were identified as potential biomarkers.