L.C.S. Ferreira

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes

Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Characterization of enterotoxigenic Escherichia coli by random amplification of polymorphic DNA

Two enterotoxigenic Escherichia coli (ETEC) strains (H10407 and 4011-1) were characterized by random amplification of polymorphic DNA (RAPD) profiles using 10-mer oligonucleotides with diverse GC content. All tested primers yielded arrays of amplified DNA products ranging in size from 200 to 3000 bp. The effects of annealing temperature, template concentration and GC content of the primers were evaluated and an optimal reaction procedure was established. Application of the RAPD analysis to ten ETEC strains belonging to five different serotypes showed that strains of the same serotype shared identical or almost identical band profiles, suggesting a similar genetic composition. The use of RAPD profiles as a tool in epidemiological analysis of ETEC is discussed.

Salmonella flagellin fused with a linear epitope of colonization factor antigen I (CFA/I) can prime antibody responses against homologous and heterologous fimbriae of enterotoxigenic Escherichia coli.

In this work, a 15-amino-acid-long peptide derived from the enterotoxigenic Escherichia coli CFA/I fimbria (11VDPVIDLLQADGNAL25) was genetically fused to the Salmonella flagellin and used to prime and boost serum antibody responses (IgG) against homologous (CFA/I) and heterologous (CS1) colonization factors (CFs) in BALB/c mice. Antibodies raised against the hybrid flagellin (Fla II) cross-reacted with CFA/I, CS1, CS2, and PCFO166 but not with CS4. Parenteral administration of Fla II primed antibody responses against both CFA/I and CS1 but boosted IgG responses only against CFA/I. These findings confirm that linear epitopes derived from the CFA/I fimbria can prime antibody responses against homologous and heterologous CFs and indicate that Salmonella flagellin represents a potential carrier for the development of broad-range peptide-based anti-colonization ETEC vaccines.

Altered expression of oligopeptide-binding protein (OppA) and aminoglycoside resistance in laboratory and clinical Escherichia coli strains.

Oligopeptide-binding protein (OppA) is the periplasmic component of the major oligopeptide transport system of enteric bacteria. Genetic and biochemical evidence suggests that OppA plays a role in the uptake of aminoglycoside antibiotics in Escherichia coli K-12. Forty-six (82%) of 56 aminoglycoside-resistant mutants of E. coli K-12 selected in vitro had reduced or undetectable OppA levels, as compared with their parent strain. Moreover, nine (36%) of 25 aminoglycoside-resistant clinical isolates of E. coli expressed reduced or undetectable levels of OppA. No decrease in OppA expression was observed among aminoglycoside-sensitive E. coli strains from patients. Twenty-three (42%) of 56 aminoglycoside-resistant mutants of E. coli K-12 and six (24%) of 25 clinical isolates also were deficient for expression of ornithine or arginine decarboxylases, or both, and these deficiencies might negatively affect OppA expression by reducing polyamine synthesis. These results support the view that reduced OppA expression is associated with aminoglycoside resistance in E. coli strains.

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil

Enterotoxigenic Escherichia coli (ETEC) strains have been implicated as important etiological agents of diarrheal disease, especially in developing countries. This group of microorganisms has been associated with a diverse range of genotypic and phenotypic markers. In the present study, 21 ETEC isolates previously defined according to the toxigenic genotypes, were characterized on the basis of O:H typing, cell adherence patterns, and colonization factors (CFs) antigens. Genetic diversity was investigated by random amplification polymorphic DNA (RAPD-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE). LT-I probe-positive isolates belonged to serotypes ONT:HNT, O7:H24, O48:H21, O88:H25, O148:H28, O159:H17 and O159:H21. ST-h probe-positive isolates belonged to serotypes O159:H17, O148:H28 and O6:H-. Serotypes O148:H28, O159:H17 and O6:H- were associated with the CS6, CFA/I and CS1 CS3 antigens, respectively. Most ETEC strains exhibited a diffuse pattern of adherence to cultured epithelial cells. In general, phenotypic and genotypic characteristics correlated well. RAPD-PCR, PFGE and MLEE showed reproducibility and good discriminatory potential. The application of molecular typing systems allowed the detection of significant diversity among the isolates, indicating a non-clonal origin and revealing intra-serotype variation overlooked by classical epidemiological approaches. The phenotypic and genotypic diversity observed lead us to recommend the use of different typing systems in order to elucidate the epidemiology of ETEC infection.

Cloning and expression of colonization factor antigen I (CFA/I) epitopes of enterotoxigenic Escherichia coli (ETEC) in Salmonella flagellin.

Oligonucleotides coding for linear epitopes of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) were cloned and expressed in a deleted form of the Salmonella muenchen flagellin fliC (H1-d) gene. Four synthetic oligonucleotide pairs coding for regions corresponding to amino acids 1 to 15 (region I), amino acids 11 to 25 (region II), amino acids 32 to 45 (region III) and amino acids 88 to 102 (region IV) were synthesized and cloned in the Salmonella flagellin-coding gene. All four hybrid flagellins were exported to the bacterial surface where they produced flagella, but only three constructs were fully motile. Sera recovered from mice immunized with intraperitoneal injections of purified flagella containing region II (FlaII) or region IV (FlaIV) showed high titres against dissociated solid-phase-bound CFA/I subunits. Hybrid flagellins containing region I (FlaI) or region III (FlaIII) elicited a weak immune response as measured in enzyme-linked immunosorbent assay (ELISA) with dissociated CFA/I subunits. None of the sera prepared with purified hybrid flagella were able to agglutinate or inhibit haemagglutination promoted by CFA/I-positive strains. Moreover, inhibition ELISA tests indicated that antisera directed against region I, II, III or IV cloned in flagellin were not able to recognize surface-exposed regions on the intact CFA/I fimbriae.

Antibody responses against flagellin in mice orally immunized with attenuated Salmonella vaccine strains.

Salmonella fIagellin has been repeatedly used as a carrier for heterologous peptide epitopes either as a parenterally delivered purified antigen or as a parenterally/orally-administered, flagellated, live, attenuated vaccine. Nonetheless, the ability to induce specific antibody responses against the flagellin moiety, fused or not with heterologous peptide, has not usually been reported in mice orally inoculated with a live, attenuated, flagellated Salmonella strain. In this work we evaluated the immunogenicity of flagellin in mice following oral inoculation with an aroA Salmonella enterica serovar Dublin SL5929 strain, which expressed plasmid-encoded recombinant hybrid flagellin fused to the CTP3 epitope (amino acids 50–64) of cholera toxin B-subunit. In contrast to parenterally immunized mice, no significant CTP3- or flagellin-specific antibody responses either in sera (IgG) or feces (IgA) were detected following repeated oral delivery of the recombinant Salmonella strain to C57BL/6 mice. Similarly, flagellin-specific antibody responses were also not detected in mice immunized with strain SL5930, which expressed a nonhybrid flagellin. The lack of flagellin-specific antibody responses was not associated with deficient Peyer patch colonization or spleen invasion. Moreover, stabilization of the flagellin-coding gene by integration into the host chromosome did not significantly improve flagellin-specific antibody responses following administration by the oral route. Taken together, these results suggest that flagellin does not represent an efficient peptide carrier for activation of antibody responses in mice orally immunized with live, attenuated Salmonella strains.

Epitope specificity of antibodies raised against enterotoxigenic Escherichia coli CFA/I fimbriae in mice immunized with naked DNA.

The cfaB gene, coding for the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), was cloned and expressed as a fusion peptide with the glycoprotein D (gD) from herpes simplex virus type 1 (HSV) in the pRE4 eukaryotic expression vector, resulting in the recombinant plasmid pRECFA. All BALB/c mice injected intramuscularly (i.m.) with a single dose (100 micrograms) of the purified plasmid developed antibodies against epitopes found on dissociated CFA/I subunits as well as other homologous ETEC fimbriae. Surface-exposed epitopes found on intact CFA/I fimbriae were also recognized by antibodies derived from DNA immunization, but they did not overlap with those generated with purified CFA/I fimbriae. None of the sera raised in mice immunizated with pRECFA were able to agglutinate bacterial cells or inhibit haemagglutination promoted by CFA/I bearing ETEC cells. These results show that pRECFA can elicit CFA/I-specific antibodies, which may have different epitope specificities and functional properties compared with those generated with purified bacterial protein.

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC)

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.

Immunoglobulin G subclass responses in mice immunized with plasmid DNA encoding the CFA/I fimbria of enterotoxigenic Escherichia coli.

Balb/c mice immunized either with a plasmid vector (pRECFA), encoding the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), or purified CFA/I fimbriae induced similar antibody responses in regard to the kinetics of serum total immunoglobulins and IgG. Nonetheless, the IgG subclass responses in mice vaccinated with DNA were composed predominantly by IgG2a (ranging from 39 to 74% of the total anti-CFA/I IgG) and, to a lesser extent, by IgG (ranging from 15 to 24% of the total anti-CFA/I IgG) during a 12 week observation period. On the other hand, mice immunized with purified CFA/I presented mainly an IgG1 antibody response (ranging from 39 to 67% of the total anti-CFA/I IgG). These results indicated that, irrespective of the immunogenic properties and-or origin of the encoded antigen, immunization with pRECFA elicited an specific IgG subclass response which may affect the ability of DNA vaccines to induce a protective immune response against CFA/I mediated colonization of ETEC bacterial cells.