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Background: Caseous Lymphadenitis (CLA) is a contagious, infectious, chronic disease caused by Corynebacterium pseudotuberculosis, which affects mainly sheep and goats. The clinical prevalence of CLA in Brazil is 30%, resulting in decreased milk production, weight loss, and unusable meat and leather. Prophylaxis is based on vaccination; however, current vaccinations do not offer effective protection against the infection, which makes the development of a new vaccine essential to control this disease., Experimental Approach: Here, we developed a recombinant vaccine based on CP40 protein (rCP40) combined with an adjuvant (Freund's complete adjuvant or saponin) and evaluated its efficacy in a murine model of CLA. Female BALB/c mice were used in an immunization assay., Key Results: rCP40 induced high levels of IgG2a and IgG2b antibodies. After challenge with a virulent strain of C. pseudotuberculosis C57 (10(4)CFU/mL), the levels of IgG2a and IgG2b were sustained, indicating a Th1 response. The groups immunized with rCP40 protein (GES and GEF groups) showed 100% protection and was statistically significant in the GES and GEF groups (p<0.037 and p<0.0952, respectively)., Conclusions: The results indicated the recombinant protein CP40 induced an specific immune response in mice that was able to afford protection after challenge, regardless the adjuvant used in the formulation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the frequency of erm genes coding for macrolide resistance among clinical isolates of non-pigmented rapidly growing mycobacteria (NPRGM) and to evaluate their importance in phenotypic resistance. Broth microdilution susceptibility testing was performed for all NPRGM tested. A PCR assay with consensus primers was used to evaluate the presence of erm genes among the 167 clinical isolates studied, which belonged to nine species of NPRGM; erm genes were detected in all nine species and 109 strains were erm-positive. The highest percentage of erm-positive isolates was found among Mycobacterium mageritense (100%) and the lowest among Mycobacterium mucogenicum (14%). The MICs of macrolides were found to be lower for erm-negative isolates (MIC(90): 2 mg/L) than for erm-positive isolates (MIC(90): 16 mg/L), although in some cases high MICs were found for erm-negative isolates. The finding that erm methylases are present in the majority of the species of NPRGM analysed in this study is not in agreement with conventional susceptibility studies. It therefore appears necessary to use a combination therapy to treat infections caused by NPRGM.

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.

Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d). In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Stimulation of the mammalian immune system by administration of plasmid DNA has been shown to be an important approach for vaccine development against several pathogens. In the present study we investigated the use of DNA vaccines to induce immune responses against an enteric bacterial pathogen, enterotoxigenic Escherichia coli (ETEC). Three plasmid vectors encoding colonization factor antigen 1 (CFA/I), an ETEC fimbrial adhesin, were constructed. Eukaryotic cells transfected with each of these plasmids expressed the heterologous antigen in different compartments: bound to the cytoplasmic membrane (pRECFA), accumulated in the cytoplasm (pPolyCFA) or secreted to the outside medium (pBLCFA). BALB/c mice were intramuscularly (i.m.) inoculated with purified plasmid DNA and the systemic, cellular and secreted CFA/I-specific immune responses were analyzed. The results showed that all three DNA vaccine formulations could elicit CFA/I-specific immune responses. Moreover, cellular location of the plasmid-encoded CFA/I seems to have an important role in the induced immune response. Taken together, these results indicate that DNA vaccines also represent a promising approach against enteric bacterial pathogens.

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.

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacIq. Treatment of the transformed strain with isopropyl-beta-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P < 0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P > 0.05) while 4/5 of the same mice developed anti-LPS IgA (P < 0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route.

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.

A monoclonal antibody (MAb 84) raised against the dissociated CFA/I fimbriae of enterotoxigenic Escherichia coli was characterized with regard to antigen binding and epitope specificity. Enzyme-linked immunosorbent assay (ELISA) showed that MAb 84 had higher affinity to CFA/I subunits than to intact CFA/I fimbriae and recognized a Salmonella flagellin carrying an insert corresponding to amino acids 32 to 45 of the CFA/I subunit. Fine epitope mapping based on the Pepscan technique showed that the peptide 39TFESY43, derived from the sequence of the mature CFA/I subunit, was specifically recognized by MAb 84. The 39TFESY43 sequence is probably not accessible on the surface of the native CFA/I fimbriae since MAb 84 did not bind to intact fimbriae as evaluated in inhibition ELISA tests. Moreover, MAb 84 did not agglutinate fimbriated ETEC cells nor inhibit CFA/I-mediated hemagglutination or the adhesion to Caco-2 cells.

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.

The affinities of six major penicillin-binding proteins (PBPs) of Yersinia pestis EV76 to different beta-lactam antibiotics were determined. The results indicate that, similar to their counterparts in Escherichia coli, PBP2 and PBP3 are the lethal targets of amdinocillin and furazlocillin, respectively. The PBP contents of four additional Y. pestis strains and the morphological effects produced by some beta-lactam antibiotics are also reported.