Masahisa Watarai

Modulation of Brucella‐induced macropinocytosis by lipid rafts mediates intracellular replication

Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft‐associated molecules such as glycosylphosphatidylinositol (GPI)‐anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP‐1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI‐anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB‐dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.

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.

Detection of anthrax spores from the air by real-time PCR

Aims: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure.

Isolation and characterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient in internalization and intracellular growth in HeLa cells

ABSTRACT Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.

Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterization of virA mutants infecting MK2 epithelial cell monolayers revealed that their invasive capacity was decreased to less than one fifth of the wild‐type level. Nevertheless, the bacteria were capable of expressing and secreting IpaB, IpaC and IpaD proteins. The virA mutants were also impaired in their ability to spread intercellularly, since the bacteria gave rise to a small number of foci in a focus‐plaque‐forming test with MK2 cells. Although virG expression was slightly decreased in the virA mutants, introduction of a cloned virG gene into a virA mutant, N1945, failed to restore spreading ability. Although, introduction of a cloned virA gene into N1945 restored invasiveness and spreading ability, the reduced virG transcription level was not affected, indicating that the reduced virG expression in virA mutants does not play a major role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp upstream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with no homology to known proteins. The VirA protein was secreted into the culture supernatant, a process that required the Mxi and Spa loci. The expression of virA was under the control of the virB gene, the positive regulator of the ipa, mxi and spa operons. These results indicate that virA is a new member of the invasion regulon directed by virB and that the VirA function is involved in invasion and intercellular spreading.

Effect of the Lower Molecular Capsule Released from the Cell Surface of Bacillus anthracis on the Pathogenesis of Anthrax

Bacillus anthracis enters the body as an endospore, and encapsulation and toxin production occur after germination. Capsule is proposed to be an antiphagocytic factor, and toxin induces cytokine production for systemic shock. The dep gene, adjacent to the cap region for the encapsulation, degrades the high-molecular weight capsule (H-capsule) to the lower-molecular weight capsule (L-capsule), which releases into the culture supernatant. This study analyzed the biological function of the cap-dep region. The dep null mutant Sm-1, which formed H-capsule but not L-capsule, was avirulent. However, Sm-1 with an intact dep gene or with purified L-capsule recovered its pathogenicity. Sm-1 was subjected to phagocytosis by macrophages more easily than its parent strain, Sm, in vitro; in vivo, it cleared without L-capsule and grew well with L-capsule, which suggests that L-capsule is essential for in vivo multiplication. Moreover, a new name, capD, might be appropriate, because of the part of the cap operon involved in both polymerization and depolymerization of the capsule.

Interferon-γ promotes abortion due to Brucella infection in pregnant mice

BackgroundThe mechanisms of abortion induced by bacterial infection are largely unknown. In the present study, we investigated abortion induced by Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in a mouse model.ResultsHigh rates of abortion were observed for bacterial infection on day 4.5 of gestation, but not for other days. Regardless of whether fetuses were aborted or stayed alive, the transmission of bacteria into the fetus and bacterial replication in the placenta were observed. There was a higher degree of bacterial colonization in the placenta than in other organs and many bacteria were detected in trophoblast giant cells in the placenta. Intracellular growth-defective virB4 mutant and attenuated vaccine strain S19 did not induce abortion. In the case of abortion, around day 7.5 of gestation (period of placental development), transient induction of IFN-γ production was observed for infection by the wild type strain, but not by the virB4 mutant and S19. Neutralization of IFN-γ, whose production was induced by infection with B. abortus, served to prevent abortion.ConclusionThese results indicate that abortion induced by B. abortus infection is a result of transient IFN-γ production during the period of placental development.

Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth

Legionella pneumophila grows in human alveolar macrophages and resides within a phagosome that initially lacks proteins associated with the endocytic pathway. Required for targeting to this unique location is the Dot/Icm complex, which is highly similar to conjugative DNA transfer apparatuses. Here, we show that exposure to three distinct inducing conditions resulted in the formation of a fibrous structure on the bacterial cell surface that contained the DotH and DotO proteins. These conditions included: (i) incubation for 2 h with mouse bone marrow‐derived macrophages; (ii) incubation for 2 h in macrophage‐conditioned media; or (iii) replication of bacteria for 22 h within macrophages. Introduction of bacteria harbouring the surface‐exposed DotH and DotO onto a fresh monolayer resulted in loss of the surface localization of DotH and DotO shortly after uptake. Treatments that resulted in the production of the fibrous structure enhanced the rate at which the bacteria were internalized, but there was no corresponding increase in the efficiency of intracellular growth compared with bacteria that had been cultured in broth using conditions that resulted in maximal intracellular growth. These data indicate that the surface‐exposed DotH and DotO on L. pneumophila may act either just before lysis from the macrophage or at the earliest stages of infection, transiently relocating in a fibrous structure on the bacterial cell surface.

An essential virulence protein of Brucella abortus, VirB4, requires an intact nucleoside- triphosphate-binding domain

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. The VirB complex, which is highly similar to conjugative DNA transfer apparatuses, is required for intracellular replication. A conserved NTP-binding domain in VirB4 suggests that one or both proteins couple energy by NTP hydrolysis to transport of putative effector molecule(s). Here it is shown that a mutant strain of B. abortus that contains an in-frame deletion in virB4 is unable to replicate in macrophages and survives in mice. Intracellular replication and virulence in mice are fully restored by expressing virB4 in trans, indicating that VirB4 is essential for intracellular replication and virulence in mice. An alteration within the NTP-binding region of VirB4 by site-directed mutagenesis abolished complementation of a virB4 mutant, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Intracellular replication was inhibited in wild-type B. abortus after introducing a plasmid expressing a mutant VirB4 altered in the NTP-binding region. The dominant negative phenotype suggests that VirB4 either functions as a multimer or interacts with some other component(s) necessary for intracellular replication. Wild-type B. abortus-containing phagosomes lack the glycoprotein LAMP-1, which is an indicator of the normal endocytic pathway. Mutant strains were found in phagosomes that co-localized with LAMP-1, indicating that VirB4 containing the intact NTP-binding region is essential for evasion of fusion with lysosomes.

Distribution of the Secondary Type III Secretion System Locus Found in Enterohemorrhagic Escherichia coli O157:H7 Isolates among Shiga Toxin-Producing E. coli Strains

ABSTRACT The ability of the complete genome sequence of enterohemorrhagic Escherichia coli O157 led to the identification of a 17-kb chromosomal region which contained a type III secretion system gene cluster at min 64.5. This locus contains open reading frames whose amino acid sequences show high degrees of similarity with those of proteins that make up the type III secretion apparatus, which is encoded by the inv-spa-prg locus on a Salmonella SPI-1 pathogenicity island. This locus was designated ETT2 (E. coli type III secretion 2) and consisted of the epr, epa, and eiv genes. ETT2 was found in enteropathogenic E. coli strains and also in some non-O157 Shiga toxin-producing E. coli (STEC) strains, but most of them contained a truncated portion of ETT2. Most O157 isolates had a complete collection of toxin-encoding genes eae and hlyA and the ETT2 locus, while most O26 strains had toxin-encoding genes eae and hlyA genes but an incomplete ETT2 locus. Thus, an intact copy of ETT2 might mark a pathogenic distinction for particular STEC strains. Therefore, the presence of the ETT2 locus can be used for identification of truly pathogenic STEC strains and for molecular fingerprinting of the epidemic strains in humans and animals.