Pathogen-associated secretion systems translocate numerous effector proteins into eukaryotic host cells to coordinate cellular processes important for infection. Spatiotemporal regulation is therefore important for modulating distinct activities of effectors at different stages of infection. Here we provide the first evidence of “metaeffector,” a designation for an effector protein that regulates the function of another effector within the host cell. Legionella LubX protein functions as an E3 ubiquitin ligase that hijacks the host proteasome to specifically target the bacterial effector protein SidH for degradation. Delayed delivery of LubX to the host cytoplasm leads to the shutdown of SidH within the host cells at later stages of infection. This demonstrates a sophisticated level of coevolution between eukaryotic cells and L. pneumophila involving an effector that functions as a key regulator to temporally coordinate the function of a cognate effector protein.

Hosts employ a combination of two distinct yet compatible strategies to defend themselves against parasites: resistance, the ability to limit parasite burden, and tolerance, the ability to limit damage caused by a given parasite burden. Animals typically exhibit considerable genetic variation in resistance to a variety of pathogens; however, little is known about whether animals can evolve tolerance. Using a bacterial infection model in Drosophila, we uncovered a p38 MAP kinase-mediated mechanism of tolerance to intracellular bacterial infection as measured by the extent to which the host's survival rate increased or was maintained despite increasing bacterial burden. This increased survival was conferred primarily by a tolerance strategy whereby p38-dependent phagocytic encapsulation of bacteria resulted in enlarged phagocytes that trap bacteria. These results suggest that phagocytic responses are not restricted to resistance mechanisms but can also be applied to tolerance strategies for intracellular encapsulation of pathogens during the invertebrate immune response.

Malaria is caused by infection with Plasmodium parasites and is a major public health concern. The CRISPR/Cas9 system is a promising technology, but still has technical problems, such as low efficiency and unexpected recombination. Here, we solved these problems by transfecting Cas9-expressing parasites with linear donor templates. The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for largescale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the postgenomic era of Plasmodium species.

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins—two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin—as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.

Background: Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, Aedes aegypti, and the canine heartworm, Dirofilaria immitis.

Bordetella species display phase modulation between Bvg þ and Bvg À phases. Because expression of known virulence factors is up-regulated in the Bvg þ phase, bacteria in this phase are considered competent for infection. However, the Bvg À phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg À phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca 2þ -dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.

Pathogenic bacteria of the genus Bartonella can induce vasoproliferative lesions during infection. The underlying mechanisms are unclear, but involve secretion of an unidentified mitogenic factor. Here, we use functional transposon-mutant screening in Bartonella henselae to identify such factor as a pro-angiogenic autotransporter, called BafA. The passenger domain of BafA induces cell proliferation, tube formation and sprouting of microvessels, and drives angiogenesis in mice. BafA interacts with vascular endothelial growth factor (VEGF) receptor-2 and activates the downstream signaling pathway, suggesting that BafA functions as a VEGF analog. A BafA homolog from a related pathogen, Bartonella quintana, is also functional. Our work unveils the mechanistic basis of vasoproliferative lesions observed in bartonellosis, and we propose BafA as a key pathogenic factor contributing to bacterial spread and host adaptation.

The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.