Studies on the type IV secretion system toxins (X-Tfes) and antitoxins (XTfis)

• Grant number: 21/08683-6
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2021
• End date: July 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Shaker Chuck Farah
• Grantee: Daniela Thais Zapata Sifuentes
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 17/17303-7 - Structure and function of bacterial secretion systems, AP.TEM

Abstract

The X. citri toxins (called XVIPs or X-Tfes) are highly diverse in size and architecture (fig. 9 of the main thematic project) but all have a conserved C-terminal domain, termed XVIPCD, which is necessary for their interaction with the VirD4 coupling protein (Alegria et al., 2005). Ten of the thirteen proteins shown in Figure 9 seem to be potentially bona-fide toxins, since, in addition to the XVIPCD, they have N-terminal domains that vary in length from 100 to 700 amino acids. Interestingly, bioinformatic analysis shows that seven Xac X-Tfes (XAC0096, XAC0466, XAC0574, XAC1918, XAC2609, XAC2885 and XAC3634) are predicted exert their toxic effects acting within the periplasm as peptidoglycan (PG)-binding proteins, PG glycohydrolases, lytic transglycosylases, PG peptidases or lipases (fig. 9, top, of the main thematic project). Furthermore, the genes of these seven proteins are found downstream to, and potentially co-transcribed with, genes that code for periplasmic lipoproteins, characterized by a predicted lipoprotein signal peptides and lipobox (fig. 9, top, of the main thematic project). Three of these lipoproteins have been shown to inhibit the activities of their cognate X-Tfes (Souza et al., 2015) and unpublished results from our laboratory). The simultaneous deletion of two or three XTfe/X-Tfi pairs produces a Xac cell with reduced lethality which requires much more time to kill E. coli cells (figure 9, bottom of the main thematic project). The activities of many X-Tfes can be predicted by bioinformatics analysis. However these predictions should be confirmed by enzymatic assays. Also, the inhibitory activities of their cognate inhibitors (X-Tfis) should also be demonstrated experimentally. These assays will be carried out in vitro using purified recombinant proteins and, in some cases, in vivo by observing the effects of genetic knockouts and mutations in the genes coding for specific X-Tfes and X-Tfis. While we have been able to determine the crystal structures of three antitoxins (X-Tfis) - XAC2610, XAC0573 and XAC2884 - we have so far not been able to determine the structures of toxins (X-Tfes) or toxin/antitoxin complexes. This difficulty is probably due to the presence of unstructured regions in the toxins that may facilitate their translocation. In order to increase the chances of successful crystallization, we will produce truncated versions of these X-Tfes that are limited to their catalytic domains. (AU)