Abortive replication of choleraphage phi 149 in Vibrio cholerae biotype el tor.

Choleraphage phi 149 adsorbed irreversibly to Vibrio cholerae biotype el tor cells, and 50% of the injected phage DNA bound to the cell membrane. Although no infectious centers were produced at any time during infection, the host macromolecular syntheses were shut off and the host DNA underwent chloramphenicol-inhibitable degradation. Synthesis of monomeric phage DNA continued similar to that observed in the permissive host. However, the concatemeric DNA intermediates produced were unstable and could not be chased to mature phage DNA. Pulse-labeling of UV-irradiated infected cells at different times during infection allowed identification of phage-specific proteins made in this nonpermissive host. Although most of the early proteins were made, only some of the late proteins were transiently synthesized.     

Experimental resistance of Vibrio cholerae el tor to nalidixic acid and fluoroquinolones]

It was shown that sensitivity of Vibrio cholerae eltor P-5879 to tetracycline, levomycetin, furazolidone, trimethoprim/sulfamethoxazole, aminoglycosides, beta-lactams, rifampicin, quinolones in vitro correlated with drugs efficacy in the treatment of experimental cholera of albino mice. Mutants of V. cholerae eltor P-5879 Nalr resistant to nalidixic acid (MIC 160-200 mg/l) formed with frequency 10(-9)-110(-8) had no cross resistance to fluoroquinolones. But the efficacy of ofloxacin, lomefloxacin, norfloxacin against these mutants in vivo reduced, though it was not changed in vitro. Mutants of V. cholerae eltor P-5879 resistant to fluoroquinolones and selected after culturing in the presence of the drugs had cross resistance to all quinolones studied. Infection caused by Cpfr mutant could not be treated with nalidixic acid and fluoroquinolones, therapeutic efficacy of rifampicin and beta-lactams, also reduced though sensitivity in vitro was not changed. The results of investigation proves the necessity of quinolones use for cholerae treatment as it is recommended for other severe enteric infections.     

Facile identification of protein sequences by mass spectrometry. B subunit of Vibrio cholerae classical biotype Inaba 569B toxin

A mass spectrometric method was applied to the B subunit of Vibrio cholerae classical biotype Inaba 569B toxin to determine its amino acid sequence and to confirm the differences in the amino acid sequences predicted from the nucleotide sequences of the genes of El Tor biotype strains 62746 and 2125 toxins. In this method, the Staphylococcus aureus protease V8 digest of the CNBr-treated B subunit of the classical biotype toxin was examined directly by fast-atom-bombardment mass spectrometry without separation of individual peptides. The values of molecular ion signals observed in the mass spectra were compared with the amino acid sequences of the classical biotype and El Tor biotype toxins. All the observed mass values coincided with those calculated from the published sequences of the B subunit except those of the sequences at positions 12-29 and 69-79. Peptides with these sequences were isolated by high-performance liquid chromatography and analyzed by Edman degradation or by combination of mass spectrometry and enzymatic degradation. The results revealed that the amino acid residues at positions 22 and 70 were Asp instead of Asn in the published sequences of classical biotype toxin. It was also found that Asn at position 44 was partially deaminated to Asp. The amino acid sequence of the classical biotype toxin was found to be different only at positions 18 (His----Tyr), 47 (Thr----Ile) and 54 (Gly----Ser) from that of El Tor biotype toxins.     

Variation in epitopes of the B subunit of El Tor and classical biotype Vibrio cholerae O1 cholera toxin

Variation in epitopes of the B subunit of cholera toxin (CT-B) produced by strains of El Tor and classical biotype Vibrio cholerae O1 was examined using monoclonal antibodies prepared to V. cholerae 569B CT. CT-B epitopes were markedly conserved for V. cholerae classical biotypes. In contrast, epitope variation was observed for El Tor biotypes, which produced both a classical-like CT-B and a unique CT-B lacking at least one epitope common to 569B CT-B. The missing epitope was located outside the GM1 ganglioside-binding site. From results of the study reported here, genetic divergence is exhibited in the El Tor biotype CT-B versus classical CT-B. Furthermore, at least five unique epitopes of V. cholerae 569B CT-B can be defined.     

A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1

A bioserogroup of halophilic vibrios, tentatively labelled bioserogroup 1875, strongly agglutinated by O1 antiserum of Vibrio cholerae is described. Cross-agglutination and agglutinin-absorption tests showed that these vibrios had antigens identical with the Ogawa and Inaba factors of V. cholerae O1, although they possessed their own specific antigen distinctive from the A factor that is the specific major antigen of the latter species. In addition, quantitative antigen variation similar to that of the Ogawa-to-Inaba type with V. cholerae O1 was recognized in this halophilic group. They were isolated from estuarine water and are considered to inhabit coastal aquatic environments. Phenotypically, however, this group is not identifiable with any of the species already recognized in the genus Vibrio .     

A bioserogroup of marine vibrios possessing somatic antigen factors in common with Vibrio cholerae O1

A bioserogroup of halophilic vibrios, tentatively labelled bioserogroup 1875, strongly agglutinated by O1 antiserum of Vibrio cholerae is described. Cross-agglutination and agglutinin-absorption tests showed that these vibrios had antigens identical with the Ogawa and Inaba factors of V. cholerae O1, although they possessed their own specific antigen distinctive from the A factor that is the specific major antigen of the latter species. In addition, quantitative antigen variation similar to that of the Ogawa-to-Inaba type with V. cholerae O1 was recognized in this halophilic group. They were isolated from estuarine water and are considered to inhabit coastal aquatic environments. Phenotypically, however, this group is not identifiable with any of the species already recognized in the genus Vibrio.     

Cell-associated hemagglutinin of classical Vibrio cholerae O1 with reference to intestinal adhesion

Abstract Extraction of Streptococcus mutans with the detergents HECAMEG and lauroyl sarcosinate preferentially extracted antigen D, previously identified as a low molecular mass wall-associated protein. Western blotting with specific antisera was used to demonstrate that this antigen is the HPr component of the sugar-phosphotransferase transport system. Non-denaturing electrophoresis indicated that HECAMEG selectively extracted only one of the two forms of HPr. It is suggested that this form of HPr may have a specific cell surface location.     

A phage protein confers resistance to the lactococcal abortive infection mechanism AbiP

Phage bIL66M1 is sensitive to the lactococcal abortive infection mechanism AbiP. No spontaneous AbiP-resistant variant could be obtained at a frequency of <10(-10). However, AbiP-resistant variants were readily obtained during infection with both bIL66M1 and the highly homologous AbiP-resistant phage bIL170. Gain of AbiP resistance was due to the acquisition of the e6 gene from bIL170.     

A three-residue signal confers localization of a reporter protein in the inner nuclear membrane

By alignment to the carboxy-terminal-deduced aa sequence of human cytomegalovirus glycoprotein B (gB), conserved hexameric aa motifs with putative function for localization in the inner nuclear membrane (INM) were identified in the nucleoplasmic tails of herpes simplex virus type 1 gB and of the cellular lamin B receptor. Fusion of the respective hexamers to CD8 as a reporter redirected transport of the chimeras into the INM, suggesting their functioning as modular signal motifs. Consecutive experiments showed that the three-residue motif RxR represents a consensus sequence which is sufficient for localization of the CD8 reporter in the INM.     

Bacterial-associated cholera toxin and GM1 binding are required for transcytosis of classical biotype Vibrio cholerae through an in vitro M cell model system

To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae, we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM1. Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre-incubation with GM1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.     

A common polymorphism in TLR3 confers natural resistance to HIV-1 infection

TLR3 recognizes dsRNA and activates antiviral immune responses through the production of inflammatory cytokines and type I IFNs. Genetic association studies have provided evidence concerning the role of a polymorphism in TLR3 (rs3775291, Leu412Phe) in viral infection susceptibility. We genotyped rs3775291 in a population of Spanish HIV-1-exposed seronegative (HESN) individuals who remain HIV seronegative despite repeated exposure through i.v. injection drug use (IDU-HESN individuals) as witnessed by their hepatitis C virus seropositivity. The frequency of individuals carrying at least one 412Phe allele was significantly higher in IDU-HESN individuals compared with that of a matched control sample (odds ratio for a dominant model = 1.87; 95% confidence interval, 1.06-3.34; p = 0.023). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Similar results were obtained: the frequency of individuals carrying at least one 412Phe allele was significantly higher compared with that of a matched control sample (odds ratio, 1.79; 95% confidence interval, 1.05-3.08; p = 0.029). In vitro infection assays showed that in PBMCs carrying the 412Phe allele, HIV-1(Ba-L) replication was significantly reduced (p = 0.025) compared with that of Leu/Leu homozygous samples and was associated with a higher expression of factors suggestive of a state of immune activation (IL-6, CCL3, CD69). Similarly, stimulation of PBMCs with a TLR3 agonist indicated that the presence of the 412Phe allele results in a significantly increased expression of CD69 and higher production of proinflammatory cytokines including IL-6 and CCL3. The data of this study indicate that a common TLR3 allele confers immunologically mediated protection from HIV-1 and suggest the potential use of TLR3 triggering in HIV-1 immunotherapy.     

Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection.

An important aspect of gene transfer into farm animals is the improvement of disease resistance. The mouse Mx1 protein is known to be sufficient to confer resistance to influenza viruses. Gene constructs containing the mouse Mx1 cDNA controlled by the human metallothionein IIA promoter (hMTIIA::Mx), the SV40 early enhancer/promoter region (SV40::Mx) and the mouse Mx1 promoter (mMx::Mx) were transferred into pigs. The results of the gene transfer experiments with the hMTIIA::Mx and the SV40::Mx constructs indicate that the permanent high-level synthesis of Mx1 might be deleterious to the organism: the gene transfer efficiency was surprisingly low, and all transgenic piglets born had rearrangements in their transgene copies that abolished protein synthesis. The use of the interferon (IFN)- and virus-inducible mMx::Mx construct resulted in normal gene transfer efficiency. Two transgenic pig lines could be established which expressed IFN-inducible mouse Mx1 mRNA. Extensive protein analysis did not detect mouse Mx1 in IFN-treated transgenic animals.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

The protective activity of tea catechins against experimental infection by Vibrio cholerae O1.

Tea catechins inhibited the fluid accumulation induced by cholera toxin in sealed adult mice. The catechins also reduced fluid accumulation by Vibrio cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea catechins may possess protective activity against V. cholerae O1.     

Interferon-inducible mouse Mx1 protein that confers resistance to influenza virus is GTPase.

The murine Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection even though the resistance mechanism is yet unclear. The Mx1 protein contains a tripartite GTP-binding domain consisting of GXXXXGKS, DXXG, and T/NKXD motifs. In the GTPase gene superfamily such as p21ras protein, signal-transducing G protein, and translation elongation factor, the GTPase activity plays a key role in each protein function. Here we show that GTPase activity is indeed associated with the intact Mx1 protein purified from Escherichia coli expressing Mx1 cDNA. Amino acid substitution within the GTP-binding motif led to significant reduction in the GTPase activity. Yeast vacuolar protein sorting (VPS1) protein and the rat microtubule-associated mechanochemical enzyme dynamin were found to be homologous to Mx1 not only in the tripartite GTP-binding motif, but also in the amino-terminal region of approximately 300 amino acids in length. The function of Mx1 is discussed in comparison with these proteins.     

A plasmid of group Q which confers resistance to trimethoprim and sulfonamides

A 5.2-Mdal plasmid, determining resistance to trimethoprim and sulfonamides, is a member of incompatibility group Q.     

Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase

170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.