SMILE chemotherapy is an effective treatment for newly diagnosed stage IV, relapsed or refractory ENKL. Myelosuppression and infection during the treatment should be carefully managed.

Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5-5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.bacteriophage ͉ genome evolution ͉ type III secretion system

Vibrio parahaemolyticus, one of the human-pathogenic vibrios, causes three major types of clinical illness: gastroenteritis, wound infections, and septicemia. Thermostable direct hemolysin (TDH) secreted by this bacterium has been considered a major virulence factor of gastroenteritis because it has biological activities, including cytotoxic and enterotoxic activities. Previous reports revealed that V. parahaemolyticus strain RIMD2210633, which contains tdh, has two sets of type III secretion system (T3SS) genes on chromosomes 1 and 2 (T3SS1 and T3SS2, respectively) and that T3SS1 is responsible for cytotoxicity and T3SS2 is involved in enterotoxicity, as well as in cytotoxic activity. However, the relative importance and contributions of TDH and the two T3SSs to V. parahaemolyticus pathogenicity are not well understood. In this study, we constructed mutant strains with nonfunctional T3SSs from the V. parahaemolyticus strain containing tdh, and then the pathogenicities of the wild-type and mutant strains were evaluated by assessing their cytotoxic activities against HeLa, Caco-2, and RAW 264 cells, their enterotoxic activities in rabbit ileal loops, and their lethality in a murine infection model. We demonstrated that T3SS1 was involved in cytotoxic activities against all cell lines used in this study, while T3SS2 and TDH had cytotoxic effects on a limited number of cell lines. T3SS2 was the major contributor to V. parahaemolyticus-induced enterotoxicity. Interestingly, we found that both T3SS1 and TDH played a significant role in lethal activity in a murine infection model. Our findings provide new indications that these virulence factors contribute to and orchestrate each distinct aspect of the pathogenicity of V. parahaemolyticus.Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that inhabits estuarine and coastal waters and can be isolated from seafood (6,7,9). It causes acute gastroenteritis in humans after they consume contaminated raw or undercooked seafood. Although this microorganism is better known for causing gastroenteritis, it also can cause wound infections and septicemia (5,23,36).Most clinical isolates of V. parahaemolyticus from patients with diarrhea show ␤-hemolysis on Wagatsuma agar (37). This phenomenon is known as the Kanagawa phenomenon (KP) and is considered a good marker to distinguish between pathogenic and nonpathogenic V. parahaemolyticus strains (25, 37). Thermostable direct hemolysin (TDH), which is responsible for the KP (28, 43), has multiple biological activities, including hemolysis, enterotoxicity, cytotoxicity, and cardiotoxicity (11-13, 26, 29, 32, 34, 38). For this reason, TDH has been considered a major virulence factor of V. parahaemolyticus.Whole-genome sequencing of a KP-positive V. parahaemolyticus strain revealed that this strain contains two sets of gene clusters for the type III secretion system (T3SS), T3SS1 and T3SS2, one on each of its two chromosomes (21). T3SS gene clusters have been detected in numerous Gram-negative animal and plant pathogens, wh...

Vibrio parahaemolyticus is a bacterial pathogen causative of food-borne gastroenteritis. Whole-genome sequencing of V. parahaemolyticus strain RIMD2210633, which exhibits Kanagawa phenomenon (KP), revealed the presence of two sets of the genes for the type III secretion system (T3SS) on chromosomes 1 and 2, T3SS1 and T3SS2, respectively. Although T3SS2 of the RIMD2210633 strain is thought to be involved in human pathogenicity, i.e., enterotoxicity, the genes for T3SS2 have not been found in trh-positive (KP-negative) V. parahaemolyticus strains, which are also pathogenic for humans. In the study described here, the DNA region of approximately 100 kb that surrounds the trh gene of a trh-positive V. parahaemolyticus strain, TH3996, was sequenced and its genetic organization determined. This revealed the presence of the genes for a novel T3SS in this region. Animal experiments using the deletion mutant strains of a gene (vscC2) for the novel T3SS apparatus indicated that the T3SS is essential for the enterotoxicity of the TH3996 strain. PCR analysis showed that all the trh-positive V. parahaemolyticus strains tested possess the novel T3SS-related genes. Phylogenetic analysis demonstrated that although the novel T3SS is closely related to T3SS2 of KP-positive V. parahaemolyticus, it belongs to a distinctly different lineage. Furthermore, the two types of T3SS2 lineage are also found among pathogenic Vibrio cholerae non-O1/non-O139 strains. Our findings demonstrate that these two distinct types are distributed not only within a species but also beyond the species level and provide a new insight into the pathogenicity and evolution of Vibrio species.Vibrio parahaemolyticus is a gram-negative halophilic marine and estuarine bacterium which is an important pathogen causative of food-borne gastroenteritis and traveler's diarrhea (1). Although most V. parahaemolyticus strains are nonpathogenic for humans, a limited population of these organisms causes human diseases. Almost all clinical V. parahaemolyticus isolates produce the thermostable direct hemolysin (TDH) and/or the TDH-related hemolysin (TRH), which are encoded by the tdh and trh genes, respectively (5, 21). The Kanagawa phenomenon (KP), a beta-type hemolysis on a special blood agar (Wagatsuma agar) (28), is known as a good marker of pathogenic strains (5, 21). V. parahaemolyticus strains which exhibit KP possess the two tdh genes tdhA (tdh2) and tdhS (tdh1) but not the trh gene (6,19,21). In contrast, KP-negative clinical V. parahaemolyticus strains possess the trh gene only or both the trh and tdh genes, while the majority of the nonpathogenic strains possess neither tdh nor trh.TDH and TRH, which have several biological activities in common (5,20,30,33), are considered to be the major virulence factors in clinical V. parahaemolyticus strains (5, 30).However, several studies have demonstrated that although the enterotoxicity was reduced in tdh-or trh-deleted mutant strains from that in the parent strains, the enterotoxic activity of these mutant strains partiall...

The Malachite Green method for determination of inorganic phosphate (Pi) (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361-366) was modified to measure Pi in the range of 0.2-15 nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6 M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2 g of sodium molybdate, 0.3 g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in 1 liter of 0.7 M HCl, and the absorbance at 650 nm is then measured after a 35-40 min incubation at 25 degrees C. Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately using relatively low concentrations of native myosin and its subfragment-1. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8-0.9 mol per site in 0.1 M KCl, 10 mM MgCl2, pH 8.0 at 25 degrees C for fresh enzyme preparations.

SummaryVibrio parahaemolyticus strain RIMD2210633 has two sets of genes encoding two separate type III secretion systems (T3SSs), called T3SS1 and T3SS2. T3SS2 has a role in enterotoxicity and is present only in Kanagawa phenomenon-positive strains, which are pathogenic to humans. Accordingly, T3SS2 is considered to be closely related to V. parahaemolyticus human pathogenicity. Despite this, the biological actions of T3SS2 and the identity of the effector protein(s) secreted by this system have not been well understood. Here we report that T3SS2 induces a cytotoxic effect in Caco-2 and HCT-8 cells. Moreover, it was revealed that VPA1327 (vopT), a gene encoded within the proximity of T3SS2, is partly responsible for this cytotoxic effect. The VopT shows approximately 45% and 44% identity with the ADPribosyltransferase (ADPRT) domain of ExoT and ExoS, respectively, which are two T3SS-secreted effectors of Pseudomonas aeruginosa. T3SS2 was found to be necessary not only for the secretion, but also for the translocation of the VopT into host cells. We also demonstrate that VopT ADP-ribosylates Ras, a member of the low-molecular-weight G (LMWG) proteins both in vivo and in vitro. These results indicate that VopT is a novel ADPRT effector secreted via V. parahaemolyticus T3SS.

Vibrio parahaemolyticus, a bacterial pathogen, causes human gastroenteritis. A type III secretion system (T3SS2) encoded in pathogenicity island (Vp-PAI) is the main contributor to enterotoxicity and expression of Vp-PAI encoded genes is regulated by two transcriptional regulators, VtrA and VtrB. However, a host-derived inducer for the Vp-PAI genes has not been identified. Here, we demonstrate that bile induces production of T3SS2-related proteins under osmotic conditions equivalent to those in the intestinal lumen. We also show that bile induces vtrA-mediated vtrB transcription. Transcriptome analysis of bile-responsive genes revealed that bile strongly induces expression of Vp-PAI genes in a vtrA-dependent manner. The inducing activity of bile was diminished by treatment with bile acid sequestrant cholestyramine. Finally, we demonstrate an in vivo protective effect of cholestyramine on enterotoxicity and show that similar protection is observed in infection with a different type of V. parahaemolyticus or with non-O1/non-O139 V. cholerae strains of vibrios carrying the same kind of T3SS. In summary, these results provide an insight into how bacteria, through the ingenious action of Vp-PAI genes, can take advantage of an otherwise hostile host environment. The results also reveal a new therapeutic potential for widely used bile acid sequestrants in enteric bacterial infections.

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound oa-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (-1,100 residues), that for glucan binding (-240 residues), and that of unknown function (-260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.Mutans streptococci produce several extracellular glucosyltransferases (GTFs) which synthesize water-soluble glucans and water-insoluble glucans (ISGs) containing co-1,6-and a-1,3-glucosyl linkages [ISG(1,6) and ISG(1,3), respectively]. The ISGs adhere to smooth tooth surfaces and facilitate aggregation of oral bacteria, so that GTFs are believed to play a key role in the formation of dental plaque (12,20).In view of this etiological importance, a number of studies of the properties of GTFs to understand the mechanism of ISG synthesis have been conducted over the past two decades (2,5,8,9,11,23,24,30,33). From the culture fluids of a strain of Streptococcus sobrinus (previously named Streptococcus mutans 6715), we purified an enzyme responsible for ISG synthesis (GTF-I) from sucrose in the presence of a-1,6 soluble glucan such as dextran T10 as a primer (9). In this reaction, sucrose is split into fructose and an enzymebound glucosyl moiety (26); the latter is transferred to the C-3 position of the glucose residue of the glucan, resulting in the formation of a-1,3-glucosyl polymer (GTF-I activity) (9). In the absence of the primer glucans, however, the glucosyl moiety is transferred to water (hydrolysis of sucrose or sucrase activity). Further studies have not been made because of a limited supply of purified GTF-I.In the meantime, the structure and function of GTFs have been studied by biochemical and recombinant DNA techniques. Glucan-binding fragments were isolated by tryptic digestion of GTF proteins from S. sobrinus, but none of them showed glucan-synthesizing activity (18,22). Of several molecular cloning studies of GTF genes from mutans streptococci (1,7,10,13,14,25...