Functional studies of the Xanthomonas citri subsp. citri type VI secretion system: secreted proteins, recruitment and regulation

Author(s)

Lucas de Moraes Ceseti

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2023-02-03

Examining board members

Cristina Elisa Alvarez Martinez; Adriano Cappellazzo Coelho; Danilo Ciccone Miguel; Maxuel de Oliveira Andrade; José Freire da Silva Neto

• Advisor: Cristina Elisa Alvarez Martinez

Abstract

Type VI Secretion Systems (T6SS) are cellular machineries used by bacteria to translocate effectors directly into eukaryotic or prokaryotic cells, mediating biological conflicts or promoting virulence. The plant pathogen Xanthomonas citri subsp. citri uses its T6SS to resist against predation by Dictyostelium discoideum, a soil amoeba that feeds on bacteria in the environment. However, the role played by the T6SS, as well as the effectors that mediate this survival mechanism remain elusive. The aim of this study was to find conditions that promote T6SS expression and activation and to identify secreted effector proteins. The results showed an increase in T6SS gene transcription after 9 hours of co-incubation with D. discoideum and during bacterial growth on the surface of leaves of the host plant Citrus sinensis. To identify proteins secreted via T6SS, the expression and secretion of the major component of the T6SS tube, Hcp, was monitored in bacterial lysates and culture supernatant samples. Overexpression of the T6SS transcriptional regulator TagK was the only condition found to promote a detectable increase in intracellular Hcp levels. T6SS activity was analyzed in strains deleted on the T6SS post-transcriptional regulators PppA and TagF carrying a vector for TagK overexpression. Hcp secretion was not detectable in culture supernatants of these strains, suggesting the occurrence of other levels of T6SS activity regulation. Therefore, other strategies for identifying T6SS effectors were used. Genes co-regulated with the T6SS and encoding proteins containing domains suggestive of a possible effector function were characterized. To evaluate their potential cytotoxic activities, candidate genes were ectopically expressed in Saccharomyces cerevisiae and D. discoideum. The expression of Xac4139 inhibited yeast growth and caused amoeba death, while Xac4125 and Xac4123 had no negative effect on S. cerevisiae growth. Interestingly, Xac4125 showed a clear association with amoeba nuclei. Co-purification assays showed that Xac4125 and Xac4123 interact with each other and, separately, with the T6SS structural component VgrG. It was also found that these two candidates are essential for the X. citri's amoeba resistance phenotype. Altogether, results of this study contribute to a deeper understanding of the role of T6SS in the biology of this important plant pathogen (AU)

• FAPESP's process: 17/02318-9 - Functional studies of the Xanthomonas citri pv. citri type 6 secretion system: secreted proteins, recruitment and regulation
• Grantee: Lucas de Moraes Ceseti
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)