Chemical Synthesis and Immunological Evaluation of Fragments of the Multiantennary Group-Specific Polysaccharide of Group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their different capsular polysaccharides. The Group B carbohydrate (GBC) is shared by all serotypes and therefore attractive be used in a glycoconjugate vaccine. The GBC is a highly complex multiantennary structure, composed of rhamnose rich oligosaccharides interspaced with glucitol phosphates. We here report the development of a convergent approach to assemble a pentamer, octamer, and tridecamer fragment of the termini of the antennae. Phosphoramidite chemistry was used to fuse the pentamer and octamer fragments to deliver the 13-mer GBC oligosaccharide. Nuclear magnetic resonance spectroscopy of the generated fragments confirmed the structures of the naturally occurring polysaccharide. The fragments were used to generate model glycoconjugate vaccine by coupling with CRM197. Immunization of mice delivered sera that was shown to be capable of recognizing different GBS strains. The antibodies raised using the 13-mer conjugate were shown to recognize the bacteria best and the serum raised against this GBC fragment-mediated opsonophagocytic killing best, but in a capsule dependent manner. Overall, the GBC 13-mer was identified to be a highly promising antigen for incorporation into future (multicomponent) anti-GBS vaccines.Bio-organic Synthesi

Inhibition of sialidase activity and cellular invasion by the bacterial vaginosis pathogen Gardnerella vaginalis

Bacterial vaginosis is a genital tract infection, thought to be caused by transformation of a lactobacillus-rich flora to a dysbiotic microbiota enriched in mixed anaerobes. The most prominent of these is Gardnerella vaginalis (GV), an anaerobic pathogen that produces sialidase enzyme to cleave terminal sialic acid residues from human glycans. Notably, high sialidase activity is associated with preterm birth and low birthweight. We explored the potential of the sialidase inhibitor Zanamavir against GV whole cell sialidase activity using methyl-umbelliferyl neuraminic acid (MU-NANA) cleavage assays, with Zanamavir causing a 30% reduction in whole cell GV sialidase activity (p < 0.05). Furthermore, cellular invasion assays using HeLa cervical epithelial cells, infected with GV, demonstrated that Zanamivir elicited a 50% reduction in cell association and invasion (p < 0.05). Our data thus highlight that pharmacological sialidase inhibitors are able to modify BV-associated sialidase activity and influence host-pathogen interactions and may represent novel therapeutic adjuncts

Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtAΔN40) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtAΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus

The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

Playing the devil’s advocate: Should we give a second chance to mTOR inhibition in renal clear cell carcinoma? – ie strategies to revert resistance to mtor inhibitors

In the last decade, the inhibition of the mechanistic target of Rapamycin (mTOR) in renal clear cell carcinoma (RCC) has disappointed the clinician’s expectations. Many clinical trials highlighted the low efficacy and unmanageable safety profile of first-generation mTOR inhibitors (Rapalogs), thus limiting their use in the clinical practice only to those patients who already failed several therapy lines. In this review, we analyze the major resistance mechanisms that undermine the efficacy of this class of drugs. Moreover, we describe some of the possible strategies to overcome the mechanisms of resistance and their clinical experimentation, with particular focus on novel mTOR inhibitors and the combina-tions of mTOR inhibitors and other anti-cancer drugs

A LONELY GUY protein of Bordetella pertussis with unique features is related to oxidative stress

The Gram-negative bacterium B. pertussis is the causative agent of whooping cough. This infection is re-emerging and new features related to Bordetella pathogenesis and microbiology could be relevant to defeat it. Therefore, we focused our attention on BP1253, a predicted exported protein from B. pertussis erroneously classified as lysine decarboxylase. We showed that BP1253 shares the highly conserved motif PGGxGTxxE and the key catalytic amino-acid residues with newly structurally characterized “LONELY GUY” (LOG) proteins. Biochemical studies have confirmed that this protein functions as a cytokinin-activating enzyme since it cleaves the N-glycosidic linkage between the base and the ribose, leading to the formation of free bases, which are the active form of plant hormones called cytokinins. Remarkably, BP1253 selectively binds monophosphate nucleotides such as AMP, GMP and CMP, showing a wider variety in binding capacity compared to other LOGs. Cytokinin production studies performed with B. pertussis have revealed 6-O-methylguanine to be the physiological product of BP1253 in agreement with the higher activity of the enzyme towards GMP. 6-O-methylguanine is likely to be responsible for the increased sensitivity of B. pertussis to oxidative stress. Although BP1253 has a primary sequence resembling the hexameric type-II LOGs, the dimeric state and the presence of specific amino-acids suggests that BP1253 can be classified as a novel type-II LOG. The discovery of a LOG along with its product 6-O-methylguanine in the human pathogen B. pertussis may lead to the discovery of unexplored functions of LOGs, broadening their role beyond plants

Expression in T-cells of the proapoptotic protein p66SHC is controlled by promoter demethylation.

p66Shc plays a key role in oxidative stress-induced apoptosis. p66Shc gene expression is tissue-specific and controlled by promoter methylation. In T-cells p66Shc expression is induced by a variety of apoptotic stimuli. We have addressed the mechanisms regulating p66Shc expression in T-cells. We show that the increase in p66Shc protein following stimulation with a Ca2+ ionophore results from enhanced gene expression, which is primarily dependent on DNA replication-independent promoter demethylation. Our data underline the role of CpG methylation in the control of p66Shc gene expression and provide evidence that Ca2+ signaling may lead to epigenetic modifications in nondividing cells. Ó 2006 Elsevier Inc. All rights reserved

Unmet clinical questions in elderly patients with locally advanced and metastatic bladder cancer

Bladder cancer (BC) is a deadly disease with high prevalence in elderly population. Several therapeutic issues are still unsolved in the clinical management of these patients. Radical surgery with or without perioperative chemotherapy represents the best therapeutic strategy in early-stage disease even though recurrence rates are high and few therapy options are available for recurrent patients. Platin-based chemotherapy is currently the standard of care for advanced disease with a poor life expectancy of about 12 months. Novel therapeutic options, including molecular-targeted agents and immunotherapy, are under preclinical and clinical evaluation with promising results.A major issue in BC care is the management of elderly patients, a population with relevant co-morbidities, increased risk of life-threatening toxicities and currently not receiving the best therapy options. This review summarizes literature data about treatment strategies in elderly BC patients and the relevance of geriatric assessment to categorize fit patients who can receive standard therapies from unfit patients who should be treated with extreme caution