Andrew S. Taft

Proteomic analysis of Schistosoma mansoni proteins released during in vitro miracidium-to-sporocyst transformation

Free-living miracidia of Schistosoma mansoni, upon penetration of the their snail intermediate host, undergo dramatic morphological and physiological changes as they transform to the parasitic sporocyst stage. During this transformation process, developing larvae release a diverse array of proteins, herein referred to as larval transformation proteins (LTPs), some of which are postulated to serve a parasite protective function. In the present study, nanoLC-tandem MS analysis was performed on all proteins represented in entire 1-dimensional SDS-PAGE-separated samples in order to gain a more comprehensive picture of the protein constituents associated with miracidium-to-sporocyst transformation and thus, their potential role in influencing establishment of intramolluscan infections. Of 127 proteins with sufficient peptide/sequence information, specific identifications were made for 99, while 28 represented unknown or hypothetical proteins. Nineteen percent of identified proteins possessed signal peptides constituting a cohort of classical secretory proteins, while 22% were identified as putative nonclassically secreted leaderless proteins based on SecretomeP analysis. Proteins comprising these groups consisted mainly of proteases/protease inhibitors, small HSPs, redox/antioxidant enzymes, ion-binding proteins including those with anti-oxidant Fe-binding activities (ferritins, heme-binding protein), and venom allergen-like (VAL) proteins. A polyclonal antibody generated against whole LTPs recognized proteins primarily associated with the cilia, ciliated epidermal plates and intercellular ridges of miracidia and the tegument of fully transformed sporocysts, identifying these structures as sources of a subset of LTPs. Thus lysis of plates and/or leakage during formation of the sporocyst syncytium likely represent significant contributors to the overall LTP makeup, especially identified nonsecretory proteins. However, as plate release/degradation and tegument formation are part of the normal developmental process, all LTPs regardless of tissue origin, would be expected at the parasite-host interface upon infection. This study significantly expands the repertoire of LTPs associated with larval transformation and identifies several, e.g., those involved in stress responses, proteolysis/inhibition, antioxidant and detoxication, and immune modulation, that may play a parasite protective role during this crucial period of transition.

Replication-incompetent influenza A viruses that stably express a foreign gene

A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.

Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus

Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans. Supplementary information The online version of this article (doi:10.1038/ncomms8491) contains supplementary material, which is available to authorized users.

The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with many physiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation of miracidia to the sporocyst stage. The Sigma-Aldrich Library of Pharmacologically Active Compounds (LOPAC) contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors, antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatly reduce or delay this transformation process during a primary screen of live miracidia. The majority of compounds inhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes. Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets of these compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting in increased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

CLONING AND FUNCTIONAL CHARACTERIZATION OF TWO CALMODULIN GENES DURING LARVAL DEVELOPMENT IN THE PARASITIC FLATWORM SCHISTOSOMA MANSONI

Abstract Schistosomiasis is endemic in over 70 countries, in which more than 200 million people are infected with the various schistosome species. Understanding the physiological processes underlying key developmental events could be useful in developing novel chemotherapeutic reagents or infection intervention strategies. Calmodulin is a small, calcium-sensing protein found in all eukaryotes and, although the protein has been previously identified in various Schistosoma mansoni stages and implicated in egg hatching and miracidia transformation, few molecular and functional data are available for this essential protein. Herein, we report the molecular cloning, expression, and functional characterization of calmodulin in the miracidia and primary sporocyst stages of S. mansoni. Two transcripts, SmCaM1 and SmCaM2, were cloned and sequenced, and a recombinant SmCaM1 protein was expressed in Escherichia coli and used to generate anti-CaM antibodies. The 2 protein sequences were highly conserved when compared to other model organisms. The alignment of the predicted proteins of both SmCaM1 and SmCaM2 exhibited 99% identity to each other and 97–98% identity with mammalian calmodulins. Analysis of steady-state transcript abundance indicate that the 2 calmodulin transcripts differ in their stage-associated expression patterns, although the CaM protein isotype appears to be constitutively expressed during early larval development. Application of RNAi to larval parasites results in a “stunted growth” phenotype in sporocysts with 30 and 35% reduction in transcript abundance for SmCaM1 and SmCaM2, respectively, and a corresponding 35% reduction in protein level after incubation in double-stranded RNA. Differential expression of CaM transcripts during early larval development and a growth defect-inducing effect associated with partial transcript and protein inhibition as a result of RNAi suggest a potentially important role of calmodulin during early larval development.

Selection of antigenically advanced variants of seasonal influenza viruses

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014–2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein

ABSTRACT Highly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptor-binding preference but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals. IMPORTANCE We have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in mammals.