Use of a nematode infection model for virulence factor screening and polymicrobial virulence testing

Introduction: Cystic Fibrosis (CF) is a debilitating genetic condition characterised by a chronic, polymicrobial biofilm infections. Pseudomonas aeruginosa is a primary pathogen in the CF lung but many other organisms are present. High throughput infection models are useful for the identification of virulence factors in a given pathogen, or for the virulence testing of polymicrobial combinations. Approach: Caenorhabditis elegans is a nematode worm that feeds on bacteria and is commonly used as an infection host. Previously, 2500 strains from a mini-Tn5-lux mutant library were screened in a nematode feeding behaviour assay to identify mutants that acted as preferred food sources. Here we tested our hypothesis that preferred food sources were less virulent. We also tested the virulence of a large panel of bacterial isolates recovered from CF patients, many of which are considered oropharyngeal (OF), non-pathogenic isolates. Results: Many of the preferred food sources had decreased virulence compared to the wild type control in the “slow killing” infection model. Preferred bacterial food sources were not due to nematode attraction or aversion of  bacterial secreted products. Many OF isolates were pathogenic to C. elegans, either individually or when in combination with P. aeruginosa. Furthermore, combinations of 2-3 isolates were often more virulent than combinations of 4-6 unique isolates. Conclusions: We developed a worm feeding behaviour and high-throughput killing assay that is amenable to testing large numbers of isolates individually and in combination. The polymicrobial infections suggest that bacteria normally considered non-pathogenic may be virulent when in combination. Future use of the nematode infection model will help examine the polymicrobial nature of many real world infections

Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition

Direct interaction with the β subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel α1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gβ1γ2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the α1 subunit, residues which are surface-exposed in the presence of the calcium channel β subunit, contribute significantly to the functional inhibition by Gβ1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gβ1γ2 subunits and with several isoforms of the auxiliary β subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel

Presynaptic calcium channels: structure, regulators, and blockers

The central and peripheral nervous systems express multiple types of ligand and voltage-gated calcium channels (VGCCs), each with specific physiological roles and pharmacological and electrophysiological properties. The members of the Ca(v)2 calcium channel family are located predominantly at presynaptic nerve terminals, where they are responsible for controlling evoked neurotransmitter release. The activity of these channels is subject to modulation by a number of different means, including alternate splicing, ancillary subunit associations, peptide and small organic blockers, G-protein-coupled receptors (GPCRs), protein kinases, synaptic proteins, and calcium-binding proteins. These multiple and complex modes of calcium channel regulation allow neurons to maintain the specific, physiological window of cytoplasmic calcium concentrations which is required for optimal neurotransmission and proper synaptic function. Moreover, these varying means of channel regulation provide insight into potential therapeutic targets for the treatment of pathological conditions that arise from disturbances in calcium channel signaling. Indeed, considerable efforts are presently underway to identify and develop specific presynaptic calcium channel blockers that can be used as analgesics

D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC

Myb overexpression synergizes with the loss of Pten and is a dependency factor and therapeutic target in T‐cell lymphoblastic leukemia

Abstract T‐lineage acute lymphoblastic leukemia (T‐ALL) is an aggressive hematological malignancy that accounts for 10%–15% of pediatric and 25% of adult ALL cases. Although the prognosis of T‐ALL has improved over time, the outcome of T‐ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, further progress in the treatment of T‐ALL requires a better understanding of its biology and the development of more effective precision oncologic therapies. The proto‐oncogene MYB is highly expressed in diverse hematologic malignancies, including T‐ALLs with genomic aberrations that further potentiate its expression and activity. Previous studies have associated MYB with a malignant role in the pathogenesis of several cancers. However, its role in the induction and maintenance of T‐ALL remains relatively poorly understood. In this study, we found that an increased copy number of MYB is associated with higher MYB expression levels, and might be associated with inferior event‐free survival of pediatric T‐ALL patients. Using our previously described conditional Myb overexpression mice, we generated two distinct MYB‐driven T‐ALL mouse models. We demonstrated that the overexpression of Myb synergizes with Pten deletion but not with the overexpression of Lmo2 to accelerate the development of T‐cell lymphoblastic leukemias. We also showed that MYB is a dependency factor in T‐ALL since RNA interference of Myb blocked cell cycle progression and induced apoptosis in both human and murine T‐ALL cell lines. Finally, we provide preclinical evidence that targeting the transcriptional activity of MYB can be a useful therapeutic strategy for the treatment of T‐ALL