Seiichi Katayama

The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne

The location of the cpe gene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenic Ciostridium perfringens strains by means of pulsed field gel electrophoresis of genomic DNA. The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, the Hin dlll repeat, and an open reading frame, ORF3, that may be part of an insertion sequence. In contrast, when the strains originated from domesticated livestock cpe was located on a large episome where it was often close to a copy of the transposable element IS 1151. In these cases, the Hin dlll repeat was not linked to the cpe gene although this was generally preceded by ORF3.

Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,β, ε, andı, in addition to the enterotoxin andλ-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins,ϑ andμ, were missing.

Transcription activation of a UV‐inducible Clostridium perfringens bacteriocin gene by a novel σ factor

Expression of the plasmid‐encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, u200aalso u200aplasmid‐encoded, u200aproved u200ato u200abe an RNA polymerase σ factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA‐independent, basal level of gene expression while the stronger, UviA‐dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of σ factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

Characterization of two putative fibronectin-binding proteins of Clostridium perfringens.

Clostridium perfringens is a Gram-positive anaerobic pathogen that causes gas gangrene and food poisoning in humans and animals. Genomic analysis of C. perfringens strain 13 revealed that this bacterium contains two genes (CPE0737 and CPE1847) that encode putative fibronectin (Fn)-binding proteins (Fbps). These genes, named fbpA and fbpB, were found to be constitutively expressed in all three strains (13, NCTC8237, CPN50) of C. perfringens, isolated from gas gangrene of human, that were tested. Both fbpA and fbpB were cloned and His-tagged, recombinant FbpA (rFbpA) and recombinant FbpB (rFbpB) were purified by Ni(2+)-Sepharose column chromatography from transformed Escherichia coli. These recombinant Fbps were shown to bind to Fn, purified from human serum, in a ligand blotting assay. Additionally, Fn bound to these rFbps in a dose-dependent manner in an enzyme-linked immunosorbent assay. Furthermore, it was found that pre-incubation of Fn with either rFbpA or rFbpB inhibited the binding of Fn to C. perfringens cells.

Phased A-tracts bind to the α subunit of RNA polymerase with increased affinity at low temperature

Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A‐tracts in a low temperature‐dependent manner. In this paper we characterize the interaction between the α subunit of C. perfringens RNA polymerase and the phased A‐tracts. Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the α subunit binds to the minor grooves of the phased A‐tracts through its C‐terminal domain with increased affinity at low temperature. The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A‐tracts.

Adhesive properties of Clostridium perfringens to extracellular matrix proteins collagens and fibronectin.

The adhesive properties of Clostridium perfringens to collagens, gelatin, fibronectin (Fn), Fn-prebound collagens, and Fn-prebound gelatin were investigated. C.xa0perfringens could bind to Fn-prebound collagen type II, type III, and gelatin, but not to gelatin or collagens except for collagen type I directly. Recombinant Fn-binding proteins of C.xa0perfringens, rFbpA and rFbpB, were used to examine Fn-mediated bacterial adherence to collagen type I. In the presence of rFbps, C.xa0perfringens adherence to Fn-prebound collagen type I was inhibited in a dose-dependent manner. Fn was not released from the coated collagen type I by the presence of rFbps, and rFbps did not bind to collagen type I. Thus, the inhibition of C.xa0perfringens binding to Fn-prebound collagen type I by rFbps could not be explained by the removal of Fn from collagen or by the competitive binding of rFbps to collagen. Instead, both rFbps were found to bind to C.xa0perfringens. These results suggest the possibility that rFbps may bind to the putative Fn receptor expressed on C.xa0perfringens and competitively inhibit Fn binding to C.xa0perfringens.

Determination of the Clostridium perfringens-binding site on fibronectin

The extracellular matrix protein fibronectin (Fn) is known to bind to the surface of Clostridium perfringens cells. Fn is a disulfide-linked homodimer protein, with each Fn polypeptide consisting of three types of repeating modules: 12 type I, 2 type II, and 15-17 type III modules. To determine the epitope on Fn recognized by C. perfringens cells, anti-Fn monoclonal antibodies (mAbs) and various Fn fragments (III2-10, rIII2-4, rIII5-7, rIII8, rIII9, rIII10) were employed. Although two C. perfringens-derived Fn-binding proteins, FbpA and FbpB, have been reported, they appear not to be the bacteriums surface Fn receptor. Moreover, both FbpA and FbpB were found to bind to C. perfringens cells. To avoid confusion, a mutant C. perfringens lacking both the fbpA and fbpB genes (MW5) was prepared using an in-frame deletion system. MW5 cells bound Fn on their surface, suggesting the presence of a putative Fn receptor(s) on C. perfringens cells. Of several anti-Fn mAbs, both HB39 and MO inhibited the binding of Fn to MW5 cells. HB39 reacted strongly with III2-10 and rIII9, and weakly with rIII2-4, rIII10 and rIII5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII9 or rIII10, but not in the presence of rIII2-4, rIII5-7, or rIII8. Binding of Fn to MW5 cells was strongly inhibited by both III2-10 and rIII9, marginally inhibited by rIII2-4, but not affected by rIII5-7, rIII8, or rIII10. Significant binding of MW5 cells to immobilized rIII9 and rIII10 as well as immobilized III2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III9 and III10.

Mode of binding of RNA polymerase α subunit to the phased A-tracts upstream of the phospholipase C gene promoter of Clostridium perfringens

Three phased A5-6-tracts lie upstream of the promoter of plc encoding the α-toxin (phospholipase C) of Clostridium perfringens. The α subunits of C.xa0perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the α subunit (αCTD). To identify the amino acid residues involved in the binding of C.xa0perfringens α subunits to the phased A-tracts, 27 amino acid residues in C.xa0perfringens αCTD were substituted with alanine. The affinities of the mutated α subunits for the phased A-tracts were examined by gel shift assays and surface plasmon resonance (SPR). The SPR analyses revealed that the phased A-tracts themselves facilitated a complex formation between the phased A-tracts and C.xa0perfringens α subunits [Kd was 6.1 (±0.3)xa0×xa010(-8)xa0M], and that Arg261, Asn264, Gly292 and Lys294 in C.xa0perfringens αCTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C.xa0perfringens αCTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli αCTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37xa0°C) indicated that the affinity of the C.xa0perfringens α subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E.xa0coli α subunit for the UP element did not. This suggests that the phased A-tracts may not simply be a subset of the UP element, and that they show specific binding activity with the RNA polymerase α subunit.

Expression of glyceraldehyde-3-phosphate dehydrogenase on the surface of Clostridium perfringens cells

During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C.xa0perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C.xa0perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C.xa0perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C.xa0perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C.xa0perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen.

Function of the phased A-tracts upstream of the phospholipase C gene promoter in Clostridium perfringens

P salmonis is a Gram-negative intracellular bacterial pathogen isolated from infected farmed salmonids which cause the salmonid piscirickettsia septicaemia (SPS). P. salmonis is a member of the class Gammaproteobacteria, classified within order Thiotrichales close to Francisella and Legionella genus. Here, we present the genome sequence analysis of pathogenic strain of P. salmonis AUS 005, in order to understand its genomic features and its pathogenic role. The P. salmonis genome consists in a single circular chromosome of 3.4 Mp with 1400 Kb in size more in comparison to Francisella noatunensis subsp. Orientalis strain Toba04. The GC content was 37.37%. The annotation resulted in a total of 2,571 protein-coding gene predictions, with 1,873 well-annotated genes which were identified in a variety of genes associated with pathogenicity, environmental adaptation, metabolic pathways and iron acquisition, as well as transposable elements and insertion sequences (ISs). The metabolic pathways prediction indicates the absence of biosynthetic and catabolic pathways of cysteine and the complete pathways of TCA cycle. Surprisingly, the genome of this immobile bacterium revealed several genes encoding components of flagellum. The availability of the P. salmonis genome represents a biotechnological opportunity to understand the molecular mechanisms of pathogenecity and the development of new therapies to counteract the piscirickettsiosis.T strains of Diaphorobacter species that could degrade 3-nitrotoluene completely, were isolated from the sludge taken from an industrial waste-water treatment plant. The complete degradation pathway was established and the first enzyme in the degradation cloned, expressed and purified from E. coli. The recombinant enzyme revealed a broad substrate specificity and could also degrade nitrobenzene, 2and 3-chloronitrobenzene and 4-nitrotoluene. Several other aromatics devoid of the nitrogroup could be biotransformed into useful chiral compounds of industrial importance by the recombinant enzyme. Comparison of the gene and protein sequence with other reported dioxygenases reveals that Diaphorobacter is a missing link in the evolution of dioxygenases.F amyloid, in the form of adhesive fimbrial proteins termed curli, was first described in Salmonella and Escherichia coli. Curli fibers adhere to various host cells and structural proteins, interact with components of the host immune system, and participate in biofilm formation. Shiga toxin-producing E. coli (STEC) cause severe hemorrhagic diarrheal disease which can progress to hemolytic uremic syndrome in certain cases. STEC typically carry stx1, stx2, or both on lambdoid prophage. Although most STEC possess all of the genes required for functional curli production, curli expression is tightly regulated resulting in great variability in curli production among different STEC strains and serotypes. Curli and the exopolysaccharide cellulose are the major components of STEC biofilms. Both are controlled by the transcriptional regulator CsgD, whose expression is dependent on the RpoS sigma factor and enhanced by the MlrA transcription factor. We have shown that prophage insertions in the coding region of mlrA, often carrying anstx gene, are major barriers that limit csgD expression and curli production. This talk will discuss differences in csgD-dependent curli expression in various STEC serotypes and present new findings regarding prophage effects on mlrA expression and curli production.C perfringens is a Gram-positive spore-forming anaerobic bacterium and a pathogen causing gas gangrene and food poisoning. Gas gangrene is mainly caused by α-toxin (phospholipase C) produced by C. perfringens. Three phased A5-6-tracts (–66 to –40) lie upstream of the promoter for plc gene encoding the α-toxin. The results of gel retardation assays and hydroxyl radical footprintings revealed that the α subunit of C. perfringens RNA polymerase binds to the minor grooves of the phased A-tracts through its C-terminal domain (αCTD). The affinity of the α subunit for the phased A-tracts was estimated by surface plasmon resonance (SPR) [Kdwas 6.1(α0.3)α10 −8 M]. To identify the amino acid residues involved in the binding of the α subunit to the phased A-tracts, 27 amino acids residues in the α CTD were substituted with alanine. SPR analyses revealed that Arg261, Asn264, Gly292 and Lys294 in the α CTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C. perfringens α CTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli α CTD with the upstream (UP) element. SPR analyses at different temperatures (15, 25 and 37°C) indicated that the affinity of C. perfringens α subunit for the phased A-tracts increased atlower temperatures, whereas that of E. coli α subunit for UP element did not. These results suggested that the phased A-tracts directly enhanced the plc gene expression in a low-temperature-dependent manner.A species, in the first place are ubiquitous fungi which ecologically tend to inhabit soil, water, vegetation and starchy products. Therefore they have an important effect on the global carbon and nitrogen reproduction chain. On the other hand, Aspergillus are well known opportunistic fungi that manipulate the immune system of human. Among the species, Aspergillus fumigatus is one of the most prevalent fungi which causes allergic forms of human disease and fatal aspergillosis It has been previously shown that A. fumigatus has the following impacts on host cells: Once the conidia are inhaled, they would be phagocytosed by the epithelial cells of respiratory tract and localize into macrophages, developing aspergillosis. They consequently as same as the other infectious particles, lead the cell entering into apoptotic phase. Also interestingly, the wild type melanotic conidia, can interfere with the intrinsic and extrinsic apoptotic pathways in macrophages and inhibit apoptosis. Nevertheless the fact that apoptosis as a major consequent of infection is a pH-dependent process; still there is a lack of knowledge about the certain role of cytosolic pH during infectious diseases development. In this study the effect of melanin derived from A. fumgatus on pH variation during the apoptosis is hyperspectrally monitored in the human monocyte. Hyperspectral imaging (also known as imaging spectroscopy) is a research technique which its application is based on collecting and processing data from multiple fluorescent dyes in quantitative means. By applying this method, the emitted spectrum from multicolored fluorescent sample, is being divided into more extended bands and consequently, the visible and graphable images are provided.Tuberculosis remains a major threat to global public health accounting for nearly two million deaths worldwide each year. The primary target cells for Mycobacterium tuberculosis (MTB) are phagocytic cells of the lung. Mycobacteria employ a number of strategies to circumvent host responses and we report novel interactions between the bacterium and human macrophages and dendritic cells that also influence the immune response. Human macrophages produce interleukin (IL)-27 following infection by MTB. IL-27 has been shown to have immunosuppressive activity toward a number of cell types including macrophages. Neutralization of IL-27 produced during infection promotes trafficking of mycobacteria to lysosomes and control of the bacterial burden in human macrophages. The mechanism involves increased protein levels and/or colocalization of vacuolar ATPase (VATPase), CD63, and cathepsin D with the bacteria. In a separate body of work, we also demonstrate nitric oxide (NO) production by human macrophages during mycobacterial infection. Interestingly, the intracellular environment in macrophages that produce NO does not inhibit but rather augments growth of MTB. Mycobacterial growth in macrophages that produce NO requires a functional nitrate reductase. Following infection by MTB, dendritic cells in the lung that have become infected or internalized mycobacterial antigens migrate to regional lymph nodes to initiate an adaptive response. This process is delayed in response to MTB as compared with other bacteria. We have demonstrated that human dendritic cells infected with MTB exhibit redistribution and decreased surface expression of heterodimeric / integrins that contain CD18 ( 2). This decrease is not recovered in infected cells and is maintained over time. Consistent with the change in surface integrins we report a significant decrease in adherence to primary lung lymphatic endothelial cells and migration through an endothelial monolayer toward lymphatic chemokines. Cumulatively, we have identified novel interactions between mycobacteria and host innate immune cells that provide additional insight into bacterial strategies to persist in the human host. Measures aimed at modulating expression of IL-27 and overcoming the MTB-mediated inhibition of 2 integrin expression and migration may have important implications in vaccination and therapeutic approaches.T virulence determinants of Gram-positive streptococci are more complex than those of Gram-negative strains. For Gram-negative bacteria, lipopolysaccharide (LPS) is a primary virulence factor. In the case of the human pathogen, Grampositive group A-Streptococcus pyogenes (GAS), several factors play various roles as virulence determinants. These include cell wall components, lipoteichoic acid and peptidoglycan, along with the cell wall capsule and exotoxins, from both the bacterial genome, e.g., the cysteine protease, streptococcal pyrogenic exotoxin (Spe)B, and from bacteriophage inserts, e.g., the DNase, streptodornase I (SdaI) and the superantigen, SpeA. Genes are also acquired by GAS strains via horizontal transfer, e.g., emm, enn, and fcR genes, as well as streptokinase (the ska gene), which functions in virulence through activation of host human plasminogen. The products of these genes attempt to defeat both innate and acquired immunity of the host. These virulence factors are under control of one-component and two-component bacterial regulatory systems, which regulate gene expression as needed at different stages during infection. This talk will detail the functions of bacterial virulence determinants and their dynamic interplay with the innate and acquired immune system of the host.B transfusion may transmit infections with serious consequences such as Hepatitis C virus (HCV). Blood banks worldwide are shifting to nucleic acid testing (NAT) for effective screening of blood donations for HCV and HIV. Since viremia precedes seroconversion by several weeks, tests that detect viral nucleic acids are more sensitive than serology and may reduce the window of infectivity by as much as 50 to 60 days for HCV. However, this has not yet been adopted nationwide in Egypt due to financial constraints. Egypt currently has the highest prevalence of HCV. Testing of pooled donor samples significantly reduces the number of tests required on a daily basis, the time to perform the testing, and the cost of testing per donation. The rise of HCV in plasma is very rapid, thus sample dilution inherent in pooling has a minimal impact on the sensitivity to detect window period viremic samples. Pooling samples makes molecular-based testing of blood donors feasible. The current study evaluates pooling schemes in a cohort of 12,000 donations to validate the possibility of avoiding the deleterious effects of transmitting HCV while maintaining cost effectiveness.G bacteria, including both commensal and pathogenic species, acquire iron with TonB-dependent uptake systems. Using fluorescence spectroscopic analyses of the Escherichia coli outer membrane (OM) protein FepA and its cell envelope partner TonB, it was described dynamic actions of both proteins during the uptake of ferric enterobactin (FeEnt). When FeEnt interacts with fluoresceinated FepA, the quenching of light emissions reflects its binding and transport process as a series of conformational motions in the receptor protein. This simple experimental system involves genetically engineering Cys sulfhydryls in any of 7 surface loops of FepA and fluorophore maleimides reacting with them. Spectroscopic or microscopic fluorescence intensity changes, ultimately mirrored cellular uptake that depleted FeEnt from solution. The TonB-ExbBD inner membrane complex transfers energy to OM iron transporters like FepA. (GFP)-TonB hybrid proteins were used to investigate its activity. Fluorescence microscopic characterization of the (GFP)-TonB hybrids revealed an unexpected, restricted localization of TonB in the central region of the cell envelope. Fluorescence polarization measurements demonstrated energy-dependent motion of TonB in living cells, which likely was rotation. The findings show that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.D caries (tooth decay) is the most prevalent chronic disease in children in the United States, affecting 28% of 2-5 year-old and 49% of 5-8 year-old children. Mutans streptococci (MS) are one group of the major caries-causing bacteria. Children with early colonization of MS had significant higher dental caries later in their life. Although MS transmission from mother to child is widely accepted as a main source of early acquisition of MS in children, studies have shown the importance of other transmission routes to children, including other caregivers and playmates. Further, a wide diversity of MS genotypes is present in the mother but only some maternal MS genotypes are transmitted. Therefore, specific virulence factors may be related to transmission of MS to children. This presentation summarizes several studies that we have conducted to investigate the transmission routes of MS in children including maternal, intergenerational and horizontal transmission. We also investigated possible virulence factors that may relate to transmission of MS including biofilm and mutancin formation. Our results confirmed that non-maternal transmission routes play an important role in MS colonization in children and mutacin activity of MS is also related to transmission. Identification of virulence factors associated with MS transmission is important in developing strategies for caries prevention. It is also important to include nonmaternal transmission routes as a part of caries prevention protocols for children.