RNAi-based discovery and validation of new drug targets in filarial nematodes

Human filarial nematodes cause river blindness and lymphatic filariasis, both of which are diseases that produce considerable morbidity. Control of these diseases relies on drug treatments that are ineffective against macrofilariae and are threatened by the development of resistance. New validated drug targets are required to allow development of new classes of antifilarial drugs. To identify and validate potential new drug targets, we propose a collaborative research strategy utilizing bioinformatic filters and assessment of gene function by RNA interference in Caenorhabditis elegans and Brugia malayi.     

Genomic filtering: an approach to discovering novel antiparasitics

Genomic filtering is a rapid approach to identifying and prioritizing molecular targets for drug discovery. For infectious disease applications, comparative genomics filters allow the selection of pathogen-specific gene products, whereas functional genomics filters, such as RNA interference (RNAi), allow the selection of gene products essential for pathogen survival. The approach is especially applicable to antiparasitic drug discovery where the phylogenetic distance between parasite and host make the likelihood of drug cross-toxicity due to conservation of molecular targets greater than for more distantly related pathogens such as prokaryotes. This article discusses some of the inherent challenges of applying genomics to the early steps of drug discovery and describes one successful comparative and functional genomics filtering strategy that has been implemented to prioritize molecular targets and identify chemical leads for nematode control.     

Role of skeletal muscle Na+-K+ ATPase activity in increased lactate production in sub-acute sepsis

Bacterial sepsis is frequently accompanied by increased blood concentration of lactic acid, which traditionally is attributed to poor tissue perfusion, hypoxia and anaerobic glycolysis. Therapy aimed at improving oxygen delivery to tissues often does not correct the hyperlactatemia, suggesting that high blood lactate in sepsis is not due to hypoxia. Various tissues, including skeletal muscle, demonstrate increased lactate production under well-oxygenated conditions when the activity of the Na+-K+ ATPase is stimulated. Although both muscle Na+-K+ ATPase activity and muscle plasma membrane content of Na+, K+-ATPase subunits are increased in sepsis, no studies in vivo have demonstrated correlation between lactate production and changes in intracellular Na+ and K+ resulting from increased Na+-K+ pump activity in sepsis. Plasma concentrations of lactate and epinephrine, a known stimulator of the Na+-K+ pump, were increased in rats made septic by E. coli injection. Muscle lactate content was significantly increased in septic rats, although muscle ATP and phosphocreatine remained normal, suggesting oxygen delivery remained adequate for mitochondrial energy metabolism. In septic rats, muscle intracellular ratio of Na+:K+ was significantly reduced, indicating increased Na+-K+ pump activity. These data thus demonstrate that increased muscle lactate during sepsis correlates with evidence of elevated muscle Na+-K+ ATPase activity, but not with evidence of impaired oxidative metabolism. This study also further supports a role for epinephrine in this process.     

Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24–144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3′- or 5′-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.     

Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca2(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca2(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca2(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca2(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca2(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.     

Regulatory T cells and control of the germinal centre response

Germinal centres (GCs) are specialised lymphoid microenvironments that form in secondary B-cell follicles upon exposure to T-dependent antigens. In the GC, clonal expansion, selection and differentiation of GC B cells result in the production of high-affinity plasma cells and memory B cells that provide protection against subsequent infection. The GC is carefully regulated to fulfil its critical role in defence against infection and to ensure that immunological tolerance is not broken in the process. The GC response can be controlled by a number of mechanisms, one of which is by forkhead box p3 expressing regulatory T (Treg) cells, a suppressive population of CD4⁺ T cells. A specialised subset of Treg cells – follicular regulatory T (Tfr) cells – form after immunisation and are able to access the GC, where they control the size and output of the response. Our knowledge of Treg cell control of the GC is expanding. In this review we will discuss recent advances in the field, with a particular emphasis on the differentiation and function of Tfr cells in the GC.     

A White Paper on Nematode Comparative Genomics

In response to the new opportunities for genome sequencing and comparative genomics, the Society of Nematology (SON) formed a committee to develop a white paper in support of the broad scientific needs associated with this phylum and interests of SON members. Although genome sequencing is expensive, the data generated are unique in biological systems in that genomes have the potential to be complete (every base of the genome can be accounted for), accurate (the data are digital and not subject to stochastic variation), and permanent (once obtained, the genome of a species does not need to be experimentally re-sampled). The availability of complete, accurate, and permanent genome sequences from diverse nematode species will underpin future studies into the biology and evolution of this phylum and the ecological associations (particularly parasitic) nematodes have with other organisms. We anticipate that upwards of 100 nematode genomes will be solved to varying levels of completion in the coming decade and suggest biological and practical considerations to guide the selection of the most informative taxa for sequencing.