Resumo

Introduction: Enterotoxigenic Escherichia coli (ETEC) colonize the human intestinal mucosa by a variety of distinct pili known as colonization factor antigens (CFAs) and Longus, a type IV pilus. Identification and distribution of CFAs and Longus among epidemic ETEC strains could be significantly important for epidemiological studies related to the design of pili-based vaccines; although many ETEC strains isolated during the course of diarrheal infections do not produce any of the known CFAs. Longus of ETEC is highly homologous to CFA/III of ETEC and the toxin-coregulated pilus of Vibrio cholerae. This type IV pilus is encoded by the structural gene lngA and is present in a considerable number of these CFA-negative strains. In addition, identification of Longus in E. coli clinical isolates would lead to the identification of a considerable number of ETEC strains. Material and Methods: A non-polar deletion in the lngA gene from the 3772 st+/lt+ strain was generated using Lambda Red Recombinase system by Datsenko and Wanner 2000. The role of Longus was evaluated in vitro using Caco-2 and HT-29 intestinal cells using 3772 st+/lt+ and 73332ΔlngA strains. Gene detection encoding for ECP, CFA/1, CS3, and Longus were performed by PCR using specific primers. Results: Quantitative and qualitative analysis showed a significant reduction of 86% (HT-29 cells) and 93% (Caco-2) when compared to 73332ΔlngA and 73332 clinical strains. Analysis by PCR of a collection of 50 ETEC clinical isolates showed that 56% (28/50) amplified lt, 18% (9/50) st, 14% (7/50) lt/st, 40% (20/50) lngA, 90% (45/50) ecpA, 12% (6/50) cs3, and 6% (3/50) cfa/I genes. Likewise, from the lngA+ strains, 25% (5/20) amplified lngA/st, 20% (4/20) lngA/lt, 20% (6/20) only lngA, 90% (18/20) lngA/ecpA, 25% (5/20) lngA/cs3 and no relationship was found with cfa/I. Inmunodetection assays showed that clinical strains of ETEC that amplified the lngA gene expressed the LngA protein. Conclusion: Our data indicated that the production of Longus by clinical strains of ETEC and their association with other virulence factors contributed to the pathogenicity of the bacteria. However, it is necessary to perform other type of assays to verify the potential role of Longus and other pili as ETEC virulence factors.