155 - Microbiología Molecular y Fisiología

A NOVEL INNER-MEMBRANE TRANSPORTER INVOLVED IN COPPER HOMEOSTASIS IN Salmonella

Sommer, Lisandro Manuel - Zhenzhen Zhao - Arguello, Jose - Soncini, Fernando -Checa, Susana Karina

Instituto de Biología Molecular y Celular de Rosario - Rosario - Santa Fe - Argentina
Contacto: lisandrosommer@gmail.com

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative, facultative anaerobic bacillus belonging to the Enterobacteriaceae family. This bacterium causes gastroenteritis in healthy individuals and systemic disease in immunocompromised or elderly people. Copper (Cu) homeostasis plays a crucial role in the interaction of Salmonella with the host. This metal, as a redox cofactor of enzymes, is essential for many biological processes but it is toxic in excess due to its ability to generate reactive oxygen and nitrogen species and to displace other metal ions from their binding sites. The host immune system exploits copper toxicity to combat infections in the phagosomes containing invading bacteria. Previously, our laboratory identified and characterized several components involved in Salmonella copper homeostasis, such as the periplasmic chaperone CueP, the inner membrane exporters CopA and GolT, and the transcriptional regulators CueR, GolS and CpxRA. Currently, we aim to elucidate how copper is mobilized from the periplasm to the cytoplasm. Recently, a Pseudomonas aeruginosa gene coding for a major facilitator superfamily (MFS) protein, was described. This gene is repressed in the presence of copper. We identified a homologue in Salmonella, that we named cuiT, and we setup a series of experiments to elucidate whether CuiT is involved in Cu import across the inner membrane. A cuiT mutant strain was generated using one-step mutagenesis method. Additionally, cuiT was cloned into an IPTG-inducible expression vector, with and without a 3XFlag tag. CuiT expression was confirmed by western blot analysis. To analyze the role of CuiT in Cu homeostasis, we tested the cuiT mutant or the CuiT-overexpressing strains for their sensitivity to CuSO₄, both in liquid and solid media, and under different growth conditions. Although the ΔcuiT did not show any growth defect, the strain overexpressing CuiT showed increased sensitivity to Cu ions compared to the wild-type strain, and this phenotype was more pronounced at lower incubation temperatures. This suggests that CuiT-overexpression promotes entry of the metal ion into the cytoplasm, increasing its toxic effects. Using atomic absorption spectroscopy, we found that in fact the CuiT-overexpressing strains accumulates more intracellular Cu than the wild-type strain. Accordingly, the mutant deleted in cuiT exhibited less Cu content than the wild-type, or the CuiT-complemented ΔcuiT strain. These results suggest that CuiT acts as the inner-membrane Cu importer of Salmonella. How this transporter integrates into the Salmonella copper homeostasis and the particularities of its regulation are matters of current investigation in our laboratory.

Palabras clave: SALMONELLA - COPPER - TRANSPORTER - GRAM NEGATIVE