N. I. Smirnova

Genome variability in the altered variants of Vibrio cholerae biovar El Tor isolated in Russia

Molecular-genetic analysis of 56 clinical strains of the Vibrio cholerae biovar El Tor isolated during the period 1965–2010 in Russia and neighboring countries was carried out. Thirty-one isolates (55.3%) bearing the gene ctxB in the genome of the prophage CTXφ in cholera vibrios of the classical biovar were found. These strains are altered variants of the cholera El Tor pathogen. The nucleotide sequence of their genes ctxB and rstR included in the prophage CTXϕ, which are related with the production of the cholera toxin, was determined. It was found that altered variants bear different alleles of these genes (ctxB1 or ctxB7; rstREl and/or rstRClass) and belong to four gene types: ctxB1, rstRClass; ctxB1, rstREl; ctxB1, rstRClass/rstREl; and ctxB7, rstRClass/rstREl. It was shown that these gene variants produce 42 to 48 times more cholera toxin than typical strains. Isolates in which the pathogenicity island VSP-2 contains a long deletion were found among altered variants. The study data are evidence of the genomic variability of altered pathogen variants of the seventh cholera pandemic and require the development of new gene-diagnostic testing systems to improve the efficiency of epidemiological control of cholera distribution.

The evolution of the Vibrio cholerae genome during the modern period

A review of the modern data concerning the phenotypical and genetic peculiarities of the recently emerged highly virulent variants of V. cholerae El-Tor biovar (the etiological agent of the current cholera pandemic) is presented. The molecular-genetic mechanisms that are considered to be a basis for V. cholerae genome evolution during the modern period are discussed. The transposon role in the increased expression of cholera toxin genes, which are constituents of the CTXφ prophage genome, is addressed. The need to work out new gene-diagnostic test-systems for the identification of natural altered variants of the cholera etiological agent is shown.

Genomic variations of CTXφ prophage of Vibrio cholerae biovar El tor induced by Tn5-Mob transposone

One of the key pathogenicity factors of the cholera pathogen is the cholera toxin (CT), which is encoded by the operon ctxAB contained in the CTXφ prophage. Variations in the CTXφ prophage genome, the core region of which contains ctxAB, zot, ace, cep, orfU, and psh genes, underlies the changes in virulent properties of cholera vibrios. The mechanisms involved in remodeling this prophage genome are still vague. In this work, we demonstrate that the insertion of Tn5-Mob (KmR) transposon into the chromosome of the Vibrio cholerae model strain MAK757 biovar El Tor, which carries two copies of the CTXφ prophage, induces the remodeling of the prophage genome, namely, the deletion of the zot, ace, cep, and orfU genes. The level of CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob, which only retain the ctxAB operon, increased more than 2000-fold as compared to the original strain. It has been shown that the increased CT production is associated with the altered structure of the chromosomal DNA region containing a copy of the ctxAB operon, encoding this protein. This mutation in the genome of CTXφ prophage induced by Tn5-Mob is unstable. Among the 600 isolated colonies obtained after screening the MAK757 chr::Tn5-Mob transposant capable of CT overproduction on a full medium free of antibiotics, 5.8% yielded clones that lost the ctxAB operon, as well as the marker KmR, thus becoming nontoxigenic. The emergence of V. cholerae mutants both that both overproduce CT and are nontoxigenic demonstrates the important role of genetic variations in the CTXφ prophage in the evolution of V. cholerae pathogenicity. The obtained plasmid-free strain of V. cholerae biovar El Tor with type-2 CToverproduction can be used as a producer of this toxin, which is used to manufacture preparations for cholera diagnostics and prevention.

Genovariants of Vibrio cholerae biovar El Tor: Construction, molecular-genetic, and proteomic analyses

Experimental modeling of the emergence of virulent Vibrio cholerae El Tor genovariants is presented. It has been shown that the obtained genovariants differed neither in phenotypic or genotypic traits from natural genetically altered strains that emerged in populations of wild-type strains. It has been established, using the PCR and sequencing methods, that the genovariants formed in the process of conjugation carried in their genome a fragment of the CTXClassφ prophage genome with the ctxB1 gene of classical-type cholera vibrios. It has been shown that changes in the prophage’s structure led to higher levels of toxigenicity and virulence in the genovariants compared to a typical recipient strain. A proteomic analysis has also revealed changes in the expression of 26 proteins performing various functions in the cell, such as metabolism, energy exchange, transport of amino acids, etc.). These data are indicative of the effect produced by the new DNA region in the genome of the genovariants on the expression level of some house-keeping genes. The obtained results confirm the idea that horizontal gene transfer is one of the mechanisms leading to the emergence of genovariants in the populations of wild-type strains.

Effect of CTXφ prophage deletion in cholera agent on expression of regulatory genes controlling virulence and biofilm formation

This work represents the results of comparative analysis of the nucleotide sequences in the chromosomal region containing CTXφ prophage in isogenic toxigenic (Tox+) and nontoxigenic (Tox–) strains of Vibrio cholerae biovar El Tor. It is shown that spontaneous mutants which have identical Tox– phenotype are formed either owing to the precise elimination of CTXφ prophage from the chromosome (deletion size is 6.9 kb) or owing to imprecise elimination with the additional loss of two other prophages (RS1φ and TLCφ), which are adjacent to CTXφ prophage (deletion size is 17.4 kb). It is revealed that, in the nontoxigenic mutants, CTXφ deletion initiates simultaneous change in several phenotypic properties, which are associated with virulence or biofilm formation, such as colonizing ability, HA/P production, VPS, and motility. For the first time, it is stated that the cause of the pleiotropic effect of the CTXφ deletion is a cascade decrease in the transcription levels of seven regulatory genes (toxR, aphA, tcpP, tcpH, toxT, vpsT, vpsR) which control virulence and biofilm formation processes in the cholera agent.

Molecular-genetic properties of Vibrio cholerae El Tor strains circulating in Africa

The review contains some brief information on cholera epidemics in Africa. Comparative analysis of the results of whole-genome sequencing of 30 clinical strains isolated in Africa in different periods of the seventh cholera pandemic (1985–2012) and data from GenBank revealed their great genetic diversity. It is demonstrated that cholera epidemics in Africa, which began in 1970, were caused for about two decades by typical strains of the pathogen brought from India and Bangladesh. Currently, cholera in Africa is caused by new variants of the pathogen, which originated in Southeast Asia as a result not only of the acquisition of new genes due to horizontal transfer but also alterations in the genomes of previously existing pathogenicity and pandemicity islands. SNP analysis of 53 strains circulating in Africa (30 strains), as well as those isolated in Southeast Asia (23 strains), made it possible to establish phylogenetic relations for the majority of African and Asian strains. The issue of the existence of strains which are apparently endemic for Africa is being discussed. The existing genetic diversity of strains with different levels of virulence and drug resistance indicates the need for continuous molecular monitoring of the causative agent of cholera in Africa.

Whole-Genome Sequencing of Vibrio cholerae O1 El Tor Strains Isolated in Ukraine (2011) and Russia (2014)

ABSTRACT Here, we present the draft whole-genome sequence of Vibrio cholerae O1 El Tor strains 76 and M3265/80, isolated in Mariupol, Ukraine, and Moscow, Russia. The presence of various mutations detected in virulence-associated mobile elements indicates high genetic similarity of the strains reported here with new highly virulent variants of the cholera agent V. cholerae.

Design and Study of Properties of Avirulent Genetically Modified Vibrio cholerae Biovar El Tor Strain with Inactivated Genes of Thermolabile Hemolysin and Effective Expression of Cloned Cholera Toxin B-subunit Gene

The results of the construction of an avirulent strain of V. cholerae genovariant of the O1 serogroup of the El Tor biovar with efficient production of the cholera toxin (CT) B-subunit, which causes the formation of antitoxic immunity during cholera infection, are presented. In the beginning of the study, a modified P18899ΔCTXφchr:TnphoA (KmRHly–) strain was obtained via nondirectional transposon mutagenesis on the basis of a nontoxigenic strain of a P18899ΔCTXφHly+ genovariant. It lost the ability to produce thermolabile hemolysin, which is an additional toxin. The ctxB gene, which encodes biosynthesis of the CT B-subunit, was further introduced into cells of this strain. To accomplish this, the cointegrative recombinant plasmid pIEM3 was used. It was generated in the process of the fusion of a conjugative plasmid pIEM1 and a nonconjugative one, pCTΔ27, which is a derivative of pBR322 (which carries the cloned gene ctxB). Via restriction analysis, it was established that disjunction of a cointegrate occurred in vibrio cholera cells, followed by the preservation of a multicopy plasmid (pCTΔ27) only. As a result, avirulent clones of KmRTcR with a high production level (5–6 μg/mL) of secreted CT B-subunit were obtained, one of which (E99) was selected for further studies. Via real-time PCR, it was discovered that the expression of the ctxB plasmid gene in the cells of a designed strain of V. cholerae, E99, doesn’t depend on the activity of the key regulatory gene toxT, which located on the chromosome. Efficient expression of the ctxB gene in E99 cells allows their use to obtain CT B-subunit in order to produce cholera immunobiological drugs.

Flow cytofluorometric monitoring of leukocyte apoptosis in experimental cholera

Flow cytofluorometric DNA analysis was applied to determine of the relative contents of proliferative (more then 2C DNA per cell) and apoptotic (less then 2C DNA per cell) leukocytes in blood of adult rabbits, challenged with 10,000 times the 50 % effective dose of Vibrio cholerae virulent strain by the RITARD technique. It has been shown that irreversible increase the percentage of cells carrying DNA in the degradation stage brings to disbalance between the genetically controlled cell proliferation and apoptosis that leads to animal death from the cholera infection. Such fatal changes were not observed in challenging of immunized animals that were not died. Thus received data show that the flow cytofluorometric measurements may be used for detection of transgressions in homeostasis during acute infection diseases, for outlet prognosis of the cholera infection.

Construction and characterization of recombinant Vibrio cholera strains carrying heterologous genes encoding non-01 antigen or cholera enterotoxin

In an attempt to study the effect of heterologous genes on the virulence of Vibrio cholerae 01 and non-01, rfb genes encoding biosynthesis of non-01 antigens were introduced by homologous recombination into the chromosome of V. cholerae 01 strain 569B (serotype Inaba, biotype classical). Recombinant strains were obtained which were not agglutinated with the diagnostic cholera 01 antiserum and were not sensitive to the cholera diagnostic bacteriophage, but produced as much cholera toxin as 569B and were highly virulent in the infant rabbit intraintestinal injection model. These data indicate that the rfb genes from the studied V. cholerae non-01 did not alter the virulence phenotype of V. cholerae 01. In contrast, cloned ctxAB genes from V. cholerae 01 encoding cholera toxin introduced into a non-pathogenic strain lead to efficient secretion of cholera toxin but to only low virulence in the infant rabbit model.