Tsuneo Uchiyama

Rickettsia japonica sp. nov., the Etiological Agent of Spotted Fever Group Rickettsiosis in Japan

We propose the name Rickettsia japonica sp. nov. (with type strain YH [= ATCC VR-1363]) for a serologically specific species of spotted fever group rickettsiae that are pathogenic for humans (J. Infect. Dis. 159:1122-1126, 1989; J. Clin. Microbiol. 28:1177-1180, 1990). The biologic and genomic characteristics of the organism (G+C content, 31.2 +/- 0.7 mol%) are essentially the same as those of other pathogenic spotted fever group rickettsiae, although the R. japonica isolates cause a persistent infection in Vero cells for many subcultures.

Rickettsial Outer-Membrane Protein B (rOmpB) Mediates Bacterial Invasion through Ku70 in an Actin, c-Cbl, Clathrin and Caveolin 2-Dependent Manner

Rickettsia conorii, an obligate intracellular tick‐borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non‐phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70–rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70‐dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB‐mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3‐kinase‐dependent activities and microtubule stability. Small‐interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c‐Cbl, clathrin and caveolin‐2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70‐dependent invasion of mammalian cells and that clathrin‐ and caveolin‐dependent endocytic events likely contribute to the internalization process.

Generation of HIV-1 derivatives that productively infect macaque monkey lymphoid cells

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is caused in part by innate cellular factors such as apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) and TRIM5α, which restrict virus replication in monkey cells. Variant HIV-1 molecular clones containing both a 21-nucleotide simian immunodeficiency virus (SIV) Gag CA element, corresponding to the HIV-1 cyclophilin A-binding site, and the entire SIV vif gene were constructed. Long-term passage in a cynomolgus monkey lymphoid cell line resulted in the acquisition of two nonsynonymous changes in env, which conferred improved replication properties. A proviral molecular clone, derived from infected cells and designated NL-DT5R, was used to generate virus stocks capable of establishing spreading infections in the cynomolgus monkey T cell line and CD8-depleted peripheral blood mononuclear cells from five of five pig-tailed macaques and one of three rhesus monkeys. NL-DT5R, which genetically is >93% HIV-1, provides the opportunity, not possible with currently available SIV/HIV chimeric viruses, to analyze the function of multiple HIV-1 genes in a broad range of nonhuman primate species.

Adherence to and invasion of Vero cells by recombinant Escherichia coli expressing the outer membrane protein rOmpB of Rickettsia japonica.

Abstract: Recombinant Escherichia coli expressing the outer membrane protein rOmpB of rickettsiae on the surface were generated. The DNA corresponding to the open reading frame of the ompB gene of a spotted fever group rickettsia, Rickettsia japonica, was amplified by polymerase chain reaction. The amplified fragment was inserted between the Sal I and the Xho I sites of the expression vector pET‐22b(+). E. coli BL21(DE3) was transformed by the constructed plasmid. The recombinant bacteria expressed a recombinant protein with a molecular size of 165 kilodaltons on the surface. The size was consistent with that of the precursor of rOmpB. The protein was reactive with monoclonal antibodies to heat‐labile epitopes of the rOmpB. This result suggested a rather native conformation of the recombinant protein. Immunofluorescence of the recombinant bacteria demonstrated the surface expression of the protein. The recombinant bacteria acquired properties to enter Vero cells. The morphological change was observed by means of transmission electron microscopy and scanning electron microscopy. Adherence triggered the generation of abundant microvilli and membrane ruffling for the cells to engulf the bacteria The manner of entry of the recombinant bacteria was similar to that of rickettsiae. Thus it is suggested that the rOmpB plays an important role in the adherence to and invasion of host cells by rickettsiae. Moreover, since even the recombinant rOmpB precursor protein expressed on the surface of the bacteria promotes adherence and invasion, the conformation of the functional domain may be similar to that of the processed mature rOmpB.

Pseudotyping human immunodeficiency virus type 1 by vesicular stomatitis virus G protein does not reduce the cell-dependent requirement of vif for optimal infectivity: functional difference between Vif and Nef.

The functions of Vif and Nef in human immunodeficiency virus type 1 (HIV-1) infection have some similarities: Vif- and Nef-dependent enhancement of HIV-1 replication is cell type-specific, and defective mutations in these genes result in restricted proviral DNA synthesis in infected cells. It has recently been shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV-1 entry to an endocytic pathway and suppresses the requirement of Nef for virus infectivity. In this study, we examined whether VSV-G pseudotyping suppresses the requirement of Vif for HIV-1 infectivity. It was found that pseudotyping HIV-1 by VSV-G did not compensate for the Vif function. Together with the findings that Vif does not influence virus binding/entry and virion incorporation of Env, it is concluded that Vif enhances HIV-1 infectivity at the post-entry step(s) independently of the Env function by a different mechanism to that of Nef.

Ultrastructural Study on Japanese Isolates of Spotted Fever Group Rickettsiae

Japanese isolates of spotted fever group rickettsiae were observed under a transmission electron microscope. In Vero cells persistently infected with Japanese isolates, small numbers of intracytoplasmic rickettsiae were seen. On the other hand, moderate numbers of rickettsiae were found in the cytoplasm of productively infected BHK cells. The electron‐lucent, halo‐like zone was found to surround organisms in the cytoplasm of their host cells, which is a prominent characteristic of spotted fever group rickettsiae. Fine structural features of the cell wall revealed thin outer and thick inner leaflets like those observed in other spotted fever group rickettsiae.

Tropism and pathogenicity of rickettsiae.

Rickettsiae are obligate intracellular parasitic bacteria that cause febrile exanthematous illnesses such as Rocky Mountain spotted fever, Mediterranean spotted fever, epidemic, and murine typhus, etc. Although the vector ranges of each Rickettsia species are rather restricted; i.e., ticks belonging to Arachnida and lice and fleas belonging to Insecta usually act as vectors for spotted fever group (SFG) and typhus group (TG) rickettsiae, respectively, it would be interesting to elucidate the mechanisms controlling the vector tropism of rickettsiae. This review discusses the factors determining the vector tropism of rickettsiae. In brief, the vector tropism of rickettsiae species is basically consistent with their tropism toward cultured tick and insect cells. The mechanisms responsible for rickettsiae pathogenicity are also described. Recently, genomic analyses of rickettsiae have revealed that they possess several genes that are homologous to those affecting the pathogenicity of other bacteria. Analyses comparing the genomes of pathogenic and non-pathogenic strains of rickettsiae have detected many factors that are related to rickettsial pathogenicity. It is also known that a reduction in the rickettsial genome has occurred during the course of its evolution. Interestingly, Rickettsia species with small genomes, such as Rickettsia prowazekii, are more pathogenic to humans than those with larger genomes. This review also examines the growth kinetics of pathogenic and non-pathogenic species of SFG rickettsiae (SFGR) in mammalian cells. The growth of non-pathogenic species is restricted in these cells, which is mediated, at least in part, by autophagy. The superinfection of non-pathogenic rickettsiae-infected cells with pathogenic rickettsiae results in an elevated yield of the non-pathogenic rickettsiae and the growth of the pathogenic rickettsiae. Autophagy is restricted in these cells. These results are discussed in this review.

A Protein from a Parasitic Microorganism, Rickettsia prowazekii, Can Cleave the Signal Sequences of Proteins Targeting Mitochondria

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the alpha and beta subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with beta-MPP but barely contained any of the glycine-rich loop region characteristic of alpha-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast beta-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.

Preparation of Virosomes Coated with the Vesicular Stomatitis Virus Glycoprotein as Efficient Gene Transfer Vehicles for Animal Cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH‐dependent membrane‐fusion) were expected to work. Plasmid DNA (pEGFP‐N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5:1:4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 μm, 2 μm, 0.65 μm, and then 0.45 μm). Assembly of the VSV G protein‐coated liposomes (VSV G‐virosomes) was performed by mixing the DNA‐encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene‐transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G‐virosomes displayed a roughly similar level of gene‐transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti‐VSV G antiserum or by addition of 20 MM NH4Cl to transfected cultures. From these results, we assume that the virosome‐mediated gene‐transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.

Murine typhus from Vietnam, imported into Japan.

To the Editor: In Vietnam, many febrile diseases such as malaria, dengue fever, Japanese encephalitis, scrub typhus, and more recently, severe acute respiratory syndrome (SARS) and avian influenza have been reported. Murine typhus cases were also reported during and before the 1960s but not thereafter (1–5). On May 3, 2003, a 54-year-old male resident of Tokushima, Japan, had onset of fever in the suburban town of Cu Chi, ≈60 km northwest of Ho Chi Minh City, Vietnam. Exanthema appeared on his trunk and limbs on May 7. He returned to Japan on May 9 and was admitted to Tokushima University Hospital on May 10. His body temperature was 39.0°C, and serum, C-reactive protein level was high (17.06 mg/dL) on admission (day 8 of illness). Unfortunately, the blood sample taken on that day was discarded. We then collected blood on days 10, 11, 12, 14, 17, and 24 of illness for diagnosis. Minocycline was administered on day 8 and resulted in a gradual decrease in fever and rash. Weil-Felix tests on day 12 showed the serum to be positive for Proteus vulgaris OX19 (titer 160); tests for P. vulgaris OX2 and OXK were negative (titer of 10 for both). We examined blood samples for possible diseases such as malaria, dengue fever, SARS, and rickettsioses. Giemsa-stained peripheral blood samples obtained on day 11 showed no malarial parasites. Results of immunoglobulin M (IgM)-capture ELISA of serum on days 10, 11, and 17 of illness were negative for dengue antibodies. Reverse transcription (RT)–PCR of the serum on day 11 was also negative. RT-PCRs of a pharyngeal swab and urine collected on day 11 were both negative for the SARS coronavirus. These specimens were also injected into Vero cells, and no cytopathic effects were generated. RT-PCR of these cultures was also negative for SARS coronavirus. Moreover, SARS antibodies were not found in serum samples on days 11 and 14 of illness. Serum was also tested for Orientia tsutsugamushi and Coxiella burnttii on day 12 to exclude scrub typhus and Q fever as diagnoses. Indirect immunofluorescence tests for etiologic agents of spotted fever, murine typhus, and epidemic typhus were then performed with serum samples collected on days 10, 14, and 24. We used Rickettsia typhi and R. prowazekii as typhus group (TG) rickettsial antigens and R. japonica and R. conorii as spotted fever group (SFG) rickettsiae. IgM antibody was detected for these antigens, indicating that the disease was a primary infection of rickettsiae (Table). When TG and SFG rickettsioses were compared, TG rickettsiae represented markedly higher elevated titers than SFG rickettsiae, which excluded a diagnosis of SFG rickettsiosis. PCR for the TG rickettsial genome in the convalescent-phase serum on day 10 was negative. Table IFA titers of the patient sera and the cross-absorption test* To demonstrate more detailed antigenic reactivity, Western immunoblotting was performed with serum on day 14 (6). The serum reacted similarly to the ladderlike lipopolysaccharide (LPS) of R. typhi and R. prowazekii. As expected from the group-specific nature of rickettsial LPS, no reaction was demonstrated to LPS of SFG rickettsiae, R. japonica and R. conorii, although weak reactivity, mainly to the major outer member protein of SFG rickettiae, rOmpB, and molecules of smaller sizes was shown (6,7). As described previously, rOmpB has cross-reactive antigenicity between TG and SFG rickettsiae (6). Compared with the trace reaction to rOmpB of SFG rickettsiae, an extremely high level of reaction was demonstrated to rOmpB of TG rickettsiae. These results confirmed the disease to be a TG rickettsiosis. To elucidate whether the disease was murine typhus or epidemic typhus, we conducted cross-absorption tests as described previously (8,9). Serum absorbed by R. typhi showed complete absorption, demonstrating no reaction to R. typhi or R. prowazekii (Table). However, the serum absorbed by R. prowazekii resulted in incomplete absorption, demonstrating no reactivity to R. prowazekii but some reactivity to R. typhi, which was left unabsorbed. Western immunoblotting with the serum absorbed by R. prowazekii showed reactivity only to the rOmpB of R. typhi but not to that of R. prowazekii. These results confirmed the diagnosis of murine typhus. This is the first serodiagnosis of murine typhus in Vietnam since the 1960s (1–5). Since rats inhabit the area where the patient acquired the illness, murine typhus seems to have occurred sporadically or endemically but to have been undiagnosed since the 1960s, maybe because it was thought to have been eradicated and thus widely forgotten. This case was the first imported into Japan since the 1940s, when many Japanese soldiers and residents who returned from abroad had the disease.