Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

Lipopolysaccharide changes in smooth-type avirulent Shigella in comparison with initial virulent strains]

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.     

Plasmid profile types of virulent and avirulent strains of phase-I Shigella sonnei and their rough phase-II and R-form variants

The study of S. sonnei in phase I, irrespective of their virulence, has revealed the existence of at least 3 types of profiles of large plasmids: (I)A having a single plasmid with a molecular weight of about 120 MD; (I)B having, alongside plasmid pSS120, a plasmid with a molecular weight of about 60 MD; (I)C, represented only by vaccine strain 6S, having three plasmids with molecular weights of about 80, 60 and 37 MD. The plasmid profiles of rough S. sonnei in phase II are characterized by the absence of large plasmids with a molecular weight of 120-80 MD, typical of bacteria in phase I, and can be in their turn subdivided, in accordance with the type of the initial culture, into three subvariants (II)A, (II)B and (II)C. The plasmid profiles of rough S. sonnei (R-forms and phase II) completely coincide. The biosynthesis of the specific antigen of S. sonnei in phase I can be determined by smaller derivatives obtained from large plasmid pSS120 by deletion (e.g., by a plasmid with a molecular weight of about 80 MD, such as plasmid pSS80).     

A library of genes of plasmid PSS120 of Shigella sonnei

The gene library of S. sonnei plasmid pSS120 was constructed with the use of plasmid pSL5 as vector. The complete restriction of the vector DNA and the partial restriction of the DNA of plasmid pSS120 were carried out by means of the enzyme EcoRI. The restricted DNA was ligated and packed in vitro into the capsid of phage lambda. The titer of negative colonies obtained after packing was 0.8 X 10(3) clones per 1 microgram of S. sonnei DNA. The total number of detected clones was 250. On the basis of the results, obtained in the analysis of the inserts of the DNA of plasmid pSS120 into the DNA of recombinant clones, the theoretical volume of the library, equal to 92 clones, was calculated. The collection of clones thus obtained will be used for checking the presence of the determinants of invasiveness and phase I antigen, localized in the DNA of plasmid pSS120.     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株的研究Ⅱ:生物学特性的研究

进一步对FS54—2、—3、—4、—5、—6、—7及—7b等双价减毒株的侵袭力、毒力、免疫原性与免疫保护性及遗传稳定性等进行了研究,证明双价减毒株仍保持了入侵肠上皮细胞的能力;小动物实验安全低毒;有很好的免疫原性,对小鼠有很好的双重免疫保护性。     

Lipopolysaccharides of Shigella sonnei

Immunobiological properties of native lipopolysaccharides (LPS) from virulent and avirulent strains of Shigella sonnei bacteria (LPS-V and LPS-A, respectively) were studied. In avirulent bacteria, LPS-V induced immunosuppressive activity specific of the virulent strain. LPS of the avirulent strain, whereas LPS-A lacked this property. Native LPS-V with immunosuppressive activity were isolated from the virulent strain by and immune affinity method. Treatment of LPS-V with phenol or TCA abolished its activity and converted it into the LPS-A form. The data showed that LPS-A can be converted back to the LPS-V form by redox treatment. This approach seems to be promising for activating LPS extracted from cells with TCA or a water-phenol mixture.     

The plasmid heterogeneity of Shigella sonnei phase-I and -II strains

The plasmid composition of S. sonnei standard strains has been studied by the method of electron microscopy of the preparations of plasmid DNA. In S. sonnei cells I-941-HP, phase I, plasmids of 2,500; 5,000; 5,600; 6,100 and 6,800 base pairs, as well as plasmids of 85,000-117,000 and 170,000-235,000 base pairs have been detected. In S. sonnei cells, phase II, plasmids of 2,500; 4,900 and 6,100 base pairs, as well as plasmids of 85,000-109,000 base pairs, have been found. Thus, virulent S. sonnei in phase I contain additional plasmids of 5,600; 6,800; 110,000-117,000 and 170,000-237,000 base pairs. The range of plasmid lengths between 85,000-117,000 and 170,000-237,000 base pairs exceeds the usual background of electron-microscopic studies, which makes it possible to come to the conclusion on the intrastrain heterogeneity of these classes of plasmids. The suggestion has been made that the transition of S. sonnei from phase I to phase II is linked with the loss of fragments of the genetic material, limited by inverted DNA repetitions.     

Large plasmid in Shigella boydii is responsible for epithelial cell penetration

A large plasmid in a virulent Shigella boydii 5 strain was transferred to plasmid-cured avirulent strains of S. boydii 5, S. boydii 12, S. sonnei form II, and Escherichia coli K12. The transconjugants acquired the ability to invade tissue culture cells, which indicated that the large plasmid in S. boydii is responsible for epithelial cell invasiveness.     

The complete sequence and analysis of the large virulence plasmid pSS of Shigella sonnei

The complete sequence of pSS, which is the large virulence plasmid of Shigella sonnei, was determined. The 214-kb plasmid is composed of segments of virulence-associated genes, the O-antigen gene clusters, a range of replication and maintenance genes, and large numbers of insertion sequence (IS) elements. Two hundred and forty-one open reading frames (ORFs) were identified, of which 117 are highly homologous to IS elements or transposases, 57 are homologous to known pathogenesis-associated proteins, and 30 are related to replication, plasmid maintenance, or other metabolic functions. Thirty-seven ORFs have no similarity to proteins with a known function, including two with no significant similarity to any hypothetical proteins. Interestingly, 10 ORFs encoding O-antigen gene clusters were identified on the plasmid and this is markedly different from most other Shigella spp. virulent plasmids. A novel toxin-antitoxin system, a series of stbDE homologs, was found on the plasmid immediately downstream of the replication region; the sole segregation stability system may be responsible for the instability of pSS. The pSS plasmid is a mixture of genes with different origins and functions. The sequence suggests a remarkable history of IS-mediated recombination and acquisition of DNA across a range of bacterial species.     

Functional characteristics of plasmids of Shigella sonnei 47 strains

The phenotypic characteristics of Shigella sonnei strain 47 containing 7 plasmids of low molecular weight and 2 plasmids 60-100 Md large have been studied. The strains of Escherichia coli containing the single plasmids or plasmid groups from Shigella sonnei have been obtained by transformation and conjugation. The comparison of phenotypes of the obtained strains has helped to find the plasmid location of the determinants for streptomycin resistance (P7), genes for colicinogenicity and colicin immunity (P5), the enzymes of host cell specificity system Sso47I (P6), Sso47II (P4), and the genes for the conjugative DNA transfer (P9). Escherichia coli strains producing individual restriction enzymes SsoI and SsoII have been isolated.     

Immunological interrelations between Shigella sonnei differing in enzymatic activity and colicinogenicity

For the first time immunological interrelations between S. sonnei differing in enzymatic activity and colicinogenicity were studied. Specific postinfectious immunity against Shigella pneumonia in intranasally infected mice was used as a model for testing S. sonnei strains, biovars II and III, with colicinogenic markers designated S5 and IE2, respectively. The development of homologous and heterologous immunity was shown to occur in the animals; immunity to the more virulent strain II S5 proved to be significantly more intense than in comparison with immunity to the less virulent strain III IE2. The unequal effectiveness of immunity to these strains (the more virulent were the strains, the more effective immunity they produced) was due to the fact that their populations contained different amounts of organisms in phase I responsible for infective action and immunity, and also, which was heretofore unknown, for the sensitivity of these bacteria to immunological action: the greater their virulence, the greater their sensitivity.     

Stimulation of the secretory IgA system in monkeys by parenteral immunization with a ribosomal Sonne dysentery vaccine

The experiment was made on 16 monkeys (rhesus macaques). Only 1 out of 12 monkeys immunized with S. sonnei ribosomal vaccine and all 4 control monkeys fell ill as the result of oral challenge with S. sonnei virulent strain. The immunized monkeys stopped excreting Shigellae earlier than the control monkeys. Antibody to lipopolysaccharide (LPS) in the serum and saliva of the monkeys were studied in the enzyme immunoassay with monospecific antibodies to human IgA, IgG and IgM. A single injection of the ribosomal vaccine in a dose of 600 micrograms was shown to lead to a considerable increase in the levels of IgA, IgG and IgM antibodies to LPS in saliva. In parenteral immunization with the ribosomal vaccine the stimulation of secretory IgA system is similar to that resulting from oral challenge with Shigella virulent strain introduced in a dose of 50 X 10(9) microbial cells. No difference in the response of monkeys to primary and booster immunization was noted.     

Comparative analysis of the protein composition of the external membrane of a virulent strain of Shigella sonnei and of genetically related avirulent bacteria

The outer membrane proteins of intact S. sonnei cells of a strain virulent in the keratoconjunctival test contain up to 24 polypeptides; one of them (with a molecular weight of about 70 KD) is absent in similar preparations of avirulent nonpenetrating shigellae in phase I, genetically related to the above strain. Study of the composition of the outer membrane proteins of S. sonnei cells in phase II has revealed that these cells, when compared with smooth bacteria, show qualitative and quantitative differences in their polypeptide fractions; of these, three fractions are characteristic of rough bacteria.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey     

The characteristics of the colonization resistance of the intestines in conventional animals with a history of clinical death]

After modeling the terminal state caused by the acute loss of blood in rats their intestinal microflora was studied, as was their resistance to colonization. Decreased resistance to colonization was registered early after resuscitation (up to 3 days), which was confirmed by the translocation of bacteria into internal organs, decreased number of lactobacilli in the contents of the small intestine and elevated level of enterobacteria in the intestine. Disturbances in resistance to colonization was also manifested by prolonged colonization of the digestive tract of the resuscitated animals by Escherichia coli indicator strain K12pSS-120 carrying Shigella sonnei (phase I) invasiveness plasmid.     

Plasmid-mediated suppression of the mutational activation of the bgl operon in Shigella sonnei

SSOR, a clinical isolate of Shigella sonnei which exhibits a Salicin-negative phenotype, is unable to mutate to give rise to Sal+ derivatives although a homolog of the Escherichia coli bgl operon is retained by the strain. This was correlated to the presence of an endogenous plasmid in the strain. A plasmid-cured derivative, AK711, could give rise to Sal+ mutants in two steps. Introduction of the plasmid DNA, extracted from SSOR, into various strains of E. coli and S. sonnei, resulted in ampicillin resistant transformants. Interestingly, the presence of the plasmid suppressed the mutational activation of the bgl operon in the transformants. This was further substantiated by the observation that, transformants that have lost the plasmid regained the ability for mutational activation of the bgl operon. Preliminary characterisation of the plasmid indicated a size of 3.8 kb with an origin of replication resembling that of ColE1 replicons and the bla gene homolog of Tn3. Observations of the mutation frequency at the srl and lac loci in the presence of the plasmid indicate that there is a reduction in the mutation frequency, suggesting an antimutator activity associated with the plasmid.     

核桃楸树皮提取物对志贺氏菌抑菌效果研究

目的：观察核桃楸树皮提取物对志贺氏茵的抑菌效果。方法：采用两倍稀释法和纸片琼脂扩散法考察桃楸树皮提取物对志贺氏茵的抑茵作用。结果：核桃楸树皮提取物对痢疾志贺氏Ⅰ型茵、痢疾志贺氏Ⅱ型菌、鲍氏志贺Ⅰ型茵、宋内氏志贺茵均为0．0313g／mL,福氏志贺Ⅱ型菌为O．0625g／mL。结论：核桃楸树皮提取物对志贺氏茵均有良好的抑菌作用,各菌对药物的敏感强弱顺序为：宋内氏志贺菌〉鲍氏志贺Ⅰ型菌〉痢疾志贺Ⅰ型菌〉痢疾志贺菌Ⅱ型菌〉福氏志贺Ⅱ型茵。     

志贺菌的质粒图谱分析

目的：分析志贺菌的质粒图谱及其与细菌药物敏感性的相关性。方法：从菌痢患者粪便标本中分离6株福氏志贺菌和4株宋内志贺菌,分别对其质粒图谱及药物敏感性进行分析。结果：不同菌株的质粒图谱具有明显的差异,但福氏志贺菌的5株以及宋内志贺菌的3株具有分子量23Kb的质粒带。各菌株的质粒图谱与其对头孢三嗪,头孢唑啉,环丙沙星,诺氟沙星,氯霉素的耐药特性无明显相关性。结论：获自患者的福氏志贺菌和宋内志贺菌具有不同的质粒图谱以及抗菌药物敏感性,提示在我市引起菌痢的志贺菌具有不同的来源。     

Differentiation of virulent strains of Shigella and entero-invasive Escherichia from their avirulent variants using modified indirect immunoenzyme analysis

The data obtained in this investigation confirm that the modified indirect enzyme immunoassay (EIA) permits the differentiation of virulent bacteria of the genus Shigella and enteroinvasive Escherichia (group 1), regularly containing virulence plasmids with a molecular weight of 120-140 MD, from their avirulent variants which have lost these plasmids (group 2). The ratio of the optic density (OD) values of the positive control samples (the OD of group 1) to the OD values of the negative ones (the OD of group 2) is significantly higher than 2.1. As revealed by EIA, the differences between groups 1 and 3 (avirulent Shigella strains and E. coli smooth strain O124, retaining high-molecular plasmids with a molecular weight of 120-140 MD or their fragments) are statistically insignificant. The ratio of the OD of group 1 to the OD of group 3 is significantly less than 2.0. Analysis of outer membrane protein (OMP) preparations isolated from S. flexneri virulent strain 2a and its isogenic avirulent plasmid-containing variant has revealed significant differences in their EIA results. The ratio of the OP of OMP preparations isolated from the virulent strain to the OD of OMP preparations from the avirulent strain exceeds 2.1.     

The stability of O-antigen plasmid is determined by a chromosomal region of Shigella dysenteriae 1

It is well established that plasmids are involved in the expression of lipopolysaccharide in certain species of Shigella. In Shigella sonnei, both the biosynthesis of oligosaccharide side chains (O antigen), and cell invasiveness are controlled exclusively by a 120 megadalton (MDa) plasmid. In Shigella dysenteriae 1, a 10 kilobase (kb) plasmid is required for O-antigen production. Shigella dysenteriae 1 strains devoid of this plasmid lose the ability to synthesize O antigen. Interestingly, this 10-kb plasmid is not stably maintained in Escherichia coli K-12 strains, where it is lost spontaneously at a high frequency. Our genetic analyses of Shigella dysenteriae 1 strain IDBM11 and its derivatives indicate that the stability of this plasmid is associated with the histidine region of the chromosome which is unique to Shigella dysenteriae. Furthermore, the 10-kb plasmid is stably maintained in wild-type IDBM11 with an intact histidine locus. However, this plasmid is not stable in IDBM11 derivatives (e.g., IDBM11-1 and IDBM11-2), in which the his locus has been substituted with the histidine region of an E. coli K-12 chromosome. The S. dysenteriae IDBM11 strain, and its derivatives (lacking a 10-kb plasmid), displayed an invasive property as demonstrated by their internalization by HeLa cells in an in vitro assay. Thus the 10-kb plasmid of Shigella dysenteriae 1 is required for O-antigen synthesis but not for cell invasion.