Nonpermissive Host Research Articles

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion.

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet

Plasmid pQBR55 (∼149 kb) represents one of five (Groups I–V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB–traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.

Two dnaB genes are associated with the origin of replication of pQBR55, an exogenously isolated plasmid from the rhizosphere of sugar beet

Plasmid pQBR55 ( approximately 149 kb) represents one of five (Groups I-V) genetically distinct transfer proficient, mercury resistance plasmid groups that have been observed in the phytosphere pseudomonad community at a single geographic location in Oxford, UK. A 4.9-kb HindIII fragment was cloned from pQBR55 (a Group III plasmid) that facilitates autonomous replication of a narrow host range cloning vector, pKIL29, in the non-permissive host Pseudomonas putida UWC1. Sequencing revealed that the fragment contains a unique tandem array of dnaB genes (one partial and one complete), a homologue of the traA gene of plasmid RP4 and several potential open reading frames with little homology to any known sequences. The fragment also contains: a short region of direct and indirect repeats, two sequences that resemble the consensus Escherichia coli DnaA box motif, an A+T rich region and evidence of a GC-skew inversion. All features associated with origins of replication. Two specific oligonucleotide primer pairs, one targeted at the tandem dnaB genes and the other at the dnaB-traA arrangement were used for PCR analysis of other plasmids isolated from the same field site. PCR generated amplification products were only amplified from Group III plasmids (defined by RFLP typing) but not from plasmids belonging to Group I, II, IV or V. The nucleotide sequences of the amplified fragments were identical to those of pQBR55, even though the other Group III plasmids had distinct RFLP patterns. The results of a more general PCR screen, using primers to amplify a 389-bp fragment of all described pseudomonad dnaB genes, suggests that closely related dnaB sequences are only associated with Group III plasmids.

Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity.

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Insect lipophorin and vertebrate lipoproteins support larval development of the endoparasitoid Venturia canescens (Hymenoptera: Ichneumonidae) as dietary lipid sources.

We previously reported that lipophorin, an insect lipoprotein, is essential for in vitro development of the larval endoparasitoid Venturia canescens from the 1st larval instar to the 2nd instar. In this study, we compared the effect of various lipoproteins from insects and vertebrates to clarify whether the growth promotion effect of lipophorin is caused by its lipid moiety. As well as the permissive host (the greater wax moth) lipophorin, lipoproteins from a nonpermissive host (the silkworm) and vertebrates (chicken and human) also promoted V. canescens development to the 2nd larval instar, suggesting that essential factors for the first larval ecdysis are ubiquitous in a wide range of animals. When total lipids from the permissive host were emulsified in a lipoprotein-free medium with multilaminar vesicles of phosphatidylcholine (liposome), V. canescens larvae underwent ecdysis in the medium. In contrast, lipophorin lipid-depleted by lipase treatment promoted larval growth less than intact lipophorin. These results indicated that the growth-promotion effect of lipophorin is mainly due to the lipid moiety of lipophorin.

Survival and differential development of Entomophaga maimaiga and Entomophaga aulicae (Zygomycetes: Entomophthorales) in Lymantria dispar hemolymph.

The closely related entomophthoralean fungi Entomophaga aulicae and E. maimaiga are both host-specific pathogens of lepidopteran larvae. However, these fungi do not have the same host range. The first objective of this study was to compare the fate of E. aulicae in the nonpermissive host Lymantria dispar with the fate of the successful pathogen E. maimaiga over the same time period. In the hemolymph of L. dispar injected with E. maimaiga protoplasts, the number of hemocytes demonstrated a decreasing trend after the first day postinjection and hemocytes completely disappeared by day 5, with the majority of larvae dying in 5.6 +/- 0.1 days. In L. dispar larvae, E. maimaiga infections developed successfully, evidenced by increasing numbers of protoplasts and hyphal bodies prior to host mortality. In contrast, at day 5 hemocytes were readily visible in hemolymph of E. aulicae-injected larvae, but E. aulicae cells did not increase in numbers, although persisting in the hemolymph for at least 16 days postinjection. For both fungal species, when hemolymph samples from injected insects were introduced to culture media viable fungal cultures were always produced. Both E. aulicae and E. maimaiga occurred in hemolymph initially after injection as protoplasts. For E. maimaiga, after day 3, <50% of fungal cells were hyphal bodies until insect death when most cells regenerated cell walls. For E. aulicae, from day 2 equal numbers of fungal cells in the hemolymph occurred as protoplasts and hyphal bodies. To investigate the cause of fungistasis in E. aulicae-injected larvae, E. aulicae cell cultures exposed to partially purified protein fractions from hemolymph of larvae infected with either fungus displayed increased lysis and decreased viability at lower concentrations of protein fractions compared with E. maimaiga cell cultures. These studies demonstrate that E. aulicae does not increase in L. dispar hemolymph, although it persists and results suggest that proteinaceous factors induced within the hemolymph may limit the capacity of E. aulicae to develop successful infections.

Cytotoxic T lymphocytes in SV40 infections.

Simian virus 40 (SV40) infects monkeys and persists in the latent form in the kidneys of this natural host. However, in nonpermissive hosts, such as rodents, the virus induces neoplasia and the outcome is controlled by the immune response of the host (1,2). Virus-neutralizing antibodies are responsible for limiting the amount of circulating virus in the natural host, leading to the establishment of latency. In contrast, the nonpermissive host undergoing tumorigenesis develops antibodies specific for the large tumor antigen, or T antigen (T-ag) encoded by SV40. In addition, the T-ag in transformed or tumor cells also serves as the target for cytotoxic T lymphocytes (CTL) that are capable of controlling tumor development (2). CTL responses directed against SV40 virion proteins have not been described.

Generation of recombinant parapoxviruses: non-essential genes suitable for insertion and expression of foreign genes

Orf virus (OV) is an epitheliotropic poxvirus and belongs to the genus Parapoxvirus (PPV). PPV, especially OV, is regarded as a promising candidate for an expression vector. Among available live vaccines only strain D1701 represents a highly attenuated OV strain with clearly reduced pathogenicity. Therefore, we started to identify potentially non-essential genes or regions of D1701, which might be suitable for insertion and expression of foreign genes. The present contribution reviews some of the progress using the vegf-e (homologue of the mammalian vascular endothelial growth factor) gene locus for the generation of recombinant D1701. The vegf-e gene of D1701 is dispensable for virus growth in vitro and in vivo, and represents a major virulence determinant of OV. It is shown that foreign genes can be inserted and functionally expressed in the vegf-e locus, also leading to the induction of a specific immune response in the non-permissive host. Furthermore, it is reported that adaptation to VERO cells led to the deletion of three further regions of the OV D1701 genome, which seems to be combined with additional virus attenuation in sheep. Molecular analysis of this OV D1701 variant allows the identification of new, potentially non-essential sites in the viral genome.

Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication.

The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.

Relationships between polydnavirus gene expression and host range of the parasitoid wasp Campoletis sonorensis

To evaluate the relationship between immune suppression and host range six lepidopteran species were parasitized by the ichneumonid parasitoid Campoletis sonorensis. Parasitism inhibited the growth of permissive hosts ( Heliothis virescens, Helicoverpa zea, and Trichoplusia ni), whereas growth of semi-permissive ( Spodoptera exigua, Agrotis ipsilon) and non-permissive hosts ( Manduca sexta) was not significantly affected. The 29–36 kDa ovarian protein (OP), responsible for transient immunosuppression in the permissive host H. virescens, bound to and was endocytosed by hemocytes of permissive and non-permissive hosts. Expression of the cysteine-rich polydnavirus gene, VHv1.4, was detected in all the hosts, but declined only in semi- and non-permissive hosts at later times after parasitization. The VHv1.4 protein bound to hemocytes of permissive and semi-permissive hosts, but did not bind to hemocytes of the non-permissive host, M. sexta. Melanization of larval hemolymph was severely inhibited by parasitism in permissive hosts, but was unaffected in M. sexta. In the semi-permissive host, A. ipsilon, hemolymph melanization was transiently inhibited while viral genes were expressed. In conclusion, C. sonorensis OP transiently inhibits encapsulation in all hosts that were tested. The host range of C. sonorensis seems to be determined by whether or not the C. sonorensis ichnovirus (CsIV) is able to establish persistent infections of parasitized larvae to provide long-term suppression of host immunity.

Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length.

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

Parapoxviruses: potential alternative vectors for directing the immune response in permissive and non-permissive hosts

Parapoxvirus (PPV) represents a genus of the poxviridae, and particularly PPV ovis (Orf virus, OV) seems to offer several potential advantages for the use of vector vaccine. Therefore, we started to investigate the genome of the highly attenuated OV strain D1701, which was only poorly characterised until now. Due to recombination of non-homologous sequences, part of the right hand end of the D1701 genome was duplicated and translocated to the opposite end of the genome. As a consequence gene deletion had occurred and the inverted terminal repeat region is increased. Results are described to identify viral genes, which are non-essential for virus replication and potentially influence viral pathogenesis, virulence, and host immunity. In more detail, we analysed the expression and functional activity of the OV-specific vascular endothelial growth factor (VEGF) gene homologue. Finally the construction and production of a D1701 mutant lacking the VEGF gene homologue is reported.

A Nonpermissive Entomophthoralean Fungal Infection Increases Activation of Insect Prophenoloxidase

Entomophaga maimaigaandEntomophaga aulicaeare entomopathogenic fungi that show species-specific infection in Lepidoptera. These fungi grow as protoplasts in the hemolymph of permissive insect hosts.E. maimaigainfects gypsy moth larvae,Lymantria dispar,andE. aulicaeinfects hemlock looper,Lambdina fiscellaria.Cross-infections do not occur and are referred to as a nonpermissive response. We circumvented cuticular barriers and injectedE. aulicaeprotoplasts intoL. disparand investigated factors which could potentially curtail entomophthoralean fungal infection in a nonpermissive host insect. There was no evidence of (i) restricted fungal growth in cell-free hemolymph of the nonpermissive host, (ii) fungal toxins specific to host insect, (iii) hemocyte encapsulation of fungal protoplasts in a nonpermissive or permissive infection, or (iv) fungal-specific induction of plasma proteins inL. dispar.However, higher levels of phenoloxidase activity for up to 96 h postchallenge, as well as a prophenoloxidase-activating trypsin activity, were observed forL. disparchallenged withE. aulicaewhen compared to anE. maimaigachallenge. Three isoforms of phenoloxidase (pI5.0–5.5) and at least six isoforms of trypsin activity (four basic trypsins pI8–10 and two acidic trypsins pI4–6) with preferences for small amino acid residues were activated inL. disparafter challenge.In vitroprophenoloxidase activation experiments showed that treatment ofL. disparhemolymph withE. aulicaeprotoplast plasma membranes consistently resulted in higher prophenoloxidase activation thanE. maimaiga.We suggest that differences in protoplastic components ofEntomophagaspecies, such as the surface glycoproteins, are implicated in activation of zymogenic trypsins in the insect which in turn activate the prophenoloxidase cascade as a nonpermissive response.

Eosinophil activation, apoptosis and pathogeny in angiostrongylus cantonensis infection in the mouse

Human cerebral angiostrongyliasis becomes an emerging disease in many parts of the world. By postmortem examination, Angiostrongylus cantonensis have been reported to cause severe pathological changes in the central nervous system. The present study was designed to determine the temporal-spatial pathological changes through experimental infections and histopathological examination of permissive (SD rats) and non-permissive (ICR mice) hosts. After infecting SD rats with 25, 50, or 100 third-stage larvae (L3) and ICR mice with 25 L3, one animal from each group was sacrificed daily from day 1 to day 30 post-infection. Each rat brain was cut into six sections and mouse brain into five sections. These sections were stained with haematoxylin and eosin and examined microscopically. Eosinophilic meningitis was found to be the most commonly pathological change and occurred on day 17 post-infection in rats with 25 L3, day 9 in the 50- or 100-L3 groups, and day 12 in infected mice. Thickness of the meninges increased 9–24 folds in infected rats and 89 folds in an infected mouse on day 28. Encephalitis, congestion, perivascular cuffing, and haemorrhage were revealed in infected mice and rats with 100 L3. Fifth-stage larvae were frequently observed in the meninges but occasionally in the parenchyma. Significant correlations between meningitis and presence of larvae in the meninges were found in the three infected rat groups but not in the infected mice. The results indicate that the clinical course of A. cantonensis infection is not self-limited but becomes more severe with the intensity of infection.

Involvement of Other Bacteriophage T4 Genes in the Blockade of Protein Synthesis and mRNA Destabilization by a Mutation of Gene61.5

Gene61.5of bacteriophage T4 is required for gene expression at late stages of infection; a mutant of gene61.5shows a reduced rate of synthesis of late proteins and accumulates short transcripts upon infection of nonpermissive host cells at 30°C (T. Kai, H. E. Sellick, and T. Yonesaki,Genetics144, 7–14, 1996). Here we describe that, although the defects are only apparent at late stages, gene61.5is expressed early after infection and that the requirement of a functional gene61.5is partially compensated by the passage of early and middle stages of infection at high temperatures, suggesting that T4 genes expressed at early and middle stages bypass the requirement for the gene61.5at high temperatures. Furthermore, we isolated five pseudorevertants of a gene61.5mutant, designated asssf1throughssf5,which partially restored the growth ability at low temperatures, stabilized mRNAs, and stimulated protein synthesis at late stages. These results strongly suggest that the products of other T4 genes interact with the product of gene61.5in stabilizing mRNAs late in T4 infection.

Comparison of the hemolymph proteins in permissive and non-permissive hosts of Euplectrus comstockii

Parasitism by Euplectrus comstockii arrested larval–larval molting in both permissive (e.g. Helicoverpa zea) and non-permissive hosts (e.g. Diatraea grandiosella, Anagasta kuehniella and Ostrinia nubilalis). Parasitized larvae of both permissive and non-permissive hosts manifested a decrease in total weight gain. However, significant alterations of the putative storage proteins occurred only in the permissive host. Therefore, the venom of Euplectrus causes some, but not all, of the same effects in non-permissive hosts as in permissive hosts. Specifically, the ability of the venom to regulate molting was shown to be distinguishable from the ability of the venom to alter the protein titer in the hemolymph of a host. The fact that parasitoid eggs did not hatch on non-permissive hosts indicated that parasitoid development was impacted prior to feeding on the host and the presence of indigenous storage proteins in the non-permissive hosts supports the concept that storage proteins may not be for the direct use by the parasitoid.

Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.

Molecular Studies on Bromovirus Capsid Protein: I. Characterization of Cell-to-Cell Movement-Defective RNA3 Variants of Brome Mosaic Virus

To evaluate the extent to which brome mosaic virus (BMV) coat protein (CP) gene is involved in the process of cell-to-cell movement,Chenopodium quinoaplants were coinoculated with BMV wild-type RNAs 1 and 2 and a RNA3 variant containing either the β-glucuronidase (GUS) gene in place of CP (B3/CP-GUS), to be subjected to GUS analysis, or a deletion in the CP gene to be analyzed by fluorescencein situhybridization (FISH). Irrespective of time postinoculation, GUS expressed from the subgenomic mRNA of B3/CP-GUS was restricted to initially infected, single epidermal cells. The defective cell-to-cell movement exhibited by B3/CP-GUS was not complementedin transwhen transcripts of B3ΔMP, a RNA3 variant capable of synthesizing functional wt CP but not movement protein (MP), were added to the inoculum. Application of FISH, a technique versatile in discriminating subliminal infections from efficient cell-to-cell spread in nonpermissive and permissive hosts, respectively, to leaves inoculated with BMV RNA3 variants defective in CP synthesis, confirmed that the resulting infections were subliminal. These data provide direct evidence for the requirement of an encapsidation-competent CP to be expressed in conjunction with a functional MP for efficient cell-to-cell spread of BMV.

The role of eosinophils in Angiostrongylus cantonensis infection.

Angiostrongylus cantonensis is the causative agent of human eosinophilic meningoencephalitis in the Pacific Islands and Southeast Asia. Prominent eosinophilia in the cerebrospinal fluid (CSF) of the patients has been used as one of the diagnostic criteria for the disease but the role(s) of the CSF eosinophils has remained to be elucidated. In this article, Kentaro Yoshimura, Hiroko Sugaya and Kazuto Ishido discuss the involvement of CSF eosinophils in the killing of intracranial worms and the damage of the central nervous system of the hosts, and consider why eosinophils in A. cantonensis infection play a more important role in nonpermissive hosts (including humans) than in the permissive rat host.

Persistent Infection of Human Erythroblastoid Cells by Poliovirus

Human erythroblastoid K562 cells have been recently described as a relatively nonpermissive host for poliovirus replication. During investigations of virus-induced cytopathic effects in this cell line, we discovered that poliovirus easily established persistently infected cultures in K562-Mu cells. In these cultures, most cells remained viable, with overall viability continuously maintained between 67 and 92% over 3 months. Infected K562 cells continued to grow, usually without any major periods of crisis in the culture or large diminutions in cell growth rate. K562-Mu cells produced a slower onset of virus production than observed in HeLa cells, and virus titers in culture supernates rapidly stabilized at levels between 105 and 105 PFU/ml. In infectious center or limiting dilution assays, only about 10% of K562 cells produced infectious virus after 2 days. However, when assays were extended to 3 to 5 days, most K562 cells in the culture scored as infectious centers, suggesting productive infection of all cells in the culture with delayed kinetics of virus production. Cultures of infected K562 cells could not be cured of virus by prolonged incubation with high-titer neutralizing antibody. Pulse-label SDS-PAGE analysis of infected cultures detected moderate levels of virus protein synthesis which peaked at 9-12 hr postinfection; however, little or no shutoff of host protein synthesis was observed at any time point during infection. Immunoblot analysis with antisera to the p220 subunit of eIF-4F demonstrated extensive but incomplete cleavage of p220 in infected K562 cells at times which correlated with peak viral protein synthesis. Taken together, the results demonstrate a persistent infection in which host cell shutoff does not occur despite significant viral protein synthesis and extensive early degradation of p220.