Protective effects of in vivo-expressed autotransporters against Bordetella pertussis infection.

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re-emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica.

Bordetella species display phase modulation between Bvg(+) and Bvg(-) phases. Because expression of known virulence factors is up-regulated in the Bvg(+) phase, bacteria in this phase are considered competent for infection. However, the Bvg(-) phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg(-) 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(2+) -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.

Detection of genes expressed in Bordetella bronchiseptica colonizing rat trachea by in vivo expressed-tag immunoprecipitation method.

Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection.

Identification of a novel protein localized to the crystalloid of the Plasmodium ookinete.

Reducing Plasmodium parasite transmission via the mosquito vector is a promising strategy for malaria control and elimination in endemic regions. In the mosquito midgut after the ingestion of an infected blood meal, malaria parasite gametes egress from erythrocytes and fertilize to develop into motile ookinetes that traverse midgut epithelial cells and transform into oocysts adjacent the basal lamina. Plasmodium ookinetes and young oocysts possess a unique organelle called the crystalloid; which has a honeycomb-like matrix structure and is indicated to be involved in sporozoite formation and maturation. In this study, we identified a novel crystalloid protein, PY17X_1113800, that is exclusively expressed in developing ookinetes. The protein possesses a signal peptide sequence, but lacks a transmembrane domain or GPI anchor signal sequence, as well as predicted adhesive domains which are characterisitic of many crystalloid proteins. The protein is highly conserved across the phylum Apicomplexa and within the greater clade Alveolata, such as Vitrella and the ciliates Paramecium and Tetrahymena, but is absent in cryptosporidia.

Activation of Imd pathway in hemocyte confers infection resistance through humoral response in Drosophila.

Upon microbial invasion the innate immune system of Drosophila melanogaster mounts a response that comes in two distinct but complimentary forms, humoral and cellular. A screen to find genes capable of conferring resistance to the Gram-positive Staphylococcus aureus upon ectopic expression in immune response tissues uncovered imd gene. This resistance was not dependent on cellular defenses but rather likely a result of upregulation of the humoral response through increased expression of antimicrobial peptides, including a Toll pathway reporter gene drosomycin. Taken together it appears that Imd pathway is capable of playing a role in resistance to the Gram-positive S. aureus, counter to notions of traditional roles of the Imd pathway thought largely to responsible for resistance to Gram-negative bacteria.

Complete genome sequences of nine Burkholderia pseudomallei strains.

We report the complete genome sequences of nine Burkholderia pseudomallei strains preserved in research facilities in Japan; GTC3P0019, GTC3P0050, GTC3P0054, GTC3P0254T (type strain), Kanagawa, Tokushima, KM376, KM390, and KM391. The genomic information of these strains may provide references for comparative studies of B. pseudomallei pathogenicity.

Legionella metaeffector exploits host proteasome to temporally regulate cognate effector.

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.

Evaluation of the Effect of Gene Duplication by Genome Editing on Drug Resistance in Plasmodium falciparum.

The emergence and spread of drug-resistant Plasmodium falciparum have compromised antimalarial efficacy and threatened the global malaria elimination campaign using artemisinin combination therapies. The impacts of amino acid substitutions in antimalarial drug resistance-associated genes on drug susceptibility have been investigated; however, the effects of amplification of those genes remain unexplored due to the lack of robust genetic approaches. Here, we generated transgenic P. falciparum parasites with an additional copy of a drug resistance-associated gene using the highly efficient CRISPR/Cas9 system and investigated their drug response. Insertion of a drug resistance-associated gene expression cassette in the genome resulted in a roughly twofold increase of mRNA levels of the target gene mdr1, which encodes multidrug resistance protein 1. The gene duplication event contributed to resistance to mefloquine, lumefantrine, and dihydroartemisinin, while the duplication of a genomic region encoding plasmepsin 2 and plasmepsin 3 did not affect resistance to antimalarial drugs, including piperaquine. We further demonstrated that mdr1 mRNA expression levels are strongly associated with mefloquine resistance in several field-derived P. falciparum lines with various genetic backgrounds. This study provides compelling evidence that mdr1 could serve as a molecular marker for the surveillance of mefloquine-resistant parasites. Long DNA integration into parasite genomes using the CRISPR/Cas9 system provides a useful tool for the evaluation of the effect of copy number variation on drug response.

Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system.

Spatial arrangement of chromosomes is responsible for gene expression in Plasmodium parasites. However, methods for rearranging chromosomes have not been established, which makes it difficult to investigate its role in detail. Here, we report a method for splitting chromosome in rodent malaria parasite by CRISPR/Cas9 system using fragments in which a telomere and a centromere were incorporated. The resultant split chromosomes segregated accurately into daughter parasites by the centromere. In addition, elongation of de novo telomeres were observed, indicating its proper function. Furthermore, chromosome splitting had no effect on development of parasites. Splitting of the chromosome is expected to alter its spatial arrangement, and our method will thus be useful for investigating its biological role related with gene expression.

Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum.

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development.

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.

Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template.

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.

AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum.

Precise subcellular localization of proteins is the key to elucidating the physiological role of these molecules in malaria parasite development, understanding of pathogenesis, and protective immunity. In Plasmodium falciparum, however, detection of proteins in the blood-stage parasites is greatly hampered by the lack of versatile protein tags which can intrinsically label such molecules. Thus, in this study, to develop a novel system that can be used to evaluate subcellular localization of known and novel proteins, we assessed the application of AGIA tag, consisting of 9 amino acids (EEAAGIARP), in P. falciparum blood-stage parasites. Specifically, AGIA-tagged ring-infected erythrocyte surface antigen (RESA-AGIA) was episomally expressed in P. falciparum 3D7 strain. The RESA-AGIA protein was detected by Western blotting and immunofluorescence assay (IFA) using recombinant rabbit anti-AGIA tag monoclonal antibody (mAb) with a high signal/noise ratio. Similarly, AGIA-tagged multidrug resistance protein 1 (MDR1-AGIA), as an example of polyptic transmembrane protein, was endogenously expressed and detected by Western blotting and IFA with anti-AGIA tag mAb. Immunoelectron microscopy of the RESA-AGIA transfected merozoites revealed that mouse anti-RESA and the rabbit anti-AGIA mAb signals could definitively co-localize to the dense granules. Put together, this study demonstrates AGIA tag/anti-AGIA rabbit mAb system as a potentially useful tool for elucidating the subcellular localization of new and understudied proteins in blood-stage malaria parasites at the nanometer-level resolution.

ADF2 is required for transformation of the ookinete and sporozoite in malaria parasite development.

Malaria parasites undergo two rounding-up transformations in their life cycle: the ookinete-to-oocyst transformation in the mosquito midgut, and the sporozoite-to-EEF (exo-erythrocytic form) differentiation in the host hepatocyte. Both events are characterized by the loss of polarity, implying that cytoskeletal reorganization is involved. In other eukaryotes, regulation of the actin skeleton is fundamental to subcellular remodeling. Although filamentous actin is well known to be involved in the motility of apicomplexan parasites, its participation in their morphological regulation is still largely unexplored. Here we describe the fundamental role of Actin depolymerization factor 2 (ADF2), a vector-stage-specific ADF isoform, in morphological changes accompanying the parasite life cycle. Among protozoan parasites, Plasmodium is unique in having two actin and two ADF genes. Disruption of the ADF2 gene in the rodent malaria parasite P. berghei had no effect on ookinete development or its subsequent invasion of the midgut. However, both the ookinete-to-oocyst and sporozoite-to-EEF transformations showed significant defects. These results indicated that Plasmodium ADF2 plays a pivotal role in transformation in the malaria parasite life cycle.

Improvement of CRISPR/Cas9 system by transfecting Cas9-expressing Plasmodium berghei with linear donor template.

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 large-scale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the post-genomic era of Plasmodium species.

Rab5b-Associated Arf1 GTPase Regulates Export of N-Myristoylated Adenylate Kinase 2 From the Endoplasmic Reticulum in Plasmodium falciparum.

Plasmodium falciparum extensively remodels human erythrocytes by exporting hundreds of parasite proteins. This remodeling is closely linked to the Plasmodium virulence-related functions and immune evasion. The N-terminal export signal named PEXEL (Plasmodium export element) was identified to be important for the export of proteins beyond the PVM, however, the issue of how these PEXEL-positive proteins are transported and regulated by Rab GTPases from the endoplasmic reticulum (ER) to the cell surface has remained poorly understood. Previously, we identified new aspects of the trafficking of N-myristoylated adenylate kinase 2 (PfAK2), which lacks the PEXEL motif and is regulated by the PfRab5b GTPase. Overexpression of PfRab5b suppressed the transport of PfAK2 to the parasitophorous vacuole membrane and PfAK2 was accumulated in the punctate compartment within the parasite. Here, we report the identification of PfRab5b associated proteins and dissect the pathway regulated by PfRab5b. We isolated two membrane trafficking GTPases PfArf1 and PfRab1b by coimmunoprecipitation with PfRab5b and via mass analysis. PfArf1 and PfRab1b are both colocalized with PfRab5b adjacent to the ER in the early erythrocytic stage. A super-resolution microgram of the indirect immunofluorescence assay using PfArf1 or PfRab1b- expressing parasites revealed that PfArf1 and PfRab1b are localized to different ER subdomains. We used a genetic approach to expresses an active or inactive mutant of PfArf1 that specifically inhibited the trafficking of PfAK2 to the parasitophorous vacuole membrane. While expression of PfRab1b mutants did not affect in the PfAK2 transport. In contrast, the export of the PEXEL-positive protein Rifin was decreased by the expression of the inactive mutant of PfRab1b or PfArf1. These data indicate that the transport of PfAK2 and Rifin were recognized at the different ER subdomain by the two independent GTPases: PfAK2 is sorted by PfArf1 into the pathway for the PV, and the export of Rifin might be sequentially regulated by PfArf1 and PfRab1b.

The Bartonella autotransporter BafA activates the host VEGF pathway to drive angiogenesis.

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.

Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor.

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.IMPORTANCEBordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

Rapid recruitment of innate immunity regulates variation of intracellular pathogen resistance in Drosophila.

Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System.

We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B. pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B. bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant mutant strain of B. pertussis. The present results demonstrate that this system successfully accomplished the above-described purpose. We consider this system to be applicable to a number of bacteria other than Bordetella.