Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method.

Paramecium spp. are a genus of free-living protists that live mainly in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium's unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

RtxA like protein contributes to infection of Francisella novicida in silkworm and human macrophage THP-1.

Tularemia is a zoonosis caused by CDC-declared Tier 1 threat agent Francisella tularensis. F. tularensis subsp. novicida (F. novicida) is virulent in mice but non-pathogenic in immunocompetent humans and serves as a potential surrogate organism. In a recent study, we established a silkworm (Bombyx mori) model of infection for F. novicida. Francisella secretes its virulence factors through various mechanisms that modify the intracellular environment to ensure its replication and survival. To identify new pathogenic factors, we focused on the type I secretory system (T1SS) of Francisella. In silico analysis revealed a RtxA (Repeats-in-toxin) like protein in the Francisella genome. The characteristics of RtxA like protein were investigated using mutant analysis. Firstly, the role of rtxA in silkworms was investigated by infecting them with F. novicida strains into the hemocoel. The rtxA mutant failed to kill the silkworms, whereas F. novicida wild-type (WT) strain killed silkworms within 3-7 days post infection. The arrested growth of the mutant strain in silkworms was observed using a whole-body CFU count assay. We also investigated the growth characteristics of the rtxA mutant in hemocytes, one of the primary multiplication sites of Francisella within silkworms. Interrupted growth of the rtxA mutant with significantly reduced cytotoxicity was observed in hemocytes via confocal microscopy. Next, we analyzed the effect of rtxA in human monocyte cell line THP-1. The mutant strain showed significantly decreased growth and reduced cytotoxicity compared with its parental strain in THP-1 cells. This study newly identified RtxA like protein of F. novicida as an important lethal pathogenic factor in silkworm and mammalian cells.

Species distribution, virulence factors, and antimicrobial resistance of Acinetobacter spp. isolates from dogs and cats: a preliminary study.

We investigated the prevalence of virulence factors and antimicrobial resistance among 67 Acinetobacter spp. isolates, consisting of 21 Acinetobacter baumannii and 46 non-baumannii Acinetobacter from companion animals. The PCR analysis showed that the most prevalent virulence gene was afa/draBC (29.9%), followed by papC (22.4%) and cvaC (20.9%). Antimicrobial susceptibility testing revealed that resistance to gentamicin (14.9%) and ciprofloxacin (11.9%) was relatively prevalent. Five gentamicin- and/or ciprofloxacin-resistant A. baumannii strains were assigned to ST25, ST149, ST164, ST203, and ST1198. All ciprofloxacin-resistant isolates harbored point mutations in gyrA and/or parC. This is the first preliminary monitoring of animal-origin Acinetobacter spp. in Japan.

Silkworm model for Francisella novicida infection.

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida, which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm (Bombyx mori) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida. We found that silkworms died within 3-7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella.

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.

Analysis of IMP-1 type metallo-ß-lactamase-producing Acinetobacter radioresistens isolated from companion animals.

IMP-1 type metallo-ß-lactamase-producing (MBL-producing) Acinetobacter radioresistens was isolated from a dog with cystitis and a cat with conjunctivitis. The MBL-producing A. radioresistens isolates were resistant to all of the ß-lactam antibiotics used in the sensitivity tests, but were susceptible to gentamicin, amikacin, and minocycline. Also, one of the two strains of A. radioresistens was susceptible to ciprofloxacin and levofloxacin. These two cases were cured by administration of tetracyclines and fluoroquinolones, which elicited a positive result in the sensitivity tests. This report of the isolation of MBL-producing A. radioresistens in companion animals is the first in the world. To prevent the proliferation of MBL-producing bacteria, veterinary hospitals need to be aware of the behavior of MBL-producing organisms.

Influence of platelet-activating factor receptor (PAFR) on Brucella abortus infection: implications for manipulating the phagocytic strategy of B. abortus.

BACKGROUND: Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. RESULTS: First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. CONCLUSIONS: This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.

Mutation of purD and purF genes further attenuates Brucella abortus strain RB51.

In the present study, transposon mutagenesis was used to further attenuate Brucella abortus RB51 vaccine strain. Two purD and purF mutants were constructed, characterized and evaluated for attenuation via intracellular survival in murine macrophage-like RAW264.7 and HeLa cells, and by clearance in BALB/c mice. The purD and purF mutants showed significantly decreased intracellular survival, and complementation of these mutants with intact copies of purD or purF genes of RB51 strain was able to restore these defects. In addition, the pur mutants presented significantly lowered persistence in mice. Immunization with purD and purF mutants protected mice against a challenge with the virulent B. abortus strain 544 at a level similar to that of the parent RB51. These data suggest that genes encoding the early stages of purine biosynthesis (purD and purF) are required for intracellular survival and virulence of B. abortus.

Interplay between clathrin and Rab5 controls the early phagocytic trafficking and intracellular survival of Brucella abortus within HeLa cells.

Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.

Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4.

Mycoplasma pneumoniae causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans. The pathogenesis of M. pneumoniae infection is attributed to excessive immune responses. We previously demonstrated that M. pneumoniae lipoproteins induced inflammatory responses through Toll-like receptor 2 (TLR2). In the present study, we demonstrated that M. pneumoniae induced strong inflammatory responses in macrophages derived from TLR2 knockout (KO) mice. Cytokine production in TLR2 KO macrophages was increased compared with that in the macrophages of wild-type (WT) mice. Heat-killed, antibiotic-treated, and overgrown M. pneumoniae failed to induce inflammatory responses in TLR2 KO macrophages. 3-Methyladenine and chloroquine, inhibitors of autophagy, decreased the induction of inflammatory responses in TLR2 KO macrophages. These inflammatory responses were also inhibited in macrophages treated with the TLR4 inhibitor VIPER and those obtained from TLR2 and TLR4 (TLR2/4) double-KO mice. Two mutants that lacked the ability to induce inflammatory responses in TLR2 KO macrophages were obtained by transposon mutagenesis. The transposons were inserted in atpC encoding an ATP synthase F0F1 ε subunit and F10_orf750 encoding hypothetical protein MPN333. These mutants showed deficiencies in cytadherence. These results suggest that cytadherence of M. pneumoniae induces inflammatory responses through TLR4 and autophagy.

Cutting Edge: Brucella abortus exploits a cellular prion protein on intestinal M cells as an invasive receptor.

Brucella abortus is a Gram-negative bacterium causing brucellosis. Although B. abortus is known to infect via the oral route, the entry site in the gastrointestinal tract has been unclear. We found that B. abortus was selectively internalized by microfold cells (M cells), a subset of epithelial cells specialized for mucosal Ag uptake. During this process, colocalization of cellular prion protein (PrP(C)) and B. abortus was evident on the apical surface as well as in subapical vacuolar structures in M cells. Internalization of B. abortus by M cells of PrP(C)-deficient (Prnp(-/-)) mice was greatly reduced compared with that in wild-type mice. Furthermore, an oral infection study revealed that translocation of B. abortus into the Peyer's patch was significantly lower in Prnp(-/-) than in wild-type mice. These observations suggest that orally infected B. abortus invades the host through M cells by using PrP(C) on the apical surface of M cells as an uptake receptor.

Toll-like receptor 4-linked Janus kinase 2 signaling contributes to internalization of Brucella abortus by macrophages.

Brucella abortus is an intracellular pathogen that uses a crafty strategy to invade and proliferate within host cells, but the distinct signaling pathways associated with phagocytic mechanisms of B. abortus remain unclear. The present study was performed to test the hypothesis that Toll-like receptor 4 (TLR4)-linked signaling interacting with Janus kinase 2 (JAK2) plays an essential role in B. abortus phagocytosis by macrophages. The effects of TLR4-JAK2 signaling on B. abortus phagocytosis in murine macrophage RAW 264.7 cells were observed through an infection assay and confocal microscopy. We determined that the uptake of B. abortus was negatively affected by the dysfunction of TLR4 and JAK2. F-actin polymerization detected by flow cytometry and F-actin assay was amplified for B. abortus entry, whereas that event was attenuated by the disruption of TLR4 and JAK2. Importantly, JAK2 phosphorylation and actin skeleton reorganization were suppressed immediately after B. abortus infection in bone marrow-derived macrophages (BMDMs) from TLR4(-/-) mice, showing the cooperation of JAK2 with TLR4. Furthermore, small GTPase Cdc42 participated in the intermediate pathway of TLR4-JAK2 signaling on B. abortus phagocytosis. Consequently, TLR4-associated JAK2 activation in the early cellular signaling events plays a pivotal role in B. abortus-induced phagocytic processes in macrophages, implying the pathogenic significance of JAK2-mediated entry. Here, we elucidate that this specific phagocytic mechanism of B. abortus might provide achievable strategies for inhibiting B. abortus invasion.

RGS2-mediated intracellular Ca2+ level plays a key role in the intracellular replication of Brucella abortus within phagocytes.

BACKGROUND: Brucella abortus can proliferate within professional and nonprofessional phagocytic host cells and thereby successfully bypass the bacteriocidal effects of phagocytes. However, the intracellular survival mechanism and factors of virulence are not fully understood. METHODS: We have investigated the role of the regulator of G protein signaling 2 (RGS2), an intracellular calcium ([Ca(2+)](i)) regulator of the host cell, in the intracellular survival of B. abortus within phagocytes. RESULTS: B. abortus infection markedly induced RGS2 messenger RNA expression in early phase and increased the [Ca(2+)](i) level up to 24 hours postinfection within macrophages from wild-type mice. The [Ca(2+)](i) level, however, was not influenced by B. abortus infection within macrophages from RGS2-deficient mice. Furthermore, B. abortus survival was reduced within RGS2-deficient macrophages, and hence bacterial proliferation was inhibited in RGS2-deficient mice. Moreover, treatment with the Ca(2+) chelator ethylenediaminetetraacetic acid (EDTA) or 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and the L-type Ca(2+) channel-blocking agent nifedipine or genistein also showed a reduced intracellular replication of B. abortus within macrophages. CONCLUSION: These results indicate that B. abortus infection induces host RGS2 expression and that up-regulation of [Ca(2+)](i) levels is an essential factor for the intracellular survival of B. abortus within phagocytes.

The association between gingivitis and oral spirochetes in young cats and dogs.

Although gingivitis frequently occurs in young cats, spirochetes are often found in the early stages of periodontal disease. This study was conducted to determine the association between gingivitis and oral spirochetes in young cats and dogs. The degree of gingivitis was evaluated in a total of 68 cats and 31 dogs under one year of age, and plaques were collected from each carnassial. To detect spirochetes or Porphyromonas gulae in plaque samples, 16S rRNA gene was amplified by polymerase chain reaction (PCR) using specific primers. All data were analyzed using Fisher's exact probability test and odds ratio (OR) with a 95% confidence interval (95% CI). The prevalence of gingivitis was significantly higher in young cats (92.6%) than in young dogs (45.2%). The positive rate of spirochetes by PCR in gingivitis cases was 85.4% in young cats and 15.4% in young dogs, and the positive rate of P. gulae was 66.7% in young cats and 15.4% in young dogs. Both results were significantly higher in young cats than in young dogs. In young cats, spirochetes were significantly associated with gingivitis (OR = 7.95; 95% CI = 1.17, 53.83; P < 0.05), but P. gulae was not (OR = 2.44; 95% CI = 0.38, 15.66; P = 0.23). These results suggest that spirochetes may be associated with the early stages of periodontal disease in cats.

Characterization of intracellular localization of PrP(Sc) in prion-infected cells using a mAb that recognizes the region consisting of aa 119-127 of mouse PrP.

Generation of an abnormal isoform of the prion protein (PrP(Sc)) is a key aspect of the propagation of prions. Elucidation of the intracellular localization of PrP(Sc) in prion-infected cells facilitates the understanding of the cellular mechanism of prion propagation. However, technical improvement in PrP(Sc)-specific detection is required for precise analysis. Here, we show that the mAb 132, which recognizes the region adjacent to the most amyloidogenic region of PrP, is useful for PrP(Sc)-specific detection by immunofluorescence assay in cells pre-treated with guanidine thiocyanate. Extensive analysis of the intracellular localization of PrP(Sc) in prion-infected cells using mAb 132 revealed the presence of PrP(Sc) throughout endocytic compartments. In particular, some of the granular PrP(Sc) signals that were clustered at peri-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrP(Sc) signals at peri-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 °C, at which temperature transport from the plasma membrane to peri-nuclear regions was impaired. Conversely, dispersed PrP(Sc) signals appeared to return to peri-nuclear regions within 30 min during subsequent incubation at 37 °C, following which PrP(Sc) at peri-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrP(Sc) is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrP(Sc) circulates between peri-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.

Francisella novicida can utilize Paramecium bursaria as its potential host.

Francisella novicida is a facultative intracellular pathogen and the causative agent of tularemia. Although cases of infection caused by exposure to contaminated water have been reported, its natural host and ecology in the environment remain unclear. In this study, we investigated in vitro the possibility that Paramecium bursaria may be a useful tool as a protist host model of F. novicida. Experimental infection with F. novicida resulted in a stable intracellular relationship within P. bursaria. This symbiotic intracellular relationship was not observed in experimental infections with other Francisella species and Legionella pneumophila. We found that F. novicida showed similar behaviour to that of the eukaryotic endosymbiont of P. bursaria, the green algae Chlorella, in the internalization process. In addition, stable intracellular localization of F. novicida was possible only when Chlorella was not present. Although we investigated the type VI secretion system of F. novicida as a candidate for the bacterial factor, we found that it was not involved in the establishment of an intracellular relationship with P. bursaria. These results suggested that P. bursaria is potentially a protist host model for F. novicida and may be a useful tool for understanding the relationship between protist hosts and their symbionts.

Distinction of Paramecium strains by a combination method of RAPD analysis and multiplex PCR.

Paramecium is employed as a valuable model organism in various research fields since a large number of strains with different characteristics of size, morphology, degree of aging, and type of conjugation can be obtained. It is necessary to determine a method for the classification and simple identification of strains to increase their utility as a research tool. This study attempted to establish a polymerase chain reaction (PCR)-based method to differentiate strains of the same species. Genomic DNA was purified from several strains of P. caudatum, P. tetraurelia, and P. bursaria used for comparison by the random amplified polymorphic DNA (RAPD)-PCR method. In P. tetraurelia and P. bursaria, it was sufficiently possible to distinguish specific strains depending on the pattern of random primers and amplification characteristics. For the classification of P. caudatum, based on the sequence data obtained by RAPD-PCR analysis, 5 specific primer sets were designed and a multiplex PCR method was developed. The comparative analysis of 2 standard strains, 12 recommended strains, and 12 other strains of P. caudatum provided by the National BioResource Project was conducted, and specific strains were identified. This multiplex PCR method would be an effective tool for the simple identification of environmental isolates or the management of Paramecium strains.

Identification of pyrC gene as an immunosuppressive factor in Francisella novicida infection.

Francisella tularensis, a bacterial causative agent of the zoonosis tularemia, is highly pathogenic to humans. The pathogenicity of this bacterium is characterized by intracellular growth in immune cells, like macrophages, and host immune suppression. However, the detailed mechanism of immune suppression by F. tularensis is still unclear. To identify the key factors causing Francisella-mediated immunosuppression, large-scale screening using a transposon random mutant library containing 3552 mutant strains of F. tularensis subsp. novicida (F. novicida) was performed. Thirteen mutants that caused stronger tumor necrosis factor (TNF)-α production in infected U937 human macrophage cells than the wild-type F. novicida strain were isolated. Sequencing analysis of transposon insertion sites revealed 10 genes, including six novel genes, as immunosuppressive factors of Francisella. Among these, the relationship of the pyrC gene, which encodes dihydroorotase in the pyrimidine biosynthesis pathway, with Francisella-mediated immunosuppression was investigated. The pyrC deletion mutant strain (ΔpyrC) induced higher TNF-α production in U937 host cells than the wild-type F. novicida strain. The ΔpyrC mutant strain was also found to enhance host interleukin-1ß and interferon (IFN)-ß production. The heat-inactivated ΔpyrC mutant strain could not induce host TNF-α production. Moreover, the production of IFN-ß resulting from ΔpyrC infection in U937 cells was repressed upon treatment with the stimulator of interferon genes (STING)-specific inhibitor, H-151. These results suggest that pyrC is related to the immunosuppressive activity and pathogenicity of Francisella via the STING pathway.

The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation.

Intracellular pathogens lose many metabolic genes during their evolution from free-living bacteria, but the pathogenic consequences of their altered metabolic programs on host immunity are poorly understood. Here, we show that a pathogenic strain of Francisella tularensis subsp. tularensis (FT) has five amino acid substitutions in RibD, a converting enzyme of the riboflavin synthetic pathway responsible for generating metabolites recognized by mucosal-associated invariant T (MAIT) cells. Metabolites from a free-living strain, F. tularensis subsp. novicida (FN), activated MAIT cells in a T-cell receptor (TCR)-dependent manner, whereas introduction of FT-type ribD to the free-living strain was sufficient to attenuate this activation in both human and mouse MAIT cells. Intranasal infection in mice showed that the ribD FT-expressing FN strain induced impaired Th1-type MAIT cell expansion and resulted in reduced bacterial clearance and worsened survival compared with the wild-type free-living strain FN. These results demonstrate that F. tularensis can acquire immune evasion capacity by alteration of metabolic programs during evolution.

Cellular prion protein promotes Brucella infection into macrophages.

The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.