Host-specific Virulence Research Articles

Comparative Patho-Genomics of Salmonella enterica Serovar Enteritidis Reveal Potential Host-Specific Virulence Factors

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most common causes of bacterial foodborne infections worldwide. It has an extensive host range, including birds and humans, making it one of the most adaptable Salmonella serovars. This study aims to define the virulence gene profile of S. Enteritidis and identify genes critical to its host specificity. Currently, there is limited understanding of the molecular mechanisms that allow S. Enteritidis to continue as an important foodborne pathogen. To better understand the genes that may play a role in the host-specific virulence and/or fitness of S. Enteritidis, we first compiled a virulence gene profile-based genome analysis of sequenced S. Enteritidis strains isolated from shell eggs in our laboratory. This analysis was subsequently used to compare the representative genomes of Salmonella serovars with varying host ranges and S. Enteritidis genomes. The study involved a comprehensive and direct examination of the conservation of virulence and/or fitness factors, especially in a host-specific manner—an area that has not been previously explored. Key findings include the identification of 10 virulence-associated clusters of orthologous genes (COGs) specific to poultry-colonizing serovars and 12 virulence-associated COGs unique to human-colonizing serovars. Virulence/fitness-associated gene analysis identified more than 600 genes. The genome sequences of the two S. Enteritidis isolates were compared to those of the other serovars. Genome analysis revealed a core of 2817 COGs that were common to all the Salmonella serovars examined. Comparative genome analysis revealed that 10 virulence-associated COGs were specific to poultry-colonizing serovars, whereas 12 virulence-associated COGs were present in all human-colonizing serovars. Phylogenetic analyses further highlight the evolution of host specificity in S. Enteritidis. This study offers the first comprehensive analysis of genes that may be unique to and possibly essential for the colonization and/or pathogenesis of S. Enteritidis in various and specific hosts.

Two homologous Zn2Cys6 transcription factors play crucial roles in host specificity of Colletotrichum orbiculare by controlling the expression of cucurbit-specific virulence effectors.

Fungal plant pathogens preferentially express a set of effector genes at specific infection stages to successfully colonize the host. However, it remains unclear how effector gene expression is regulated during host infection. This study identified a Zn2Cys6 transcription factor, TFV1 (Transcription Factor for Virulence 1), whose deletion weakened virulence of Colletotrichum orbiculare on its cucurbit hosts. The additional deletion of a TFV1 paralog gene, TVL1 (TFV1-like 1), resulted in a further reduction in virulence on the cucurbits. Notably, TFV1 TVL1 double mutants retained wild-type virulence on the Solanaceae host Nicotiana benthamiana. Expression of putative effector genes, including four cucurbit host-specific virulence effectors (effector protein for cucurbit infection, EPC1-4), was commonly downregulated in the TFV1 knockout mutants. Yeast one-hybrid assays suggested that TFV1 binds to the putative promoter regions of EPC2, EPC3, and EPC4, indicating the importance of TFV1 for the induced expression of key effector genes in cucurbit infection. Among the effector-like genes whose expression was affected by TVL1 deletion, a novel LysM effector gene, EPC5, was identified as being specifically required for virulence on cucurbit hosts. Our study extends the knowledge of the regulatory mechanisms governing host- and stage-specific effectors in C. orbiculare.

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites resulted in cross-fertilization and the formation of "yellow" oocysts, which contain 4 haploid sporozoites that are the product of meiosis. Recombinant oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. The most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

Comparative Genomic Analysis of Shrimp-Pathogenic Vibrio parahaemolyticus LC and Intraspecific Strains with Emphasis on Virulent Factors of Mobile Genetic Elements.

Vibrio parahaemolyticus exhibits severe pathogenicity in humans and animals worldwide. In this study, genome sequencing and comparative analyses were conducted for in-depth characterization of the virulence factor (VF) repertoire of V. parahaemolyticus strain LC, which presented significant virulence to shrimp Litopenaeus vannamei. Strain LC, harboring two circular chromosomes and three linear plasmids, demonstrated ≥98.14% average nucleotide identities with 31 publicly available V. parahaemolyticus genomes, including 13, 11, and 7 shrimp-, human-, and non-pathogenic strains, respectively. Phylogeny analysis based on dispensable genes of pan-genome clustered 11 out of 14 shrimp-pathogenic strains and 7 out of 11 clinical strains into two distinct clades, indicating the close association between host-specific pathogenicity and accessory genes. The VFDB database revealed that 150 VFs of LC were mainly associated with the secretion system, adherence, antiphagocytosis, chemotaxis, motility, and iron uptake, whereas no homologs of the typical pathogenic genes pirA, pirB, tdh, and trh were detected. Four genes, mshB, wbfT, wbfU, and wbtI, were identified in both types of pathogenic strains but were absent in non-pathogens. Notably, a unique cluster similar to Yen-Tc, which encodes an insecticidal toxin complex, and diverse toxin-antitoxin (TA) systems, were identified on the mobile genetic elements (MGEs) of LC. Conclusively, in addition to the common VFs, various unique MGE-borne VFs, including the Yen-Tc cluster, TA components, and multiple chromosome-encoded chitinase genes, may contribute to the full spectrum of LC virulence. Moreover, V. parahaemolyticus demonstrates host-specific virulence, which potentially drives the origin and spread of pathogenic factors.

Virulence Mechanisms of Staphylococcal Animal Pathogens.

Staphylococci are major causes of infections in mammals. Mammals are colonized by diverse staphylococcal species, often with moderate to strong host specificity, and colonization is a common source of infection. Staphylococcal infections of animals not only are of major importance for animal well-being but have considerable economic consequences, such as in the case of staphylococcal mastitis, which costs billions of dollars annually. Furthermore, pet animals can be temporary carriers of strains infectious to humans. Moreover, antimicrobial resistance is a great concern in livestock infections, as there is considerable antibiotic overuse, and resistant strains can be transferred to humans. With the number of working antibiotics continuously becoming smaller due to the concomitant spread of resistant strains, alternative approaches, such as anti-virulence, are increasingly being investigated to treat staphylococcal infections. For this, understanding the virulence mechanisms of animal staphylococcal pathogens is crucial. While many virulence factors have similar functions in humans as animals, there are increasingly frequent reports of host-specific virulence factors and mechanisms. Furthermore, we are only beginning to understand virulence mechanisms in animal-specific staphylococcal pathogens. This review gives an overview of animal infections caused by staphylococci and our knowledge about the virulence mechanisms involved.

Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen.

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C.orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C.orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C.orbiculare to cucurbit hosts, but not to the Solanaceae host N.benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C.orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Horizontal transfer of the rfb cluster in Leptospira is a genetic determinant of serovar identity.

Leptospira bacteria comprise numerous species, several of which cause serious disease to a broad range of hosts including humans. These spirochetes exhibit large intraspecific variation, resulting in complex tabulations of serogroups/serovars that crisscross the species classification. Serovar identity, linked to biological/clinical phenotypes, depends on the structure of surface-exposed LPS. Many LPS biosynthesis-encoding genes reside within the chromosomic rfb gene cluster. However, the genetic basis of intraspecies variability is not fully understood, constraining diagnostics/typing methods to cumbersome serologic procedures. We now show that the gene content of the rfb cluster strongly correlates with Leptospira serovar designation. Whole-genome sequencing of pathogenic L. noguchii, including strains of different serogroups, reveals that the rfb cluster undergoes extensive horizontal gene transfer. The rfb clusters from several Leptospira species disclose a univocal correspondence between gene composition and serovar identity. This work paves the way to genetic typing of Leptospira serovars, and to pinpointing specific genes within the distinct rfb clusters, encoding host-specific virulence traits. Further research shall unveil the molecular mechanism of rfb transfer among Leptospira strains and species.

Integrative Pathogenicity Assay and Operational Taxonomy-Based Detection of New Forma Specialis of Fusarium oxysporum Causing Datepalm Wilt.

Pathogenicity-associated genes are highly host-specific and contribute to host-specific virulence. We tailored the traditional Koch’s postulates with integrative omics by hypothesizing that the effector genes associated with host-pathogenicity are determinant markers for virulence, and developed Integrative Pathogenicity (IP) postulates for authenticated pathogenicity testing in plants. To set the criteria, we experimented on datepalm (Phoenix dactylifera) for the vascular wilt pathogen and confirmed the pathogen based on secreted in xylem genes (effectors genes) using genomic and transcriptomic approaches, and found it a reliable solution when pathogenicity is in question. The genic regions ITS, TEF1-α, and RPBII of Fusarium isolates were examined by phylogenetic analysis to unveil the validated operational taxonomy at the species level. The hierarchical tree generated through phylogenetic analysis declared the fungal pathogen as Fusarium oxysporum. Moreover, the Fusarium isolates were investigated at the subspecies level by probing the IGS, TEF1-α, and Pgx4 genic regions to detect the forma specialis of F. oxysporum that causes wilt in datepalm. The phylogram revealed a new forma specialis in F. oxysporum that causes vascular wilt in datepalm.

Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton–Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Expression and Functional Analysis of the Type III Secretion System Effector Repertoire of the Xylem Pathogen Erwinia tracheiphila on Cucurbits.

The predicted repertoire of type III secretion system effectors (T3SEs) in Erwinia tracheiphila, causal agent of cucurbit bacterial wilt, is much larger than in xylem pathogens in the closely related genera Erwinia and Pantoea. The genomes of strains BHKY and SCR3, which represent distinct E. tracheiphila clades, encode at least 6 clade-specific and 12 shared T3SEs. The strains expressed the majority of the T3SE genes examined in planta. Among the shared T3SE genes, eop1 was expressed most highly in both strains in squash (Cucurbita pepo) and muskmelon (Cucumis melo) but the clade-specific gene avrRpm2 was expressed 40- to 900-fold more than eop1 in BHKY. The T3SEs AvrRpm2, Eop1, SrfC, and DspE contributed to BHKY virulence on squash and muskmelon, as shown using combinatorial mutants involving six T3SEs, whereas OspG and AvrB4 contributed to BHKY virulence only on muskmelon, demonstrating host-specific virulence functions. Moreover, Eop1 was functionally redundant with AvrRpm2, SrfC, OspG, and AvrB4 in BHKY, and BHKY mutants lacking up to five effector genes showed similar virulence to mutants lacking only two genes. The T3SEs OspG, AvrB4, and DspE contributed additively to SCR3 virulence on muskmelon and were not functionally redundant with Eop1. Rather, loss of eop1 and avrB4 restored wild-type virulence to the avrB4 mutant, suggesting that Eop1 suppresses a functionally redundant effector in SCR3. These results highlight functional differences in effector inventories between two E. tracheiphila clades, provide the first evidence of OspG as a phytopathogen effector, and suggest that Eop1 may be a metaeffector influencing virulence. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Complete Genome Sequences of Four Strains ofErwiniatracheiphila: A Resource for Studying aBacterial Plant Pathogen with a Highly Complex Genome.

Complete Genome Sequences of Four Strains ofErwiniatracheiphila: A Resource for Studying aBacterial Plant Pathogen with a Highly Complex Genome.

In Silico Exploration of Mycobacterium tuberculosis Metabolic Networks Shows Host-Associated Convergent Fluxomic Phenotypes.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is composed of several lineages characterized by a genome identity higher than 99%. Although the majority of the lineages are associated with humans, at least four lineages are adapted to other mammals, including different M. tuberculosis ecotypes. Host specificity is associated with higher virulence in its preferred host in ecotypes such as M. bovis. Deciphering what determines the preference of the host can reveal host-specific virulence patterns. However, it is not clear which genomic determinants might be influencing host specificity. In this study, we apply a combination of unsupervised and supervised classification methods on genomic data of ~27,000 M. tuberculosis clinical isolates to decipher host-specific genomic determinants. Host-specific genomic signatures are scarce beyond known lineage-specific mutations. Therefore, we integrated lineage-specific mutations into the iEK1011 2.0 genome-scale metabolic model to obtain lineage-specific versions of it. Flux distributions sampled from the solution spaces of these models can be accurately separated according to host association. This separation correlated with differences in cell wall processes, lipid, amino acid and carbon metabolic subsystems. These differences were observable when more than 95% of the samples had a specific growth rate significantly lower than the maximum achievable by the models. This suggests that these differences might manifest at low growth rate settings, such as the restrictive conditions M. tuberculosis suffers during macrophage infection.

Differential virulence of infectious hematopoietic necrosis virus (IHNV) isolated from salmonid fish in Gangwon Province, Korea

The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed <60% cumulative mortality in Atlantic salmon and coho salmon. The expression of G genes in the kidney was higher than that in the spleen-infected fish, with the highest expression observed in the kidneys of rainbow trout. The relative expression levels of innate immunity genes were higher in rainbow trout than in Atlantic salmon and coho salmon. The expression level of immunoglobulin M increased until day 7, and the expression of type I interferon was higher in the spleen than in other tissues. The expression of Mx-1 was higher in the kidney and liver than other tissues. These results indicate that IHNV isolates from Gangwon province show host-specific virulence in rainbow trout and that their virulence and replication were higher in JRt-Shizuoka type than in JRt-Nagano type isolates.

Fusarium oxysporum f. sp. passiflorae infecting passionfruit in New Zealand in a changing taxonomic landscape

Fusarium oxysporum f. sp. passiflorae (FOP) is reported for the first time in Northland, New Zealand. The identity of this host-specific pathogen was confirmed by pathogenicity testing, morphological characters, and DNA sequencing. Pathogenic strains of Fusarium oxysporum secrete unique proteins or effectors, ‘secreted in xylem’ (SIX), which are likely to contribute to host-specific virulence. Sequence analysis of the EF-1a gene, β-tubulin and the effector genes SIX6 and SIX9 confirmed that New Zealand isolates belong to FOP. This study confirmed that the three New Zealand EF-1α haplotypes of FOP had identical SIX6 and SIX9 sequences, indicating that the same homolog of each gene, SIX6a and SIX9a, is shared by both haplotypes of FOP. SIX genes are rarely detected in non-pathogenic strains of Fusarium oxysporum species complex (FOSC) and pathogenicity tests are necessary to confirm its pathogenicity status.

Staphylococcus aureus Host Tropism and Its Implications for Murine Infection Models.

Staphylococcus aureus (S. aureus) is a pathobiont of humans as well as a multitude of animal species. The high prevalence of multi-resistant and more virulent strains of S. aureus necessitates the development of new prevention and treatment strategies for S. aureus infection. Major advances towards understanding the pathogenesis of S. aureus diseases have been made using conventional mouse models, i.e., by infecting naïve laboratory mice with human-adapted S. aureus strains. However, the failure to transfer certain results obtained in these murine systems to humans highlights the limitations of such models. Indeed, numerous S. aureus vaccine candidates showed promising results in conventional mouse models but failed to offer protection in human clinical trials. These limitations arise not only from the widely discussed physiological differences between mice and humans, but also from the lack of attention that is paid to the specific interactions of S. aureus with its respective host. For instance, animal-derived S. aureus lineages show a high degree of host tropism and carry a repertoire of host-specific virulence and immune evasion factors. Mouse-adapted S. aureus strains, humanized mice, and microbiome-optimized mice are promising approaches to overcome these limitations and could improve transferability of animal experiments to human trials in the future.

Screening of Lactococcal Adhesion Genes and Two Pneumococcal Genes as Genetic Determinants of Virulence in Lactococcus garvieae Strains

Nine lactococcal adhesin and two pneumococcal virulence genes were characterized in twenty Lactococcus garvieae strains obtained from rainbow trout, and also one human isolate by PCR. Findings showed that all strains carried adhPsaA (522 bp), LPxTG-1 (947 bp), adhCI (490 bp), and purB (864 bp) while some of the strains had adhPav (1048 bp), LPxTG-2 (767 bp), LPxTG-3 (231 bp), adhCII (732 bp), adh (398 bp) and SP_0121 (966 bp). High nucleotide homologies (85-99%) of adhCI, adhCII, adh, adhPav, adhPsaA, LPxTG-1, LPxTG-3 involved in bacterial adhesion were determined between L. garvieae strains and reference Lg2. Genes containing polymorphisms among strains were not considered to be directly involved in bacterial pathogenesis. The amplification of LPxTG-3 only in fish isolates showed that it might be responsible for coding the host-specific virulence factor. However, undetermined amplicons demonstrated that LPxTG-4 could not be used as a host-specific gene marker. Pneumococcal purB and SP_0121 have been experimentally detected in L. garvieae genome for the first time. Consequently, both purB and SP_0121 can be used as a virulence marker for L. garvieae. Findings provided valuable knowledge about L. garvieae pathogenesis and, will contribute to identify the appropriate genomic targets to develop new therapeutics against the lactococcosis.

Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens.

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa- ). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.

Molecular Targets for Coevolutionary Interactions Between Pacific Oyster Larvae and Their Sympatric Vibrios.

Bacteria of the Vibrio genus are the most predominant infectious agents threatening marine wildlife and aquaculture. Due to the large genetic diversity of these pathogens, the molecular determinants of Vibrio virulence are only poorly understood. Furthermore, studies tend to ignore co-evolutionary interactions between different host populations and their locally encountered Vibrio communities. Here, we explore the molecular targets of such co-evolutionary interactions by analyzing the genomes of nine Vibrio strains from the Splendidus-clade showing opposite virulence patterns towards two populations of Pacific oysters introduced into European Wadden Sea. By contrasting Vibrio phylogeny to their host specific virulence patterns, we could identify two core genome genes (OG1907 and OG 3159) that determine the genotype by genotype (G × G) interactions between oyster larvae and their sympatric Vibrio communities. Both genes show positive selection between locations targeting only few amino acid positions. Deletion of each gene led to a loss of the host specific virulence patterns while complementation with OG3159 alleles from both locations could recreate the wild type phenotypes matching the origin of the allele. This indicates that both genes can act as a genetic switch for Vibrio-oyster coevolution demonstrating that local adaptation in distinct Vibrio lineages can rely on only few genes independent of larger pathogenicity islands or plasmids.

The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout.

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (Oncorhynchus mykiss). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout.IMPORTANCE Rainbow trout farming is a major food source industry worldwide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) protein.