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Biomedical subjects

Sou-ichi Makino

Publications and source records attributed to Sou-ichi Makino.

At least 19 recordsLinked to original sources
PubMed2006

Enhancement of mice susceptibility to infection with Listeria monocytogenes by the treatment of morphine.

The effect of morphine on the susceptibility of BALB/c mice to diarrheagenic Escherichia coli, Shigella flexneri, Listeria monocytogenes, Salmonella Enteritidis, Yersinia enterocolitica, was examined via the intraperitoneal inoculation. Morphine treatment increased the susceptibility to S. Enteritidis and L. monocytogenes, resulting in bacteremia and central nervous system (CNS) invasion (for L. monocytogenes), while the infection with other bacteria did not show the systemic dissemination in the morphinetreated mice. Notably, L. monocytogenes infection caused 100% mortality with a mean survival time (MST) of 1.3 days in morphine-treated mice, but untreated mice did not die. The present data suggested that individuals using heroin or treated with morphine derivatives might be at high risk for listeriosis, especially those who are immunocompromised. Recent increasing consumption of morphine may propose the necessity for further epidemiological surveillance on infectious diseases.

Animals↗
PubMed2006

The sigma factor RpoN (sigma54) is involved in osmotolerance in Listeria monocytogenes.

Listeria monocytogenes is able to grow under conditions of high osmolarity. We constructed a deletion mutant of rpoN, encoding the alternative sigma factor RpoN, and analyzed its response to osmotic stress. In a minimal medium with 4% NaCl and 1 mM betaine, the mutant showed a similar growth to that of the parental strain, EGD. In the same medium with 4% NaCl and 1 M carnitine, the growth rate of the mutant was greatly reduced, when the optical density at 600 nm (OD600) at the starting point of growth, was 0.15. However, when growth of the culture was started at an OD600 of 0.025, the growth of the mutant was similar to that of EGD. The mutant's expression of two betaine transporter genes, betL and gbuB, and the carnitine transporter gene opuCA, was osmotically induced at a level similar to EGD, and its rate of carnitine uptake was similar to that of EGD. These results suggest that the growth defect from the rpoN mutant is caused not by the transcriptional regulation of opuCA or by a decrease in carnitine uptake, but possibly by larger amounts of carnitine being needed for growth of the mutant in minimal medium when NaCl is present.

Bacterial Proteins↗
PubMed2005

Quantitative analysis of Staphylococcus aureus in skimmed milk powder by real-time PCR.

A large-scale outbreak of food poisoning caused by consumption of skimmed milk powder contaminated with staphylococcal enterotoxin A (SEA) occurred in Japan. No viable Staphylococcus aureus was detected in the skimmed milk powder, however, sea and nuc genes of S. aureus were detected in it by PCR. The number of S. aureus in skimmed milk powder was estimated by quantitative real-time PCR.

Animals↗
PubMed2004

Changes in Salmonella enterica serovar Oranienburg viability caused by NaCl-induced osmotic stress is related to DNA relaxation by the H-NS protein during host infection.

The NaCl sensitivity of Salmonella enterica serovar Oranienburg strains depends on their origin. We found previously that food- and patient-origin isolates in an outbreak were, respectively, NaCl-resistant and NaCl-sensitive, and the NaCl-resistant strain of food-origin isolates became NaCl-sensitive after passage of the strain through mice [FEMS Microbiol. Lett. 212 (2002) 87]. Here, we report that this phenotypic difference is mainly dependent on topological changes regulated by H-NS, a bacterial histone-like nucleoid protein that binds non-specifically to DNA. That is, this phenotypic difference was caused by changes in DNA topology during infection of the host. Based on these findings, we propose this mechanism has a key role in promoting the survival of Salmonella under osmotic stress.

Animals↗
PubMed2004

Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus.

Brucella abortus is a facultative intracellular bacterium that can survive inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to the conjugative DNA transfer system. In this study, we showed that a class A scavenger receptor (SR-A) of macrophages is required to internalize B. abortus and contributes to the establishment of bacterial infection in mice. Macrophages from SR-A-deficient mice inhibited internalization and intracellular replication of both wild type strain and the virB4 mutant, and that bacterial proliferation was inhibited in SR-A-deficient mice. Adding lipopolysaccharide from B. abortus and Salmonella enterica serovar Typhimurium, but not from Escherichia coli, to macrophages inhibited bacterial internalization. VirB-dependent bacterial internalization induced localization of SR-A into detergent-resistant membrane lipid rafts. These results indicate that B. abortus internalizes into macrophages by using SR-A as a receptor and that the VirB type IV secretion system of B. abortus regulates signal transduction dependent on SR-A to form replicative phagosomes, and which is mediated by lipid rafts.

Animals↗
PubMed2004

Epidemiological and serological survey of brucellosis in Mongolia by ELISA using sarcosine extracts.

Brucellosis is an important zoonosis, and serological surveillance is essential to its control. However, cross-reactions of attenuated live cells of Brucella abortus strain S-19 and B. melitensis strain Rev-1 with Yersinia enterocolitica O9 or vaccinated animal sera interfere with accurate serological diagnosis by the Rose Bengal test (RBT). Therefore, we used ELISA with sarcosine extracts from the virulent B. abortus strain 544 to eliminate false-positives among RBT positive-sera. A total of 697 serum samples were collected in Mongolia from humans and animals in 23 nomadic herds. The herds were classified into three groups as brucellosis-endemic (BE), brucellosis-suspected (BS), or Brucella-vaccinated (BV). The number of 295 animals (43.0%) was positive by RBT, but 206 (69.8%) of these were positive according to ELISA; therefore, 30.2% of the RBT-positive sera were found to be false positives. The false positive samples for RTB represent 4.1%, 27.4%, and 68.2% of the animals from the BE, BS, and BV herds, respectively. In addition, 32% of RBT-positive human sera were also false positives. Thus, our ELISA would be more specific than RTB and useful for epidemiological surveillance for brucellosis.

Animal Husbandry↗
PubMed2004

Isolation of mini-Tn5Km2 insertion mutants of Salmonella enterica serovar Oranienburg sensitive to NaCl-induced osmotic stress.

We previously reported that the viability of Salmonella Oranienburg strains under NaCl stress was variable and depended on the strain's origin; food strains were resistant and patient strains sensitive to NaCl. Therefore, we mutagenized a food strain with a mini-Tn5Km2 transposon. Of 2,400 mutants screened, 15 NaCl-sensitive mutants were isolated, and 7 genes associated with NaCl-sensitivity were identified. The intact genes complemented their own food-strain mutants, but not patient-strain mutants, suggesting that the difference in NaCl-sensitivity between food and patient S. Oranienburg strains might not arise from a single gene mutation, but from change in multiple osmoregulatory mechanisms in Salmonella.

Base Sequence↗
PubMed2003

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.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores.

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.

Air Microbiology↗
PubMed2003

Enzyme-linked immunosorbent assay to differentiate the antibody responses of animals infected with Brucella species from those of animals infected with Yersinia enterocolitica O9.

Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.

Animal Diseases↗
PubMed2003

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

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.

ATP-Binding Cassette Transporters↗
PubMed2002

Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation from the viable but non-culturable state.

An outbreak caused by dried processed squids contaminated with Salmonella Oranienburg occurred in Japan in 1999. Isolates obtained from the causative food were resistant to NaCl osmotic stress, but isolates from the patients were sensitive to NaCl. Although strains from both sources were almost identical in their virulence in mice, a NaCl-resistant strain from food (Sa9911T) became NaCl-sensitive after passage in mice and a NaCl-sensitive strain from one patient (Sa99004) retained NaCl sensitivity after such passage. When dried squid was contaminated experimentally with both strains during processing, only Sa9911T was recovered directly from the final product. Nevertheless, the viability of the Sa99004 cells was over 90% found by fluorescent staining. We suggested that Sa99004 might become viable but non-culturable (VNC) by NaCl stress. This hypothesis was confirmed by resuscitation by efficient enrichment. We concluded that VNC S. Oranienburg would be potentially dangerous contaminants of NaCl-preserved foods and that measures to ensure their detection should be taken at the time of food inspection.

Animals↗
PubMed2002

[The serotype and Shiga toxin type of Shiga toxin-producing Escherichia coli from humans and various animals].

To clear the route of STEC (Shiga toxin-producing Escherichia coli) infection to humans, we examined the serotype. Shiga toxin genotype and eae gene of STEC strains from humans and various animals. The most predominant serotype originated from humans was O157:H7, followed by O26:H11, and other serotypes were O91:H21, O103:H2, O111:NM, O121:H19, etc. The eae gene was found in 79 of 93 strains from human origin. The serotypes of STEC from cattle were significant by similar to that of STEC from humans. The eae gene was found in 44 of 87 strains from cattle. Shiga toxin genotypes possessed O157 strains from humans and cattle, were divided into six groups, stx1, stx2, stx2c, stx1 + stx2, stx1 + stx2c and stx2 + stx2c. Moreover, frequency rates of Shiga toxin genotypes of O157 were also similar to both human and cattle origins. The serotypes of STEC from sheep were also a little similar to that of STEC from humans. Seven of 8 strains from deer possessed stx2d gene that the strains from humans seldom possessed, and none of the strains possessed eae gene. In STEC originated from swine, 15 of 25 strains were O139:H1 that Shiga toxin genotype was stx2e. It was thought that the sources of STEC infection to human are cattle and sheep, and deer and swine had little possibility to human STEC infection.

Animals↗
PubMed2002

Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene.

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.

Amino Acid Sequence↗
PubMed2002

Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice.

Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. Intracellular replication of B. abortus requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of B. abortus and also contributes to the establishment of bacterial infection in mice. The internalization of B. abortus was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL). Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of B. abortus. Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of B. abortus in macrophages. The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted B. abortus infection. NPC1-deficient mice were resistant to the bacterial infection. Molecules associated with cholesterol-rich microdomains, "lipid rafts," accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of B. abortus. Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of B. abortus infection.

Acetanilides↗