| Congresso Brasileiro de Microbiologia 2023 | Resumo: 55-1 | ||||
Resumo:The Escherichia albertii species comprises the newest member of the genus Escherichia and is responsible for sporadic cases, as well as outbreaks of diarrhea in several countries worldwide. One of the main virulence features observed in this pathogen is the formation of the attaching and effacing (AE) lesion, characterized by intimate adherence of the bacteria to the epithelial cells, effacement of the microvilli and formation of a pedestal-like structure that is rich in F-actin and other eukaryotic cytoskeleton elements. Previous studies carried out with the Brazilian prototype of E. albertii 1551-2, showed this pathogen has the capacity to recruit F-actin for the AE lesion formation using both the Nck-dependent pathway, which requires the phosphorylation of the Tyrosine residue 474 in the Tir receptor protein, as well as the Nck-independent pathway, which uses the TccP (Tir-cytoskeleton coupling protein) adaptor protein. In a recently published study a novel TccP, named TccP3, was identified in the 1551-2 isolate. However, TccP3 was not considered essential for F-actin polymerization during AE lesion formation in infected cells. Despite TccP3, genomic analyses have identified a gene-encoding an additional TccP adaptor protein in the E. albertii 1551-2 isolate. Thus, our main objectives were to evaluate the association of this novel adaptor protein with the other TccP proteins described so far, as well as to evaluate the participation of this novel TccP in the AE lesion formation in a Nck-independent pathway. A maximum likelihood tree was constructed comparing the amino acid sequences of the different TccP and the sequence of the novel TccP uncovered in the 1551-2 isolate. Subsequently, mutagenesis and in trans complementation of the gene responsible for encoding this novel adaptor protein were performed in the 1551-2ΔtccP3 mutant background. Sanger sequencing and Immunoblotting, with anti-Myc serum, were performed to confirm mutagenesis and complementation, respectively. Further, quantitative FAS (Fluorescent actin staining) assays were performed in MEF (Mouse Embryonic Fibroblast) cells lacking Nck (-/-), infected with the distinct bacteria to be evaluated. The novel TccP protein identified in this study, occupied an exclusive clade in the maximum likelihood tree and showed less than 80% identity compared to other TccP proteins. These results indicate that it is a novel TccP, that was termed TccP4 in the present study. The tccP4 gene deletion in the 1551-2ΔtccP3 mutant background was confirmed by sequencing, and the production of the TccP4-Myc recombinant protein in the complemented isolate was demonstrated by Immunoblotting. Quantitative FAS assays showed that 1551-2 and 1551-2ΔtccP3 were very efficient in the AE lesion formation, since 72.7% and 74.57% of adherent bacteria co-localized with polymerized F-actin in the MEF infected cells, respectively. On the other hand, only 2.4% of the E. albertii 1551-2 lacking tccP4 (1551-2ΔtccP3/ΔtccP4) preserved this ability, with this difference being considered very significant (P<0.0001). Complementation restored the ability of the 1551-2ΔtccP3/ΔtccP4 (pTccP4) to recruit F-actin as efficiently as the wild-type (72.7% vs. 71.74%; P=0.9944). In conclusion, our data led to the identification of a novel TccP, named TccP4. Moreover, we showed that TccP4 is crucial for F-actin recruitment, in the Nck-independent pathway, during AE lesion formation in host cells infected by this pathogen. Palavras-chave: E. albertii, AE lesion, virulence, TccP4 Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo | |||||