Krisana Asano

Identification and Characterization of a Novel Staphylococcal Emetic Toxin.

ABSTRACT Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.

Competition between trans-translation and termination or elongation of translation

The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3′-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3′-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.

Mouse peptidoglycan recognition protein PGLYRP-1 plays a role in the host innate immune response against Listeria monocytogenes infection.

ABSTRACT The role of mouse peptidoglycan recognition protein PGLYRP-1 in innate immunity against Listeria monocytogenes infection was studied. The recombinant mouse PGLYRP-1 and a polyclonal antibody specific to PGLYRP-1 were prepared. The mouse PGLYRP-1 showed antibacterial activities against L. monocytogenes and other Gram-positive bacteria. PGLYRP-1 mRNA expression was induced in the spleens and livers of mice infected with L. monocytogenes. The viable bacterial number increased, and the production of cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) was reduced in mice when mice had been injected with anti-PGLYRP-1 antibody before infection. The levels of IFN-γ and TNF-α titers in the organs were higher and the viable bacterial number was reduced in mice injected with recombinant mouse PGLYRP-1 (rmPGLYRP-1) before infection. PGLYRP-1 could directly induce these cytokines in spleen cell cultures. The elimination of intracellular bacteria was upregulated in NMuLi hepatocyte cells overexpressing PGLYRP-1. The enhancement of the elimination of L. monocytogenes from the organs was observed in IFN-γ−/− mice by rmPGLYRP-1 administration but not in TNF-α−/− mice. These results suggest that PGLYRP-1 plays a role in innate immunity against L. monocytogenes infection by inducing TNF-α.

Attenuation of collagen-induced arthritis in mice by salmon proteoglycan.

Rheumatoid arthritis (RA) is a serious autoimmune disease caused by chronic inflammation of connective tissues. The basic principle of RA treatment is aimed to reduce joint inflammation. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in different mouse inflammatory diseases. In this study, we investigated the prophylactic effect of PG on the progression of RA using an experimental mouse model, collagen-induced arthritis (CIA). Clinical and histological severity of CIA was attenuated by daily oral administration of PG. In the joints of PG-administered mice, infiltration of macrophages and neutrophils and also osteoclast accumulation were limited. In comparison to nonadministered mice, anti-collagen antibodies in the sera of PG-administered mice did not alter. On the other hand, local expression of interleukin-17A (IL-17A), IL-6, IL-1β, interferon-γ (IFN-γ), C-C chemokine ligand 2 (CCL2), C-X-C chemokine ligand 1 (CXCL1), and CXCL2 in the joints of PG-administered mice decreased. Moreover, in the response of type II collagen- (CII-) restimulation ex vivo, IL-17A and IFN-γ production by splenocytes from PG-administered mice was less than that of control mice. These data suggested that daily ingested PG attenuated CIA pathogenesis by modulating immune response of splenocytes to CII stimulation and local production inflammatory cytokines and chemokines in the joints.

Adipose Tissue-Derived Mesenchymal Stem Cells Attenuate Staphylococcal Enterotoxin A-Induced Toxic Shock

ABSTRACT Adipose tissue-derived stem cells (ASCs), which are mesenchymal stromal cells isolated from adipose tissues, exhibit immunomodulatory effects that are promising for several applications, including the therapeutics of inflammatory diseases. In the present study, the effect of ASCs on bacterial toxin-induced inflammation was investigated. Intraperitoneal administration of ASCs rescued mice from lethal shock induced by staphylococcal enterotoxin A (SEA) potentiated with lipopolysaccharide. In the sera and/or spleens of mice administered ASCs, the production of proinflammatory cytokines, including interferon gamma, tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-2 was reduced. By quantitative real-time PCR, the expression of Foxp3 in the mice administered ASCs was not altered. On the other hand, the expression of IL-12 receptor and STAT4 was decreased with ASC administration. These results imply that the effect of ASCs is not involved in the lineage of regulatory T cells but that these cells may modulate TH1 differentiation. This information provides evidence that ASCs have properties that are effective to attenuate SEA-induced toxic shock and should prompt further exploration on other inflammatory diseases caused by bacterial toxins or bacterial infections.

Vaccination with non-toxic mutant toxic shock syndrome toxin-1 induces IL-17-dependent protection against Staphylococcus aureus infection

Toxic shock syndrome toxin-1 (TSST-1) is one of superantigens produced by Staphylococcus aureus. We have previously demonstrated that vaccination with non-toxic mutant TSST-1 (mTSST-1) develops host protection to lethal S. aureus infection in mice. However, the detailed mechanism underlying this protection is necessary to elucidate because the passive transfer of antibodies against TSST-1 fails to provide complete protection against S. aureus infection. In this study, the results showed that interleukin-17A (IL-17A)-producing cells were increased in the spleen cells of mTSST-1-vaccinated mice. The main source of IL-17A in mTSST-1-vaccinated mice was T-helper 17 (Th17) cells. The protective effect of vaccination was induced when the vaccinated wild type but not IL-17A-deficient mice were challenged with S. aureus. Gene expression of chemokines, CCL2 and CXCL1, and infiltration of neutrophils and macrophages were increased in spleens and livers of vaccinated mice after infection. The IL-17A-dependent immune response was TSST-1 specific because TSST-1-deficient S. aureus failed to induce the response. The present study suggests that mTSST-1 vaccination is able to provide the IL-17A-dependent host defense against S. aureus infection which promotes chemokine-mediated infiltration of phagocytes into the infectious foci.

Autolysin amidase of Listeria monocytogenes promotes efficient colonization of mouse hepatocytes and enhances host immune response

Listeria monocytogenes is an intracellularly growing pathogen which is able to infect and to spread from cells to cells. It produces several virulence factors required for invasion and intracellular niche colonization. Endogenous peptidoglycan hydrolases which are important for survival of bacteria have been shown to be involved in pathogenesis. An autolysin amidase (Ami)-deficient mutant of L. monocytogenes (Δami) is attenuated in virulence as evidenced by a reduction in mortality of infected mice. We showed that Ami is not essential for bacterial growth and protein secretion. Histopathological analysis suggests that Ami promotes bacterial colonization of hepatocytes. By using cultured eukaryotic cells, we present evidence that a critical function of Ami in pathogenesis is to promote an efficient listerial adherence and internalization into mouse hepatocytes. Simultaneously, the peptidoglycan hydrolase activity of Ami linked to the release of immunologically active cell wall components enhances production of tumor necrosis factor (TNF)-α and interleukin 6. In the early phase of infection, interferon-γ and TNF-α production of Δami-infected mice is significantly less than that of wild-type controls, suggesting a contribution of Ami to enhance the host innate immune response to listerial infection.

Suppression of starvation-induced autophagy by recombinant toxic shock syndrome toxin-1 in epithelial cells.

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced from Staphylococcus aureus, has been reported to bind directly to unknown receptor(s) and penetrate into non-immune cells but its function is unclear. In this study, we demonstrated that recombinant TSST-1 suppresses autophagosomal accumulation in the autophagic-induced HeLa 229 cells. This suppression is shared by a superantigenic-deficient mutant of TSST-1 but not by staphylococcal enterotoxins, suggesting that autophagic suppression of TSST-1 is superantigenic-independent. Furthermore, we showed that TSST-1-producing S. aureus suppresses autophagy in the response of infected cells. Our data provides a novel function of TSST-1 in autophagic suppression which may contribute in staphylococcal persistence in host cells.

Disinfection and healing effects of 222-nm UVC light on methicillin-resistant Staphylococcus aureus infection in mouse wounds

UVC radiation is known to be highly germicidal. However, exposure to 254-nm-UVC light causes DNA lesions such as cyclobutane pyrimidine dimers (CPD) in human cells, and can induce skin cancer after long-term repeated exposures. It has been reported that short wavelength UVC is absorbed by proteins in the membrane and cytosol, and fails to reach the nucleus of human cells. Hence, irradiation with 222-nm UVC might be an optimum combination of effective disinfection and biological safety to human cells. In this study, the biological effectiveness of 222-nm UVC was investigated using a mouse model of a skin wound infected with methicillin-resistant Staphylococcus aureus (MRSA). Irradiation with 222-nm UVC significantly reduced bacterial numbers on the skin surface compared with non-irradiated skin. Bacterial counts in wounds evaluated on days 3, 5, 8 and 12 after irradiation demonstrated that the bactericidal effect of 222-nm UVC was equal to or more effective than 254-nm UVC. Histological analysis revealed that migration of keratinocytes which is essential for the wound healing process was impaired in wounds irradiated with 254-nm UVC, but was unaffected in 222-nm UVC irradiated wounds. No CPD-expressing cells were detected in either epidermis or dermis of wounds irradiated with 222-nm UVC, whereas CPD-expressing cells were found in both epidermis and dermis irradiation with 254-nm UVC. These results suggest that 222-nm UVC light may be a safe and effective way to reduce the rate of surgical site and other wound infections.

The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable causative agents of emetic food poisoning in humans. New types of SEs and SE‐like (SEl) toxins have been reported. Several epidemiological investigations have shown that the SEs and SEl genes, particularly, SEK, SEL, SEM, SEN and SEO genes, are frequently detected in strains isolated from patients with food poisoning. The purpose of the present study was to evaluate the emetic activity of recently identified SEs using a small emetic animal model, the house musk shrew. The emetic activity of these SEs in house musk shrews was evaluated by intraperitoneal administration and emetic responses, including the number of shrews that vomited, emetic frequency and latency of vomiting were documented. It was found that SEs induce emetic responses in these animals. This is the first time to demonstrate that SEK, SEL, SEM, SEN and SEO possess emetic activity in the house musk shrew.