Noboru Nakasone

Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor.

A method that stimulates cholera toxin (CT) production by Vibrio cholerae O1 biotype El Tor (El Tor vibrios) to the level of several micrograms per ml in the culture fluid was established. Such a large amount of CT was obtained by the following method: El Tor vibrios were cultured in AKI medium (1.5% Bacto peptone, 0.4% yeast extract‐Difco, 0.5% NaCl, 0.3% NaHCO3) at 37 C for 4 hr in a stationary test tube and then for 16 hr in a shaken flask, with inoculum sizes of 105 to 107/ml. With this method, 35 strains out of 60 examined produced 2 to 16 μg/ml of CT as determined by the reversed passive latex agglutination test (RPLA). Thirty‐three randomly selected strains out of the 60 produced reasonable amounts of rabbit skin vascular permeability factor, reflecting the amount of CT titrated with RPLA.

Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

ABSTRACT A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

Antibiotic resistance conferred by a class I integron and SXT constin in Vibrio cholerae O1 strains isolated in Laos

ABSTRACT Changes in the drug susceptibility pattern were observed in Vibrio cholerae O1 isolated in the Lao Peoples Democratic Republic during 1993 to 2000. In this study, 50 V. cholerae O1 strains were selected during this period for studying the presence of class I integron and SXT constin. Twenty-four streptomycin-resistant strains out of 26 isolated before 1997 contained a class I integron harboring the aadA1 gene cassette. Twenty-four strains isolated after 1997 contained an SXT constin (a large conjugative element). Twenty of the strains were resistant to chloramphenicol, tetracycline, streptomycin, and trimethoprim-sulfamethoxazole, while four strains were susceptible to the antibiotic tested. The resistance genes included in the SXT constins were floR, tetA, strAB, and sulII, which encode resistance to chloramphenicol, tetracycline, streptomycin, and sulfamethoxazole, respectively. The antibiotic resistance gene cluster was found to be deleted in the four susceptible strains. SXTLAOS did not contain dfrA1 or dfr18, which confer resistance to trimethoprim in SXTET and SXTMO10, respectively. A hot spot region of SXTLAOS was sequenced, and we identified two novel open reading frames showing homology to sO24 (exonuclease) and sO23 (helicase) of the genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104. Analysis of SXTLAOS showed that there is a continuous flux of genes among V. cholerae SXT constins which should be carefully monitored.

Pathogenic Vibrio Activate NLRP3 Inflammasome via Cytotoxins and TLR/Nucleotide-Binding Oligomerization Domain-Mediated NF-κB Signaling

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The β form of pro–IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of β form of pro–IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-κB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-κB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-κB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-κB.

Inflammasome activation via intracellular NLRs triggered by bacterial infection

Members of the nucleotide‐binding, oligomerization domain (NOD)‐like receptor (NLR) proteins assemble into a multiprotein platform, known as the inflammasome, to induce caspase‐1 activation followed by the subsequent secretion of IL‐1β and IL‐18. In this review, we focus on the role of NLRs in inflammasome activation as part of the host defence against bacterial pathogens. One of activators of the NLRC4 inflammasome is bacterial flagellin secreted through type III or IV secretion systems, which are important for the pathogenicity of many Gram‐negative bacteria. The NLRP3 inflammasome is mainly activated by a large number of bacterial pore‐forming toxins. Despite our knowledge of inflammasome activation upon bacterial infection, the function of antibacterial defence under in vivo conditions remains to be elucidated. Further understanding of NLR function should provide new insights into the mechanisms of host pro‐inflammatory responses and the pathogenesis of bacterial infections.

Vibrio parahaemolyticus effector proteins suppress inflammasome activation by interfering with host autophagy signaling.

Bacterial pathogens utilize pore-forming toxins or sophisticated secretion systems to establish infection in hosts. Recognition of these toxins or secretion system by nucleotide-binding oligomerization domain leucine-rich repeat proteins (NLRs) triggers the assembly of inflammasomes, the multiprotein complexes necessary for caspase-1 activation and the maturation of inflammatory cytokines such as IL-1β or IL-18. Here we demonstrate that both the NLRP3 and NLRC4 inflammasomes are activated by thermostable direct hemolysins (TDHs) and type III secretion system 1 (T3SS1) in response to V. parahaemolyticus infection. Furthermore, we identify T3SS1 secreted effector proteins, VopQ and VopS, which induce autophagy and the inactivation of Cdc42, respectively, to prevent mainly NLRC4 inflammasome activation. VopQ and VopS interfere with the assembly of specks in infected macrophages. These data suggest that bacterial effectors interfere with inflammasome activation and contribute to bacterial evasion from the host inflammatory responses.

Pili of an Aeromonas hydrophila Strain as a Possible Colonization Factor

Aeromonas hydrophila Ae6 had 2 morphologically distinctive kinds of pili. One appeared rigid and straight with a diameter of 9 nm (R‐pili). The other appeared wavy and flexible with a diameter of 7 nm (W‐pili). W‐pili were very few on the cell as compared with R‐pili. In this study, W‐pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 21 kDa as estimated by SDS‐PAGE. There was no immunological cross‐reaction between W‐pili and other cellular components. The strain Ae6 and its purified W‐pili adhered to human and rabbit intestine and agglutinated human and rabbit erythrocytes. Organisms pretreated with the Fab fraction of anti‐pilus antibody failed to adhere to the intestine. Pretreatment of intestine with purified W‐pili blocked adherence of the organisms to the intestine. These results suggest that the W‐pili are the colonization factor of A. hydrophila Ae6.

Pili of Aeromonas hydrophila: purification, characterization, and biological role.

Aeromonas hydrophila (Ae6) has 2 morphologically distinctive kinds of pili. One appeared rigid, channeled, and straight with a diameter of 9 nm (Ae6‐R pili). The other looked flexible, wavy, and having helical structure with a diameter of 7 nm (Ae6‐W pili). Ae6‐R pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 18 kDa as estimated by SDS‐PAGE, and contained 42.3% hydrophobic amino acids and one cysteine residue. The pilus was solubilized to 18 kDa subunit protein by 2‐mercaptoethanol, dithiothreitol, hydrochloric acid, or heating at 120 C for 5 min. The organism Ae6 was strongly adhesive to rabbit intestines as well as human intestines, and agglutinated erythrocytes. Anti‐pili antibody (Fab fraction) did not block the adhesion. Purified Ae6‐R pili did not adhere to the intestine or to the erythrocytes. However, the anti‐pili Fab inhibited pellicle formation of the organisms cultured in broth, and also inhibited salt agglutination with ammonium sulfate. From these results, Ae6‐R pili are not likely a colonization factor but probably play a role in the autoaggregation of the organisms.

Quantitative Evaluation of Colonizing Ability of Vibrio cholerae O1

A new method to evaluate the adhesive ability of Vibrio cholerae O1 was proposed. Broth cultured V. cholerae O1 and a piece of formalin‐fixed rabbit intestinal wall were incubated together in KRT buffer and the number of adhered organisms was counted under a scanning electron microscope. This method was much less laborious than other methods that have been used so far, and most significantly, constant results were obtained in repeated experiments. The adhesive properties of toxigenic V. cholerae O1 evaluated by this method correlated well with its observed experimental pathogenicity.

Lose the battle to win the war: bacterial strategies for evading host inflammasome activation

The inflammasome is composed of nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins, and leads to caspase-1 activation and subsequent secretion of the proinflammatory cytokines interleukin 1β (IL-1β) and interleukin-18 (IL-18). After certain pathogenic bacteria infect host cells, such as macrophages, NLR-mediated inflammasome activation is triggered to form part of the host defenses against the invading pathogens. However, recent evidence has shown that bacteria have strategies for evading inflammasome activation in host cells. In this review, we focus on NLR-mediated inflammasome activation and bacterial evasion of the inflammasome as part of the battle between the host defenses and pathogens.