RESUMO
AIMS: Characterization of the aggregation-promoting factor (APF) of the human intestinal isolate Lactobacillus crispatus M247 and its homologous nonaggregating mutant Mu5. METHODS AND RESULTS: Western blot analysis revealed that the supernatant of both M247 and Mu5 contains a 28-kDa protein which cross reacts with the antiserum produced against the APF of Lact. gasseri 4B2. The apf genes of M247 and Mu5 strains were identical and were shown to be 672 nucleotides in length and encoding a protein of 223 amino acids with a predicted molecular weight of 24.0 kDa. CONCLUSION: Our results shows that the lost of aggregation in Mu5 is not related to a defect in secretion of the APF protein or a mutation in the apf gene. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the mutation in Mu5 may be contained in another molecule involved in aggregation such as a possible receptor for APF.
Assuntos
Proteínas de Bactérias/genética , Lactobacillus/genética , Sequência de Aminoácidos , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Sequência de Bases , Western Blotting/métodos , Meios de Cultura , DNA Bacteriano/genética , Genes Bacterianos/genética , Lactobacillus/metabolismo , Mutação , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
A wild-type Lactobacillus crispatus, showing a cell aggregation phenotype and its spontaneous nonaggregating mutant were compared for their in vitro adhesion properties to human ileal mucus and to a cultured human colonic cell line (Caco2) and for their in vivo colonization and adhesion potential with colonoscopy patients as volunteers in feeding trials. The wild-type strain adhered better to mucus or to Caco2 cells than did the mutant. Altogether, three human trials with the wild type and two with the mutant strain were performed. In two of the trials, the wild type could be recovered from either fecal samples or biopsies taken from the colon, while the mutant strain could not be demonstrated in either of the trials where it was used. The L. crispatus colonies recovered from the trials were often mixed, and several enterococci and lactobacillus strains coaggregating with L. crispatus wild type could be isolated. The results indicate that the surface-mediated properties, such as aggregation, of lactobacilli can have a role in adhesion and colonization.
Assuntos
Mucosa Intestinal/microbiologia , Lactobacillus/genética , Lactobacillus/fisiologia , Aderência Bacteriana , Biópsia , Células CACO-2 , Células Cultivadas , Colo/microbiologia , Fezes/microbiologia , Humanos , Lactobacillus/isolamento & purificação , Mutação , Fenótipo , ProbióticosRESUMO
Lactobacillus gasseri 4B2 is a human isolate characterised by a strong autoaggregating phenotype mediated by APF (aggregation-promoting factor), a secreted protein. Two primer pairs were developed for simultaneous amplification of a specific fragment of the APF gene and a highly conserved region of the 16S rRNA gene. The specificity of this protocol was checked in DNA samples isolated from single and mixed cultures of Lactobacillus. The same amplification protocol was successfully used directly adding whole bacterial cells to PCR reaction tubes. The suitability of this method for in vivo studies was investigated through feeding L. gasseri 4B2 to mice and analysing colony forming units obtained by plating faecal samples on selective medium. The methodology allows a fast and reliable identification of the target strain without any DNA extraction procedure.
