Contribution of the tdh1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin.

Kanagawa phenomenon-positive strains of Vibrio parahaemolyticus contain two copies of the tdh gene (tdh 1 and tdh 2) encoding thermostable direct hemolysin (TDH). Previous studies suggested that the tdh 2 gene, but not the tdh 1 gene, was responsible for production of extracellular TDH. In this study, a tdh 2-deficient isogenic mutant of Kanagawa phenomenon-positive strain AQ3815 was constructed by a suicide vector-mediated in vivo recombination method. The intact tdh 1 gene in the mutant contributed little to Kanagawa phenomenon on Wagatsuma agar but produced TDH in broth media, accounting for 0.5-9.4% of total extracellular TDH of AQ3815.     

Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin.

Vibrio parahaemolyticus produces a thermostable direct hemolysin (TDH) that has been implicated in the pathogenesis of diarrheal disease caused by this organism. However, previous studies attempting to demonstrate the contribution of the hemolysin to virulence have been inconclusive. We investigated this putative virulence factor by using an isogenic TDH-negative (TDH-) strain constructed by specifically inactivating the two copies of the tdh gene encoding TDH. The enterotoxigenicities of the parent strain (AQ3815) and the mutant strain were tested by adding sterile culture supernatants to rabbit ileal tissue mounted in Ussing chambers. The culture filtrate of the parent strain produced a significant increase in short-circuit current (Isc), compared with the change induced by the TDH- mutant. The capacity of the culture filtrate of AQ3815 to increase the Isc was reduced by neutralization with anti-TDH serum, and the return of the cloned tdh gene to the TDH- mutant restored the ability to increase the Isc. These results were corroborated by rabbit ileal loop assays in which AQ3815 caused fluid accumulation but the TDH- mutant did not. No microscopic damage was seen in mucosal tissues exposed to the culture filtrate of either strain. These results indicate that TDH has an enterotoxigenic effect on rabbit small intestine and could be responsible for the watery diarrhea seen with V. parahaemolyticus.     

The glucose-dependent transactivation activity of ABF1 on the expression of the TDH3 gene in yeast

Autonomously replicating sequence binding factor 1 (ABF1) has been implicated in the control of a variety of gene expressions in Saccharomyces cerevisiae. In this paper evidence is presented that ABF1 is involved in the glucose-dependent expression of the TDH3 gene which encodes glyceraldehyde-3-phosphate dehydrogenase. ABF1 binds to consensus sites located between-420 and-250, and between +77 and +200, and acts as a transactivator in an orientation-independent manner on both upstream and downstream sites. TDH3-lacZ fusions having an ABF1 consensus motif showed glucose-dependent expression of TDH3, whereas in the abf1 mutant strain JCA35 glucose-dependent expression disappeared. These findings suggest that ABF1 functions as a glucose-dependent transactivator for the expression of the TDH3 gene.     

Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766.

Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized. The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns. The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically. The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs. The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced. Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1%. At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I. Two amino acid differences were observed between TrhX and Trh. Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously. This is the first evidence that a strain of V. parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.     

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and application of the antibodies for enzyme-linked immunosorbent assay

A total nine hybridoma cell lines that produced monoclonal antibodies against thermostable direct hemolysin (Vp-TDH), a possible pathogenic toxin, of Kanagawa phenomenon-positive Vibrio parahaemolyticus was isolated and characterized. These monoclonal antibodies (mAbs) were divided into a minimum of five different specificity groups, including mAbs specific to Vp-TDH and common to Vp-TDH and Vp-TRH, a Vp-TDH-related hemolysin produced by Kanagawa phenomenon-negative V. parahaemolyticus. An enzyme-linked immunosorbent assay (ELISA) using mAb-1-D, a mAb specific for Vp-TDH, was developed for specific detection of Vp-TDH. On the other hand, the ELISA using mAb-9-D, and mAb common to both Vp-TDH and Vp-TRH, could be used for detection of both Vp-TDH and Vp-TRH. Thus, by combining these two ELISAs differential detection of Vp-TDH and Vp-TRH can be performed. Hence, the two ELISAs were applied for various strains of V. parahaemolyticus and it was found that most Kanagawa phenomenon-positive and -negative clinical isolates produced Vp-TDH and Vp-TRH, respectively, but all environmental strains, that were Kanagawa phenomenon-negative, produced neither toxin.Supported by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Developmental Scientific Research from the Ministry of Education, Science, and Culture of Japan     

Cloning and molecular analysis of theIsi1(rfaF) gene ofNeisseria meningitidiswhich encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells

Neisseria meningitidis, but notHaemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. TheIsi1gene, which had been shown to be involved in LPS biosynthesis ofNeisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology withIsi1ofN. gonorrhoeaeand therfaFgene ofSalmonella typhimuriumwhich encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the clonedIsi1gene, as a functionalrfaFhomologue, was confirmed by the complementation of aS. typhimurium rfaFmutant using a P22 phage sensitivity test. AnIsi1mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type,Isi1, andgalEstrains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galEmutant) or the majority (Isimutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity.     

NADH-reductive stress in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (<Emphasis Type="Italic">TDH1</Emphasis>)

A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2 strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2 strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2 strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.H. Valadi and Å. Valadi contributed equally to this work     

The frequency of fim genes among Salmonella serovars

Salmonella serovars were examined for the presence of fim gene sequences using specific DNA probes. All strains, regardless of their ability to express surface-associated fimbriae, retain a considerable amount of DNA homologous to the gene probes used. The phenotypically non-fimbriate FIRN and non-FIRN strains of S. typhimurium retain detectable amounts of fim gene sequences and, therefore, may not be genotypically non-fimbriate. The MS adhesin can be expressed by type 2 fimbriate bacteria when they are transformed with discrete regions of the fim gene cluster. However, this conversion to a hemagglutinating phenotype is not associated with a small region of DNA. Therefore, the inability of type 2 fimbria-producing strains of Salmonella to mediate hemagglutination does not appear to be due to a small deletion in a single fim gene.     

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma — Next step toward reliable non-invasive prenatal diagnostics

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n = 51) or female (n = 19) fetus sampled throughout gestation and absent in non-pregnant individuals (n = 29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ = 0.66, P < 0.001), total RASSF1A and GLO (ρ = 0.65,P < 0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ = 0.62, P < 0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.     

Systemic Infection Following Intravenous Inoculation of Mice with Candida albicans int1 Mutant Strains

The Candida albicans gene INT1 is associated with epithelial adhesion, hyphal formation, and virulence. C. albicans strains carrying two, one, or no functional INT1 alleles were used to assess the association between mortality and C. albicans persistence in the liver and kidney of intravenously inoculated mice. Mice were injected with 10⁵C. albicans CAF2 (parent strain, INT1/INT1), C. albicans CAG3 (homozygous disruptant, Int1/int1), or C. albicans CAG5 (heterozygous reintegrant, int1/int1 + INT1). Mortality was monitored and mice were sacrificed on Days 1, 7, 14, and 21 for quantitative analysis of kidney and liver microbes, with histologic analysis of these tissues as well. Mortality was highest for mice injected with the wild-type strain CAF2 (INT1/INT1) and lowest for mice injected with the homozygous disruptant CAG3 (int/int1). Yeast were readily cleared from the liver of all mice injected with any of the three C. albicans strains. Although the mutant strains CAG3 and CAG5 are defective for hyphal formation in vitro, there was histological evidence of abundant hyphal formation in the renal pelvis of mice injected with these strains. Compared to the wild-type strain, mutant strains were associated with reduced mortality but increased C. albicans persistence in the kidney. Thus, the absolute ability to form hyphae in the kidney did not appear to modulate either C. albicans-induced mortality or the course of progressive infection in the kidney. In addition, reduced virulence was paradoxically associated with increased, not decreased, persistence of C. albicans in the kidney.     

Investigation of Batten Disease with the YeastSaccharomyces cerevisiae

TheCLN3gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned theSaccharomyces cerevisiaehomolog to the humanCLN3gene, designatedBTN1,whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p,btn1-Δ deletion yeast strains, are more resistant to -(−)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the humanCLN3gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced intoBTN1,altered the degree of ANP resistance. Severity of Batten disease due to mutations inCLN3and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.     

The A312L 5′-UTR of Chlorella virus PBCV-1 is a translational enhancer in Arabidopsis thaliana

PBCV-1 (Paramecium bursaria Chlorella virus) is a large double stranded DNA virus that replicates in certain eukaryotic chlorella like green algae. The PBCV-1 A312L gene encodes a 33-kDa protein whose function currently is unknown. The 5′-UTR of the A312L mRNA is 153 nucleotides, longer than the 5′-UTR in any other PBCV-1 gene. The sequence 5′-AAAC was repeated 17 times within 156 bp 5′ to the A312L gene start codon and this sequence was repeated 13 times continuously in the 5′-UTR of the mRNA. Recombinant genes were constructed in vector pBI121 that contained the A312L 5′-UTR, in both the forward and inverse-complement orientations, fused to the GUS gene under the control of the CaMV 35S promoter. These constructs were introduced into Arabidopsis thaliana and the results indicated that the A312L 5′-UTR functions as a translational enhancer only in the forward orientation. Overall, the ratio of GUS enzyme activity to GUS mRNA was 15-fold higher in constructs derived from the A312L 5′-UTR in the forward orientation as compared to constructs containing the 5′-UTR in the inverse-complement orientation or those lacking the A312L 5′-UTR.     

Defective responses to oxidative stress in protein l-isoaspartyl repair-deficient Caenorhabditis elegans

We have shown that Caenorhabditis elegans lacking the PCM-1 protein repair l-isoaspartyl methyltransferase are more sensitive to oxidative stress than wild-type nematodes. Exposure to the redox-cycling quinone juglone upon exit from dauer diapause results in defective egg-laying (Egl phenotype) in the pcm-1 mutants only. Treatment with paraquat, a redox-cycling dipyridyl, causes a more severe developmental delay at the second larval stage in pcm-1 mutants than in wild-type nematodes. Finally, exposure to homocysteine and homocysteine thiolactone, molecules that can induce oxidative stress via distinct mechanisms, results in a more pronounced delay in development at the first larval stage in pcm-1 mutants than in wild-type animals. Homocysteine treatment also induced the Egl phenotype in mutant but not wild-type nematodes. All of the effects of these agents were reversed upon addition of vitamin C, indicating that the developmental delay and egg-laying defects result from oxidative stress. Furthermore, we have demonstrated that a mutation in the gene encoding the insulin-like receptor DAF-2 suppresses the Egl phenotype in pcm-1 mutants treated with juglone. Our results support a role of PCM-1 in the cellular responses mediated by the DAF-2 insulin-like signaling pathway in C. elegans for optimal protection against oxidative stress.     

Genetic analysis of a host determination mechanism of bromoviruses in Arabidopsis thaliana

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.     

A high capacity Alphavirus heterologous gene delivery system

A novel replication competent Sindbis virus based gene delivery vector has been developed for the introduction of genetic cargo into cell lines in vitro and potentially, animal models in vivo. This delivery system expands the previous uses of Sindbis virus as a gene delivery system in that no replicons are required and the resulting cargo containing virus particles are infectious. The heterologous vector is based on a morphological mutant in C, Ser180/Gly183 which produces larger than the normal size T = 4 virus particles of 70 nm in size. This mutant produced particles up to 205 nm in size equal to a triangulation number of 36. It was postulated that because the Ser180/Gly183 mutant was capable of assembling such large particles, that increasing the size of the RNA genome incorporated into this mutant capsid protein would favor the assembly of larger than T = 4 wild type sized virions. The first generation prototype larger vehicle, described here, carries a ~ 18 kb cDNA insert, however it is conceivable that RNA as large as 32 kb could be transcribed and packaged. The large variant produces a high virus titer of ~ 10⁹ pfu/ml from either mammalian or insect cells in culture. Multiple passages of the virus show no loss of the inserted genetic material.     

Dihydropteroate synthase gene mutations in Pneumocystis jiroveci strains isolated from immunocompromised patients

The emergence of Pneumocystis jiroveci drug resistance has been suggested recently by the mutations in the gene encoding dihydropteroate synthase (DHPS). The aim of the present study was to determine the prevalence of DHPS mutations in P. jiroveci strains isolates from bronchoalveolar lavages (BAL) and sputum samples of 21 immunocompromised patients. We used the touchdown-PCR for amplification of DHPS gene and the restriction fragment length polymorphism (RFLP) technique for discrimination of wild and mutant DHPS genotypes. The DHPS amplification was positive in 17 patients (81%). The association of wild genotype and mutant genotype was detected in two patients after the enzymatic digestion of the PCR products by AccI and HaeIII. No mutations in the DHPS gene were seen in 15 patients. In addition, no variation was observed in DHPS genotypes detected in the repeated specimens (BAL and sputum) from some patients. The touchdown PCR-RFLP technique is a simple and rapid method for revelation of DHPS gene mutations in P. jiroveci strains. It could be advantageously used in clinical laboratory to control the prevalence of mutations associated with sulfa resistance.     

In vivo detection of lamellocytes in Drosophila melanogaster

Drosophila has recently become a powerful model organism for studies of innate immunity. The cellular elements of innate immunity in Drosophila, the hemocytes, have been characterized by morphological criteria, molecular markers, and cell-type-specific immunological markers. Here we suggest that an MiET1 GFP-reporter element insertion in the untranslated region of a gene (l1-atilla) – expressed in a subset of hemocytes, the lamellocytes – allows in vivo investigations of lamellocyte differentiation and facilitates genetic screens.