Cholera Vibrio Virulence Strategy and Ways of its Realization (Scientific Review)

The basic ways of Vibrio cholerae virulence strategy realization through acquisition and expression of genes of various toxic substances are discussed. Considered are molecular mechanisms responsible for interaction between host organism and cholera vibrios, as well as for genetic information exchange, for accumulation and loss of determinants of factors, which are non-identical structurally but functionally similar. Based on the analysis of literature data and personal observations put forward is a conception of pathogenicity factor intersubstitutability allowing for restoration and maintenance of pathogenetic potential of severe diarrheal disease agents among the strains deprived of cholera toxin genes

Cholera Vibrios of nonO1/nonO139 Serogroups in Etiology of Acute Intestinal Infections: Current Situation in Russia and Around the World

Cholera vibrios of nonO1/nonO139 serogroups (NAG vibrios) are well known as natural inhabitants of aquatic environments and as agents of acute intestinal infections (AII) with different degree of severity. On the background of the current vehement dissemination of the new highly virulent Vibrio cholerae El Tor strains, the moral and economic damage from NAG vibrios is not that evident, but still, not less real and rather significant. In spite of rare association with large outbreaks, NAG vibrios rank in the etiology of AII all over the world and pose a potential threat for the population health in our country. The paper reviews the present-day morbidity as regards NAG infections in various countries, including Russia; as well as the molecular premises of the agents’ pathogenicity and drug resistance. Analysis of the published data has revealed an utter heterogeneity of NAG populations, circulating in certain territories, by reference to the presence/absence of genetic determinants of pathogenicity, persistence, housekeeping and antibiotic resistance factors. Nevertheless, the joint gene-pool of the population includes rather a wide set of genes and can expand due to importations of strains, which differ from resident ones in the genotypes, from other regions. This brings about the danger of emergence of clones with higher pathogenic and, probably, even epidemic potential driven by gene exchange, and the consequences of their occurrence are unpredictable. Therefore, the NAG vibrios demand proper attention from investigators and sanitary-epidemiological institutions of the Russian Federation

MSHA-like pili of non-toxigenic Vibrio cholerae strains

Aim. In this study, we set out to identify the homologues of genes from the msh-cluster in the genomes of non-toxigenic V cholerae, to perform the bioinformatics analysis of their products, as well as to study the adhesive properties of strains containing altered genes. Materials and methods. We analysed 17 clinical strains of non-O1/non-O139 V cholerae and 2 strains of the O1 serogroup isolated from water bodies. Genes belonging to the msh-cluster were identified in the whole genomes using the BLASTN 2.2.29 and BioEdit 7.2.5 programs. Gene translation, comparative analysis of their nucleotide sequences and the amino acid sequences of deduced products were performed using the Vector NTI Advance 11 (Invitrogen). Results and discusssion. In 18 out of the 19 studied genomes we identified gene clusters responsible for production of adhesion pili (mshH-Q) represented by diverse alleles, the majority of which differed from the prototype genes of the msh-cluster in nucleotide composition but had the same localization and arrangement. Only one strain had a cluster that was close to that of the prototype. A bioinformatics analysis of their deduced products indicated that the amino acid sequence of the major MshA pilus subunit is homologous to the prototype only in a short N-terminal region (1-41) while sharing no similarities with the rest of the sequence. Nevertheless, this protein, similar to VcfA described by Kuroki H. et al. (2001) and designated by us as MshA-like, retained a putative pilus domain. A similar pattern was observed in the minor subunits designated as MshC-like. Other minor subunits also retained their characteristic domains. All of the strains agglutinated human erythrocytes (group O) and chicken erythrocytes, and in isolates harboring modified mshA-like and mshC-like genes the reaction was not inhibited by mannose. Since most of the studied strains were isolated from hospitalized patients, it is possible that in non-toxigenic V. cholerae lacking the pathogenicity island VPI, MSHA-like pili may serve as a colonization factor of the human intestine, in contrast to VPI-positive strains. The obtained information provides a basis for experimental verification of this assumption

Escherichia coli Strain – Super-Producer of Vibrio cholerae Hemolysin

Objective of this work was the cloning of Vibrio cholerae hlyA gene in a plasmid vector providing expression of foreign genes under the control of T5 promoter, and construction of E. coli strain – super-producer of Vibrio cholerae recombinant hemolysin. Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplified, cloning was carried out by means of conventional methods, productivity of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Results and conclusions. A recombinant plasmid pHlyA, expressing the cloned hlyA gene of Vibrio cholerae El Tor under the control of T5 promoter after IPTG induction, has been constructed. Carrying this plasmid strain E. coli M15[pREP4]pHlyA is the super-producer of hemolysin: the content of the product in whole cell lysates is up to 13 %, and in inclusion bodies – up to 17 % of the total cell proteins. The product of the cloned gene, in spite of the absence of proteolytic processing and presence of the hexahistidine block (6His-tag) at its N-terminus, possesses hemolytic activity towards sheep erythrocytes. 6His-tag will provide for obtaining a purified preparation on specific sorbents with a view to create diagnosticums as well as to study the significance of hemolysin as a pathogenicity/persistence factor. The advantages of this producer are the high output of the required protein, inability of synthesis of any accessory biologically active substances, short-term period of biomass growing (4–6 h including induction) and possibility of culturing without sticking to the guidelines for work with the agents of particularly dangerous infections

Analysis of Genetic Determination of <I>Vibrio cholerae</I> Tweenase Activity

Studied are Vibrio cholerae of different serogroups on the presence of cef (CHO cell elongating factor) gene and activity against tweens and tributyrin using HDS-agar, prepared on the basis of bakery yeast pancreatic digest. Determined is the fact that all cef-positive strains hydrolyze tweens 20, 40, 60, 80, 85 and all but toxigenic V.cholerae O139, hydrolyze tributyrin. In contrast, non-toxigenic cef-negative strains of O139 serogroup, are active only against the latter. Apparently, the tweenase activity of Vibrio cholerae is provided, partly, by Cef, and the ability to hydrolyze tributyrin is the result of combined activity of Cef and other ferments. Shown is the efficiency of HDS-agar application to determine these characteristics

Recombinant Escherichia coli Strain with Enhanced Production of Vibrio cholerae Neuraminidase

Objective of this work was cloning of the Vibrio cholerae nanH gene as part of a plasmid vector, providing expression of foreign genes under the control of T5 promoter, and construction of a E. coli strain – producer of V. cholerae recombinant neuraminidase.Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplifi ed, the cloning was carried out by means of conventional methods, performance of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Neuraminidase activity was identifi ed by fl uorescence in ultraviolet light after incubation with specifi c substrate (4-methylumbelliferyl-N-acetylneuraminic acid).Results and discussion. Recombinant plasmid pNanH, containing the cloned gene nanH V. cholerae, has been constructed. The gene is inserted into BamHI-PstI sites of the polylinker of pQE30. Expression of the cloned gene in the producer strain E. coli JM103pNanH occurs under the control of T5 promoter after isopropyl-1-thio-β-D-galactopyranoside (IPTG) induction. The strain shows neuraminidase activity. The recombinant NanH protein is accumulated in the producer’s cells in two forms. The fi rst form, with molecular mass (MM) of 89.5 kDa, is an unprocessed protein with the hexahistidine block (6His-tag) at its N-terminus, it is located in inclusion bodies. Its percentage is 5.6–6.6 % of the total cell proteins. The second one, with MM of 83 kDa, is found in the periplasmic space and corresponds to the mature NanH, its percentage being 3.4–3.8 %. The total percentage of both forms is 9–10 % of total cell proteins, which allows to consider the strain E. coli JM103pNanH to be a super-producer of the required protein. The strain may be used for purifi cation of NanH preparations for construction of specifi c diagnostic, therapeutic and pharmaceutical preparations as well as for investigation of the protein as a virulence/persistence factor of the pathogen

Variability of cef Genes in Toxigenic and Non-Toxigenic Vibrio cholerae O1 Strains

Objective of the investigation was a comparative bioinformatics analysis of Vibrio cholerae О1 Cef (CHO cell elongating factor) genes and proteins. Materials and methods. 36 Vibrio cholerae О1 strains from the Rostov-on-Don Research Anti-Plague Institute collection have been utilized. DNA sequencing was conducted on the MiSeq platform (Illumina); gene identification and analysis was carried out by means of BioEdit 7.2.5, BLASTN 2.2.29, BLASTP, MEGA 7, Vector NTI Advance 11 software programs. Results and conclusions. The data obtained confirmed Cef to be rather conserved in choleragenic strains (carrying cholera toxin genes ctxAB as a part of genome-integrated CTX prophage): all of them shared closely related prototype alleles cefС or cefЕ1. The Е1 allele was also revealed in ctxAB– strains carrying the pre-CTX prophage and in a single strain lacking both prophages. In the rest of CTX–/pre-CTX– V. cholerae four novel cef variants, that were not previously described, have been identified, two of which (E2 and E3), belonging to the Russian isolates, appeared to be unique, while for the two others absolute homologues were found in NCBI. In this connection several strains which caused severe cholera-like diseases in humans were placed in the group of cefЕ4 host strains. Since Cef is one of pathogenicity/persistence factors of cholera vibrios, we presume that conservation of its altered variants in the course of natural selection embodies a certain biological sense in respect of possible acquisition of qualities, significant for realization of both pathogenic and persistence potential

CLINICAL SIGNIFICANCE OF CYTOKINES SERUM LEVELS IN CHILDREN WITH CHICKEN POX

The serum levels of cytokines IL-1β, IL-8, IL-6, IFNα, IFNγ, IL-4, IL-10 were tested in ELISA in 74 children with different courses of chicken pox. Moderate severity course of chicken pox was accompanied by significant increase of IL-1β, IL-8, IFNα, IFNγ, IL-10 levels, but severe course of infection was associated with cytokine response reduction. Manifestation of varicella-zoster virus encephalitis was accompanied by the rise of IL-1β, IL-6, IFNγ and IL-10 levels during the second week of disease

Major and minor lymphocytes subpopulations in peripheral blood and cerebrospinal fluid of children with meningitis

Introduction. The analysis of current publications indicates at our insufficient understanding of subpopulation composition of lymphocytes in peripheral blood and cerebrospinal fluid (CSF) during pediatric neuroinfectious diseases. It has been found that the main lymphocyte populations are divided into many small (minor) subpopulations.The purpose of this research was to assess percentage of major and minor blood and CSF lymphocyte subsets in children with aseptic viral meningitis (AM) or bacterial purulent meningitis (BM).Materials and methods. Phenotyping of blood and CSF lymphocytes of children aged from 4 months to 17 years diagnosed with AM (n = 86) and BM (n = 39) was carried out by using flow cytometry. As a comparison group, we analyzed peripheral blood and CSF samples collected from children with acute respiratory viral infections (ARVIs) associated with syndrome of meningism (n = 27). There was evaluated percentage of the major cell subpopulations (CD3+ T-lymphocytes, T-helpers — CD3+CD4+ Th, cytotoxic T-lymphocytes — CD3+CD8+ CTL, natural killer cells — CD3-CD16+CD56+ NK, B-cells — CD3-CD19+), as well as minor lymphocyte subsets (double positive (DP) (CD3+CD4+CD8+), double negative (DN) (CD3+CD4-CD8-) T-cells, NKT (CD3+CD16+CD56+), CD3-CD8+ NK, CD3+CD8dim and CD3+CD8 8bright).Results. It was found that the acute period of BM and AM vs. the comparison group (ARVI) was characterized by significant differences in the blood and CSF composition of major and minor lymphocyte subsets. In particular, blood T-cells, Th, CTL, NK, NKT, DN, CD3-CD8+ NK, CD3+CD8bright and CD3+CD8dim dominated in parallel with significantly lowered B-cell frequency in AM vs. BM. In the CSF of children with AM, T-cells and Th prevailed, whereas count of B-cells and CD3-CD8+ NK was lower compared to those in BM. In addition, further differences were revealed in CSF and blood cell subset composition depending on nosological entity, while maintaining differences in some major and minor lymphocyte subpopulations lacked in the comparison group. Calculating the CSF/blood ratio for the major and minor lymphocyte subsets uncovered the prevalence for the majority of cell subpopulations (the coefficients ranged from 1.2 to 16.4) in the CSF of the comparison group (ARVI), except B-cells, NK and CD3-CD8+ NK (coefficients ranged from 0.07 to 0.31). AM and BM were featured with various changes in the CSF/blood ratio found for most of the studied subpopulations in the acute period as well as the recovery phase highlighted with characteristic traits for each nosological form.Conclusion. The data obtained indicate about finding specific features in the activation of systemic and intrathecal immune response during viral and bacterial meningitis in children, which may be used as an additional differential diagnostic criterion

Cholera: Epidemiological Situation around the World in 2005-2014, and Prognosis for 2015

Cholera epidemiological situation around the world (2005-2014) has been assessed. Distribution of infection in the territory of African, Asian, and American countries, as well as in the Caribbean Region has been shown. Interstate and inter-continental importations of cholera in Europe, Australia, and America, including USA and Canada have taken place during this period. Epidemic process is spatially disseminated (with involvement and affection of new countries and administrative territories) and temporally chronic (America, the Caribbean Region; Africa) due to occurring of natural or social emergency situations. Alongside epidemics and outbreaks of the disease, caused by genetically altered variants of V. cholerae O1 El Tor and strains with multiple drug resistance, outbreaks with isolation of clinical strains of V. cholerae O139 serogroup take place in the Southeast Asia (China) on an annual basis. The forecast for 2015, as regards cholera in the world, stays unfavorable, which in its turn allows for the possibility to import this infection in the territory of the Russian Federation