Hideaki Nagamune

Correlation between Resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds and Expression of Outer Membrane Protein OprR

ABSTRACT The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated. A P. aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method. It was revealed that P. aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride. Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P. aeruginosa. Moreover, the P-12-resistant strain exhibited cross-resistance to BAC. Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa. The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR. A correlation between the level of expression of OprR and the level of resistance of P. aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain. The knockout mutant recovered susceptibility not only to P-12 but also to BAC. These results suggested that OprR significantly participated in the adaptation of P. aeruginosa to QACs, such as P-12 and BAC.

Intermedilysin Is Essential for the Invasion of Hepatoma HepG2 Cells by Streptococcus intermedius

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human‐specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep‐seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep‐seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post‐infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface‐bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein.

Intermedilysin is a pore‐forming cytolysin belonging to the streptolysin O gene family known as the ‘Cholesterol‐binding/dependent cytolysins’ and is unique within the family in that it is highly human‐specific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome‐embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome‐binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human‐specific step of intermedilysin action, that the host cell membrane‐binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human‐specific action step and the cell‐binding site of domain 4 lies within the 56 amino acids of the C‐terminal, excluding the undecapeptide region.

The essentiality and involvement of Streptococcus intermedius histone‐like DNA‐binding protein in bacterial viability and normal growth

Streptococcus intermedius histone‐like DNA‐binding protein (Si‐HLP) is a homodimeric protein and, conserved with Escherichia coli HU, a well‐documented nucleoid‐associated protein (NAP). In E. coli, HU plays important roles as both structural and regulatory factors, but it is not essential for E. coli viability. Streptococcal HLP has been found to bind host cells and induce cytokine production, but its physiological role remains poorly defined. In the present study, using gene insertion knockout and tetracycline‐regulated antisense RNA expression techniques, we determined whether Si‐HLP is essential for bacterial viability and normal growth in S. intermedius. The Si‐HLP‐downregulated S. intermedius strain showed alterations in its morphology and surface properties. Downregulation of Si‐HLP led to an expanded nucleoid to fill the intracellular space. Transcription levels of several genes, including virulence‐associated factors, were found to be activated or repressed in the antisense Si‐hlp RNA‐expressing strain by real‐time PCR and reverse‐transcription PCR. Collectively, these data suggest that Si‐HLP serves as an essential NAP governing the nucleoid architecture and controlling the gene transcription profile in S. intermedius.

Effect on polymorphonuclear cell function of a human-specific cytotoxin, intermedilysin, expressed by Streptococcus intermedius

ABSTRACT Streptococcus intermedius is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin fromStreptococcus intermedius on PMN function. Active ILY at a concentration of 40 ng/μl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis ofStaphylococcus aureus. These findings may have implications for the role of ILY in deep-seated abscesses.

Uncoupling of oxidative phosphorylation by divalent cationic cyanine dye. Participation of phosphate transporter

The trinuclear cationic cyanine dye tri-S-C4(5) was found to be an uncoupler of oxidative phosphorylation. Its uncoupling required inorganic phosphate (Pi) or arsenate, which is transported into mitochondria via the Pi transport system, and was abolished by the Pi-transport inhibitor N-ethylmaleimide or mersalyl. The dye stimulated Pi uptake into mitochondria, and its uncoupling action was accompanied by swelling of the mitochondria. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of Pi, the dye did not cause uncoupling, but its uptake was much greater than in the presence of Pi. The cyanine dye is suggested to induce uncoupling by acting on the membrane, rather than after its electrophoretic transfer into the mitochondria.

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep‐seated purulent infections in major organs, such as the brain and liver. Histone‐like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro‐inflammatory cytokine inductions in THP‐1 cells by stimulation with recombinant HLP of S. intermedius (rSi‐HLP). rSi‐HLP stimulation‐induced production of pro‐inflammatory cytokines (IL‐8, IL‐1β and TNF‐α) occurred in a time‐ and dose‐dependent manner. In contrast with the heat‐stable activity of DNA binding, the induction activity of rSi‐HLP was heat‐unstable. In subsequent studies, rSi‐HLP acted cooperatively with lipoteichoic acid, the synthetic Toll‐like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that rSi‐HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun N‐terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si‐HLP can trigger a cascade of events that induce pro‐inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.

The lipophilic weak base (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) is a potent protonophore type cationic uncoupler of oxidative phosphorylation in mitochondria

The lipophilic weak base AU-1421 acts as a simple protonophoric uncoupler of oxidative phosphorylation in rat liver mitochondria judging from the following observations. In the absence of any carrier lipophilic anions or P(i), AU-1421 stimulated the rate of state 4 respiration maximally about 7-fold at a concentration of 30 nmol/mg mitochondrial protein. At the same maximum effective concentration, it also inhibited ATP synthesis, released oligomycin-inhibited state 3 respiration, dissipated the proton motive force in the energized state, and activated latent H(+)-ATPase. AU-1421 also allowed proton conduction in both mitochondrial membranes and liposomes. These actions of AU-1421 resemble those of the typical anionic uncoupler SF6847. A marked difference between the two was, however, that ATPase activation by AU-1421 was not suppressed at higher concentrations of AU-1421, whereas ATPase activated by SF6847 was suppressed on increase of the SF6847 concentration. The finding that this simple protonophoric cation acts as an uncoupler at a micromolar concentration is significant, because all true (i.e., protonophore type) uncouplers known so far are anionic not cationic. Thus, AU-1421 is a unique uncoupler of the protonophore type.

Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ32 in E. coli, although S. intermedius does not utilize σ32 for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

Developmental expression of a novel Kexin family protease, PACE4E, in the rat olfactory system.

Abstract PACE4 is a mammalian Kexin family protease that is involved in the maturation of precursor proteins. Four PACE4 isoforms have been identified. We identified a novel PACE4 isoform, PACE4E, from a human cerebellum cDNA library, which possesses a hydrophobic cluster in its C-terminus participating in membrane association. The size of PACE4E mRNA from adult rat brain was estimated by Northern blotting to be 4.4 kb. In situ hybridization histochemistry revealed that the highest level of PACE4E mRNA was expressed in the mitral cells of the adult rat olfactory bulb (OB). The OB is a unique sensory organ in that it has a lifelong regenerating capacity and it affects brain development. We further analyzed the expression of PACE4E mRNA in the developing olfactory system. On day 13.5 of gestation, PACE4E mRNA was expressed at high levels in the neuroepithelium of the forebrain vesicle (FV), olfactory epithelium, and cells in the fiber bundles projecting to the FV. As development proceeded, PACE4E mRNA was expressed in developing mitral cells but decreased in the olfactory epithelium. In the newborn, its expression was confined to the mitral cells in both the main and accessory OB and in some periglomerular cells, as shown in adult rats. The spatio-temporal expression of PACE4E suggests that it plays a role in the establishment and maintenance of the olfactory receptor system.