ROP18 and ROP5 alleles combinations are related with virulence of T. gondii isolates from Argentina

The allelic combination of ROP18/ROP5 genes of Toxoplasma gondii has been shown to be highly predictive of mouse virulence in canonical isolates and strains. The aims of this study were to analyze the alleles present in the ROP18/ROP5 genes from T. gondii isolates obtained in Argentina, to associate the results with the virulence registered in mouse model, and to compare with other isolates and reference strains using a phylogenetic network. Fourteen T. gondii isolates from Argentina were analyzed by nPCR-RFLP for ROP18/ROP5. Phylogenetic network analysis was inferred using the ToxoDB genotypes and the ROPs molecular markers. All isolates and reference strains were categorized as lethal or non-lethal. As results, combinations 2/2, 3/3 and 4/3 for ROP18/ROP5 were detected in 12 isolates, whereas only alleles 1 and 2 of ROP5 were detected in 2 isolates. The majority of the isolates had a mouse virulence matching to that predicted by the ROP18/ROP5 allele combination. The 3 isolates that differed from the expected virulence presented non-clonal genotypes. ROPs incorporation increased the accuracy of the phylogenetic network relations among the T. gondii samples, prevailing the clustering according to regions. Our results indicate a predominance of type 3 allele in both ROP18 and ROP5 markers and an association of allelic profiles 3/3 and 4/3 of non-clonal genotypes from Argentina, both with virulent and avirulent profiles in mice.     

The Rhoptry Proteins ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine,But Not the Human,Interferon-Gamma Response

The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans.     

Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.     

Characterization of ROP18 alleles in human toxoplasmosis

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis.     

Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Background

Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission.

Methodology/Principal Findings

Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7).

Conclusion/Significance

A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.     

The Polymorphic Pseudokinase ROP5 Controls Virulence in Toxoplasma gondii by Regulating the Active Kinase ROP18

Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δrop5 and Δrop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3^−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.     

Toxoplasma gondii Virulence Factor ROP18 Inhibits the Host NF-κB Pathway by Promoting p65 Degradation

The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.     

Two viable Toxoplasma gondii isolates from red-necked wallaby (Macropus rufogriseus) and red kangaroo (M. rufus)

Wallabies and kangaroos are susceptible to Toxoplasma gondii. However, little information concerning T. gondii infection in captive macropods is available. Three dead macropods collected from a zoo exhibited no clinical symptoms associated with toxoplasmosis. Heart fluids were tested for T. gondii antibodies using a modified agglutination test. T. gondii DNA samples derived from macropod tissues were tested by Polymerase Chain Reaction. Viable T. gondii were isolated from myocardium of macropods via mouse bioassay. Tissues (brain, lungs, or mesenteric lymph nodes) from T. gondii-positive mice were seeded into Vero cell culture flasks. The virulence of the isolated T. gondii strains was evaluated in Swiss mice. The DNA from T. gondii tachyzoites obtained from cell cultures was characterized by 10 PCR-RFLP markers and the virulence genes, ROP18 and ROP5. T. gondii antibodies were identified in two of the three macropods (Macropod^#5 and ^#7). T. gondii DNA was obtained from the heart and lungs of Macropod^#7. Two viable T. gondii strains were isolated from the myocardium of Macropus rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) via mouse bioassay and designated as TgRooCHn2 and TgRooCHn3, respectively. TgRooCHn2 was ToxoDB genotype^#3, and TgRooCHn3 was ToxoDB genotyp^#2. Both 10⁴ TgRooCHn2 and TgRooCHn3 tachyzoites had intermediate virulence in mice. M. rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) may have been in the initial stages of toxoplasmosis, due to a recent T. gondii infection with oocysts. This study is the first to document the T. gondii ToxoDB^#3 isolate in macropods. T. gondii infection in captive macropods indicates the urgent need to control the transmission of this parasite in the environment, food and water of zoo animals.     

Clonal and atypical Toxoplasma strain differences in virulence vary with mouse sub-species

The severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.     

Characterization of a spontaneous cyst-forming strain of Toxoplasma gondii isolated from Tokachi subprefecture in Japan

Apicomplexan parasite Toxoplasma gondii has three distinct clonal lineages: high, medium and low virulent strains, type I, II and III, respectively. T. gondii avoids the immune response by transforming from fast multiplying tachyzoite to slow multiplying bradyzoite, and establishing a chronic infection. In the present study, we isolated a new strain of T. gondii from cat feces in the Tokachi subprefecture, Hokkaido, Japan and named it as TgCatJpObi1 (Obi1) strain. Genotyping analysis of 12 loci revealed atypical characters close to type II, genotype 4 according to ToxoDB classification. Phenotypically, Obi1 strain shows slow growth rate and the ability of spontaneous cyst formation in both human foreskin fibroblast (HFFs) and mouse peritoneal macrophages in vitro without bradyzoite induction. Parasite virulence was assessed by means of mouse survival upon infection with either Obi1 or ME49 strains. Obi1 strain displayed no mortalities in comparison to type II clonal lineage, ME49 at LD₅₀ to LD₁₀₀ range (1 × 10³–10⁶ tachyzoites). Although virulence of Obi1 strain is significantly lower than that of ME49, nucleotide sequences analyses revealed that genes of virulence factors such as Gra15, Rop5, 16, 17, and 18 in Obi1 strain were 100% identical to those in the type II strain. Thus, characterization of a newly isolated strain, Obi1, is crucial to clarify the development of toxoplasmosis in both humans and animals.     

Phenotypic and Gene Expression Changes among Clonal Type I Strains of Toxoplasma gondii

Toxoplasma gondii has an unusual population structure consisting of three clonal lineages that predominate in North America and Europe. This simple pattern has encouraged the use of only a few laboratory isolates that are representative of each lineage. Principle among these is the type I RH strain, originally isolated from a child with encephalitis some 70 years ago. Comparison of different passages of the RH strain that have been propagated differently over the intervening time period revealed that the commonly used clonal line called RH-ERP was not representative of natural isolates of the type I lineage. Notably, RH-ERP formed much larger plaques than other type 1 strains, including a separate, earlier derived isolate of the RH strain. The RH-ERP variant also showed enhanced extracellular survival, faster growth, and decreased differentiation compared to the prototype type I strain GT1. Comparison of gene expression differences in the RH-ERP line revealed that several ABC transporters were upregulated, which may provide a growth advantage in vitro. These findings illustrate that dramatic phenotypic changes can arise in laboratory strains, emphasizing the need for comparison with recent clinical isolates.Toxoplasma gondii is a widespread protozoan parasite in the phylum Apicomplexa, an ancient group of protozoan parasites containing more than 5,000 species (27). T. gondii infects a wide range of warm-blooded animals and, while not part of the natural transmission, human infections can lead to severe disease in immunocompromised individuals (19). Using a unique form of actin-based motility, T. gondii penetrates host cells, where it rapidly multiplies within a nonfusigenic vacuole and eventually causes lysis of the host cell (44). During the acute phase of the infection, repeated cycles of cell invasion, replication, and lysis contribute to rapid spread and tissue damage (2, 28). In addition to this rapid lytic form of growth, asexual replication by slow-growing bradyzoites contributes to long-term chronic infection in a variety of hosts (52). In contrast, sexual development occurs only in the intestinal epithelial cells of cats (11). Despite this sexual phase in the life cycle, T. gondii maintains a highly clonal population structure that consists of three lineages type I, II and III, which predominate in North America and Europe (1, 17). These three clonal lineages are the result of recent expansion from either a genetic bottleneck or selective sweep that occurred within the last 10,000 years (48). Within each lineage, all strains are thought to be genetically identical except for random mutations that have occurred since their recent origin. Differences between the three clonal lineages of T. gondii are also limited, and they display only 1 to 2% divergence at the nucleotide level for most loci (6). Despite this similarity, the different clonal lineages differ dramatically in biological traits such as growth (37), migration (2), and pathogenesis in laboratory mice (28). The type I lineage has been shown to exclusively contain those strains of T. gondii that are acutely virulent in laboratory mice (43, 49). The lethal dose of type I strains in outbred mice is estimated to be a single organism based on limiting dilution studies, whereas types II and III have 50% lethal dose that are ∼4 logs higher than this. It has been suggested that the virulence of type I strains may be due to continuous laboratory passage (12). However, more than 20 natural isolates that share this clonal genotype and acute virulence phenotype have been described (43, 49), indicating that acute virulence is genetically encoded and not simply a consequence of passage history. Consistent with this, genetic mapping has recently revealed that the acute virulence of type I strains in the mouse model is controlled by the rhoptry protein ROP18, which encodes a secretory serine threonine kinase (50). ROP18 shows dramatic levels of polymorphism and, although highly expressed in the type I and II lineages, it is expressed at very low levels in the type III lineage owing to the presence of an ancestral upstream region that influences expression (23).The commonly used type I strain known as RH was originally isolated by Albert Sabin from a child who died with toxoplasmic encephalitis in 1939 (40). The RH strain was reported to be virulent on primary inoculation in laboratory mice (40). Since then, it has been serially passaged in mice by many laboratories. The RH strain was adapted for in vitro culture ca. 1977 by Elmer Pfefferkorn, who generated a clonal line that has also been widely distributed (36). This RH-ERP line was used to develop procedures for plaque formation on monolayers of host cells, providing a very useful in vitro assay that captures a combination of invasion, replication, egress, and spread. The ability of T. gondii to form such plaques has been used to generate temperature sensitive mutants (36), define the molecular basis of various chemically derived mutants (33-35), test drug sensitivity of parasite strains (29, 30), develop genetic transformation (45), and to test the phenotype of gene knockouts (20).In the 70 plus years since its isolation, the RH strain has been widely distributed and become the most commonly used laboratory strain. Previous genetic analysis has shown that, whereas type I strains are highly similar, isolates of RH obtained from different laboratories show minor genetic differences in some polymorphic DNA markers (18). This likely reflects minor genetic drift; however, the significance of these relatively small differences is unclear. To determine whether long-term in vitro passage may have affected growth, we compared a number of type I strains by using a plaque formation assay. Surprisingly, this comparison revealed that the clonal isolate RH-ERP differed substantially in plaque size from other type I strains. Additional differences were apparent in extracellular survival after egress, growth rate, differentiation, and changes in gene expression. Our findings indicate that the isolation of cloned lines can give rise to variants that are no longer representative of natural isolates, demonstrating the plasticity of phenotypic traits and limiting the utility of laboratory isolates.     

Guanylate-binding Protein 1 (Gbp1) Contributes to Cell-autonomous Immunity against Toxoplasma gondii

IFN-γ activates cells to restrict intracellular pathogens by upregulating cellular effectors including the p65 family of guanylate-binding proteins (GBPs). Here we test the role of Gbp1 in the IFN-γ-dependent control of T. gondii in the mouse model. Virulent strains of T. gondii avoided recruitment of Gbp1 to the parasitophorous vacuole in a strain-dependent manner that was mediated by the parasite virulence factors ROP18, an active serine/threonine kinase, and the pseudokinase ROP5. Increased recruitment of Gbp1 to Δrop18 or Δrop5 parasites was associated with clearance in IFN-γ-activated macrophages in vitro, a process dependent on the autophagy protein Atg5. The increased susceptibility of Δrop18 mutants in IFN-γ-activated macrophages was reverted in Gbp1^−/− cells, and decreased virulence of this mutant was compensated in Gbp1^−/− mice, which were also more susceptible to challenge with type II strain parasites of intermediate virulence. These findings demonstrate that Gbp1 plays an important role in the IFN-γ-dependent, cell-autonomous control of toxoplasmosis and predict a broader role for this protein in host defense.     

Fitness of Virulent Meloidogyne incognita Isolates on Susceptible and Resistant Cowpea

A study of life-history traits was made to determine factors associated with the fitness of Meloidogyne incognita isolates virulent to resistance gene Rk in cowpea. Egg hatch, root penetration, egg mass production, and fecundity (eggs per egg mass) of avirulent and virulent phenotypes were compared among M. incognita isolates, isofemale lines, and single descent lines over multiple generations on resistant and susceptible cowpea. Variation (P ≤ 0.05) in both hatch and root penetration rates was found among isolates at a given generation. However, this variation was not consistent within nematode lines among generations, and there was no correlation with level of virulence, except for penetration and virulence on resistant cowpea at generation 20. Resistant and susceptible cowpea roots were penetrated at similar levels. Differences in reproductive factors on resistant plants were correlated with levels of virulence expression. In some isofemale lines, single descent lines, and isolates, lower (P ≤ 0.05) rates of egg mass production and fecundity on susceptible cowpea were associated with virulence to Rk, indicating a trade-off between reproductive fitness and virulence. Other virulent nematode lines from the same isolates did not have reduced reproductive ability on susceptible cowpea over 27 generations. Thus, virulent lineages varied in reproductive ability on susceptible cowpea, contributing to adaptation and maintenance of virulence within M. incognita populations under stabilizing selection.     

Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X‐ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate‐binding site. ROP kinases contain a typical bilobate kinase fold and a novel N‐terminal extension that both stabilizes the N‐lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N‐terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N‐terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP‐binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence

At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence.     

Abundance of the Quorum-Sensing Factor Ax21 in Four Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor.