The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding beta-subunit (CD18) of beta2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.

The EG95 antigen of Echinococcus spp. contains positively selected amino acids, which may influence host specificity and vaccine efficacy

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.

Are cryptic host species also cryptic to parasites? Host specificity and geographical distribution of acanthocephalan parasites infecting freshwater Gammarus

Many parasites infect multiple host species. In coevolving host–parasite interactions, theory predicts that parasites should be adapted to locally common hosts, which could lead to regional shifts in host preferences. We studied the interaction between freshwater Gammarus (Crustacea, Amphipoda) and their acanthocephalan parasites using a large-scale field survey and experiments, combined with molecular identification of cryptic host and parasite species. Gammarus pulex is a common host for multiple species of Acanthocephala in Europe but, in Switzerland, is less common than two cryptic members of the Gammarus fossarum species complex (type A and type B). We found that natural populations of these cryptic species were frequently infected by Pomphorhynchus tereticollis and Polymorphus minutus. Four additional parasite species occurred only locally. Parasites were more common in G. fossarum type B than in type A. Infection experiments using several host and parasite sources confirmed consistently lower infection rates in G. pulex than in G. fossarum type A, suggesting a general difference in susceptibility between the two species. In conclusion, we could show that cryptic host species differ in their interactions with parasites, but that these differences were much less dramatic than differences between G. fossarum (type A) and G. pulex. Our data suggest that the acanthocephalans in Switzerland have adapted to the two most common Gammarus species in this region where host species frequencies differ from near-by regions in Europe.

Presence of polydnavirus transcripts in an egg-larval parasitoid and its lepidopterous host

The parasitoid Chelonus inanitus (Braconidae, Hymenoptera) oviposits into eggs of Spodoptera littoralis (Noctuidae, Lepidoptera) and, along with the egg, also injects polydnaviruses and venom, which are prerequisites for successful parasitoid development. The parasitoid larva develops within the embryonic and larval stages of the host, which enters metamorphosis precociously and arrests development in the prepupal stage. Polydnaviruses are responsible for the developmental arrest and interfere with the host's endocrine system in the last larval instar. Polydnaviruses have a segmented genome and are transmitted as a provirus integrated in the wasp's genome. Virions are only formed in female wasps and no virus replication is seen in the parasitized host. Here it is shown that very small amounts of viral transcripts were found in parasitized eggs and early larval instars of S. littoralis. Later on, transcript quantities increased and were highest in the late last larval instar for two of the three viral segments tested and in the penultimate to early last larval instar for the third segment. These are the first data on the occurrence of viral transcripts in the host of an egg-larval parasitoid and they are different from data reported for hosts of larval parasitoids, where transcript levels are already high shortly after parasitization. The analysis of three open reading frames by RT-PCR revealed viral transcripts in parasitized S. littoralis and in female pupae of C. inanitus, indicating the absence of host specificity. For one open reading frame, transcripts were also seen in male pupae, suggesting transcription from integrated viral DNA.

RTX toxins in Pasteurellaceae

RTX toxins (repeats in the structural toxin) are pore-forming protein toxins produced by a broad range of pathogenic Gram-negative bacteria. In vitro, RTX toxins mostly exhibit a cytotoxic and often also a hemolytic activity. They are particularly widespread in species of the family Pasteurellaceae which cause infectious diseases, most frequently in animals but also in humans. Most RTX toxins are proteins with a molecular mass of 100-200 kDa and are post-translationally activated by acylation via a specific activator protein. The repeated structure of RTX toxins, which gave them their name, is composed of iterative glycine-rich nonapeptides binding Ca2+ on the C-terminal half of the protein. Genetic analysis of RTX toxins of various species of Pasteurellaceae and of a few other Gram-negative bacteria gave evidence of horizontal transfer of genes encoding RTX toxins and led to speculations that RTX toxins might have originated from Pasteurellaceae. The toxic activities of RTX toxins in host cells may lead to necrosis and apoptosis and the underlying detailed mechanisms are currently under investigation. The impact of RTX toxins in pathogenicity and the immune responses of the host were described for several species of Pasteurellaceae. Neutralizing antibodies were shown to significantly reduce the cytotoxic activity of RTX toxins. They constitute a valuable strategy in the development of immuno-prophylactics against several animal diseases caused by pathogenic species of Pasteurellaceae. Although many RTX toxins possess cytotoxic and hemolytic activities toward a broad range of cells and erythrocytes, respectively, a few RTX toxins were shown to have cytotoxic activity only against cells of specific hosts and/or show cell-type specificity. Further evidence exists that RTX toxins play a potential role in host specificity of certain pathogens.

Evidence of species-specific neighborhood effects in the dipterocarpaceae of a Bornean rain forest

Although accumulating evidence indicates that local intraspecific density-dependent effects are not as rare in species-rich communities as previously suspected, there are still very few detailed and systematic neighborhood analyses of species-rich communities. Here, we provide such an analysis with the overall goal of quantifying the relative importance of inter- and intraspecific interaction strength in a primary, lowland dipterocarp forest located at Danum, Sabah, Malaysia. Using data on 10 abundant overstory dipterocarp species from two 4-ha permanent plots, we evaluated the effects of neighbors on the absolute growth rate of focal trees (from 1986 to 1996) over increasing neighborhood radii (from 1 to 20 m) with multiple regressions. Only trees 10 cm to < 100 cm girth at breast height in 1986 were considered as focal trees. Among neighborhood models with one neighbor term, models including only conspecific larger trees performed best in five out of 10 species. Negative effects of conspecific larger neighbors were most apparent in large overstory species such as those of the genus Shorea. However, neighborhood models with separate terms and radii for heterospecific and conspecific neighbors accounted for more variability in absolute growth rates than did neighborhood models with one neighbor term. The conspecific term was significant for nine out of 10 species. Moreover, in five out of 10 species, trees without conspecific neighbors had significantly higher absolute growth rates than trees with conspecific neighbors. Averaged over the 10 species, trees without conspecific neighbors grew 32.4 cm(2) in basal area from 1986 to 1996, whereas trees with conspecific neighbors only grew 14.7 cm(2) in basal area, although there was no difference in initial basal area between trees in the two groups. Averaged across the six species of the genus Shorea, negative effects of conspecific larger trees were significantly stronger than for heterospecific larger neighbors. Thus, high local densities within neighborhoods of 20 m may lead to strong intraspecific negative and, hence, density-dependent, effects even in species rich communities with low overall densities at larger spatial scales. We conjecture that the strength of conspecific effects may be correlated with the degree of host specificity of ectomycorrhizae.

Potential for virus transfer between the honey bees Apis mellifera and A. cerana

Viruses seem to play a key role in European honey bee, Apis mellifera health, and have a much broader host spectrum than previously thought. Few studies have investigated interspecific virus transfer within the genus Apis. The introduction of A. mellifera into Asia exposed endemic Apis species to the risk of obtaining new viruses or viral strains and vice versa. To investigate the potential for host shifts, virus prevalence and sequences were monitored over three years in single and mixed-species apiaries hosting introduced A. mellifera and endemic Apis cerana. Deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), and sacbrood virus (SBV) were found, but not KBV, VDV-1, ABPV, or CBPV. Virus infections and prevalence were generally lower in A. cerana compared to A. mellifera, and varied over the years. The sequence data provided evidence for interspecific transfer of IAPV, BQCV, and DWV, but SBV strains seem to be species specific. Prevalence and sequence results taken together indicate that interspecific transfers of viruses are rare, even if honey bees are kept in close proximity. We discuss the pattern observed in the context host specificity and resistance. Our understanding of the extent of these exchanges is limited by a lack of knowledge on the mechanisms of adaptation of viruses to different hosts.

Human TOP1 residues implicated in species specificity of HIV-1 infection are required for interaction with BTBD2, and RNAi of BTBD2 in old world monkey and human cells increases permissiveness to HIV-1 infection

Host determinants of HIV-1 viral tropism include factors from producer cells that affect the efficiency of productive infection and factors in target cells that block infection after viral entry. TRIM5 restricts HIV-1 infection at an early post-entry step through a mechanism associated with rapid disassembly of the retroviral capsid. Topoisomerase I (TOP1) appears to play a role in HIV-1 viral tropism by incorporating into or otherwise modulating virions affecting the efficiency of a post-entry step, as the expression of human TOP1 in African Green Monkey (AGM) virion-producing cells increased the infectivity of progeny virions by five-fold. This infectivity enhancement required human TOP1 residues 236 and 237 as their replacement with the AGM counterpart residues abolished the infectivity enhancement. Our previous studies showed that TOP1 interacts with BTBD1 and BTBD2, two proteins which co-localize with the TRIM5 splice variant TRIM5 in cytoplasmic bodies. Because BTBD1 and BTBD2 interact with one HIV-1 viral tropism factor, TOP1, and co-localize with a splice variant of another, we investigated the potential involvement of BTBD1 and BTBD2 in HIV-1 restriction.

Host and viral genetic correlates of clinical definitions of HIV-1 disease progression

Various patterns of HIV-1 disease progression are described in clinical practice and in research. There is a need to assess the specificity of commonly used definitions of long term non-progressor (LTNP) elite controllers (LTNP-EC), viremic controllers (LTNP-VC), and viremic non controllers (LTNP-NC), as well as of chronic progressors (P) and rapid progressors (RP).

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.

HLA-B⁎27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope

Background & Aims: HLA-B⁄27 is associated with spontaneous HCV genotype 1 clearance. HLA-B⁄27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B⁄27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B⁄27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Methods: Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B⁄27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B⁄27:02 and 05. Results: The NS5B2820 epitope is only restricted by the HLA-B⁄27 subtype HLA-B⁄27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B⁄27 subtype B⁄27:05. Indeed, the epitope is very dominant in HLA-B⁄27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B⁄27:02+ chronically infected patients. Conclusions: The NS5B2820 epitope is immunodominant in the context of HLA-B⁄27:02, but is not restricted by other HLA-B⁄27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines.

Detection and quantification of leptospires in urine of dogs: a maintenance host for the zoonotic disease leptospirosis.

Leptospirosis is a global zoonotic disease. Pathogenic Leptospira species, the causative agent of leptospirosis, colonize the renal tubules of chronically infected maintenance hosts such as dogs, rats and cattle. Maintenance hosts typically remain clinically asymptomatic and shed leptospires into the environment via urine. In contrast, accidental hosts such as humans can suffer severe acute forms of the disease. Infection results from direct contact with infected urine or indirectly, through contaminated water sources. In this study, a quantitative real-time PCR specific for lipL32 was designed to detect the urinary shedding of leptospires from dogs. The sensitivity and specificity of the assay was evaluated using both a panel of pathogenic Leptospira species and clinical microbial isolates, and samples of urine collected from experimentally infected rats and non-infected controls. The lower limit of detection was approximately 3 genome equivalents per reaction. The assay was applied to canine urine samples collected from local dog sanctuaries and the University Veterinary Hospital (UVH) at University College Dublin. Of 525 canine urine samples assayed, 37 were positive, indicating a prevalence of urinary shedding of leptospires of 7.05%. These results highlight the need to provide effective canine vaccination strategies and raise public health awareness.

Evaluation of genome-wide loci of iron metabolism in hereditary hemochromatosis identifies PCSK7 as a host risk factor of liver cirrhosis

Genome-wide association studies (GWAS) have revealed genetic determinants of iron metabolism, but correlation of these with clinical phenotypes is pending. Homozygosity for HFE C282Y is the predominant genetic risk factor for hereditary hemochromatosis (HH) and may cause liver cirrhosis. However, this genotype has a low penetrance. Thus, detection of yet unknown genetic markers that identify patients at risk of developing severe liver disease is necessary for better prevention. Genetic loci associated with iron metabolism (TF, TMPRSS6, PCSK7, TFR2 and Chr2p14) in recent GWAS and liver fibrosis (PNPLA3) in recent meta-analysis were analyzed for association with either liver cirrhosis or advanced fibrosis in 148 German HFE C282Y homozygotes. Replication of associations was sought in additional 499 Austrian/Swiss and 112 HFE C282Y homozygotes from Sweden. Only variant rs236918 in the PCSK7 gene (proprotein convertase subtilisin/kexin type 7) was associated with cirrhosis or advanced fibrosis (P = 1.02 × 10(-5)) in the German cohort with genotypic odds ratios of 3.56 (95% CI 1.29-9.77) for CG heterozygotes and 5.38 (95% CI 2.39-12.10) for C allele carriers. Association between rs236918 and cirrhosis was confirmed in Austrian/Swiss HFE C282Y homozygotes (P = 0.014; ORallelic = 1.82 (95% CI 1.12-2.95) but not in Swedish patients. Post hoc combined analyses of German/Swiss/Austrian patients with available liver histology (N = 244, P = 0.00014, ORallelic = 2.84) and of males only (N = 431, P = 2.17 × 10(-5), ORallelic = 2.54) were consistent with the premier finding. Association between rs236918 and cirrhosis was not confirmed in alcoholic cirrhotics, suggesting specificity of this genetic risk factor for HH. PCSK7 variant rs236918 is a risk factor for cirrhosis in HH patients homozygous for the HFE C282Y mutation.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro.

Pestivirus N(pro) is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N(pro) blocks the host׳s interferon response by inducing degradation of interferon regulatory factor-3. N(pro׳)s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N(pro)-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N(pro) proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N(pro׳)s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N(pro) does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus.