RESUMO
Avian pathogenic Escherichia coli (APEC), widely spread among poultry, is well-known to cause colibacillosis in chickens, which results in significant losses in poultry industry. The ability to uptake iron in the extra-intestinal environment is prerequisite for APEC survival. For adaptation to the low-iron environments, the bacteria have evolved multiple iron acquisition systems to ensure optimal iron uptake. However, many components of these iron acquisition pathways are still not clearly known. An in silico analysis of the genome of a septicemic APEC O1 strain E516 identified two putative iron transport genes homologous to the c2515 and c2516 genes from uropathogenic E. coli CFT073. In this study, we constructed the single and double gene deletion mutants, and studied their biological characteristic and pathogenic traits through in vitro and in vivo assays. Reverse transcriptase PCR (RT-PCR) analyses demonstrated that the mutations destroying the reading frame of the target genes abolished their transcription. Deletion of the single or double genes of c2515 and c2516 in APEC E516 weakened its ability to produce siderophore. Consistently, the mutants exhibited growth defect under iron-depleted conditions and the intracellular iron levels in the mutants were decreased in comparison with that of the wild-type (WT). Cell infection assays showed that the iron uptake defective mutants were more easily eliminated by the macrophage. Inactivation of the c2515 and c2516 genes affected bacterial colonization of chicken tissues, as well as the 50% lethal dose levels compared with the WT strain. Moreover, the expression levels of several iron uptake-related genes were significantly decreased in the double-deletion mutant. In total, the c2515 and c2516 may involve in siderophore-mediated iron uptake and participate in the pathogenesis of APEC O1 strain E516.
RESUMO
Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.