Interaction via the N terminus of the type IV secretion system (T4SS) protein VirB6 with VirB10 is required for VirB2 and VirB5 incorporation into T-pili and for T4SS function

Abstract

Many bacterial pathogens employ multicomponent protein complexes such as type IV secretion systems (T4SSs) to transfer virulence factors into host cells. Here we studied the interaction between two essential T4SS components: the very hydrophobic inner membrane protein VirB6, which may be a component of the translocation channel, and VirB10, which links the inner and outer bacterial membranes. To map the interaction site between these two T4SS components, we conducted alanine scanning and deleted six-amino acid stretches from the N-terminal periplasmic domain of VirB6 from Brucella suis. Using the bacterial two-hybrid system to analyze the effects of these alterations on the VirB6–VirB10 interaction, we identified the amino acid regions 16–21 and 28–33 and Leu-18 in VirB6 as being required for this interaction. SDS-PAGE coupled with Western blotting of cell lysates and native PAGE of detergent-extracted membrane proteins revealed that the corresponding VirB6 residues in Agrobacterium tumefaciens (Phe-20 and amino acids 18–23 and 30–35) modulate the stability of both VirB6 and VirB5. However, the results from immuno-EM and super-resolution microscopy suggested that these regions and residues are not required for membrane association or for polar localization of VirB6. The six-amino acid deletions in the N terminus of VirB6 abolished pilus formation and virulence of A. tumefaciens, and the corresponding deletions in the VirB6 homolog TraD from the plasmid pKM101-T4SS abrogated plasmid transfer. Our results indicate that specific residues of the VirB6 N-terminal domain are required for VirB6 stabilization, its interaction with VirB10, and the incorporation of VirB2 and VirB5 into T-pili.

Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

“…The levels of VirB proteins (VirB2, VirB5 and VirB8) were analyzed after the separation of samples by SDS-PAGE on 15% Tricine gels (32) and western blotting with specific antibodies as described (13, 14, 33). VirB2 and TraM were identified by silver staining of SDS gels, excised from the gel, followed by proteomics analysis (proteolytic digestion followed by LC-MS/MS) in the institutional service facility at the Institut de recherche en immunologie et cancérologie (IRIC) at Université de Montréal.…”

Comparative Genomic Analysis Confirms Five Genetic Populations of the Select Agent, Rathayibacter toxicus

“…TXSScan also used HMMER3 [64] to identify flagellin, type-IV pili, tight adherence (Tad), T1SS, T2SS, T3SS, T4SS (types B, C, F, G, I and T), pT4SSi, pT4SSt, T5SS (a, b and c type), T6SS (types i, ii and iii), and T9SS genes. virB6 (polytopic inner membrane essential for substrate secretion) [146][147][148] of the T4SS type-T was the only gene identified in all representative strains of the five R. toxicus populations, except RT-IV strain CS36, and the R. tritici strain NCPPB 1953 genome. TXSScan identified 3070, 2999, 3001, 3048 and 1806 virB6 domains within the genomes of SA03-04 (RT-I), WAC7056 (RT-II), WA40-23C (RT-III), CS39 (RT-V) and R. tritici strain NCPPB 1953, respectively.…”

“…Microorganisms 2020, 8, x FOR PEERREVIEW 12 of 37 essential for substrate secretion)[146][147][148] of the T4SS type-T was the only gene identified in all representative strains of the five R. toxicus populations, except RT-IV strain CS36, and the R. tritici strain NCPPB 1953 genome. TXSScan identified 3070, 2999, 3001, 3048 and 1806 virB6 domains within the genomes of SA03-04 (RT-I), WAC7056 (RT-II), WA40-23C (RT-III), CS39 (RT-V) and R. tritici strain NCPPB 1953, respectively.…”

Pathogenesis of brucellosis: analysis of the immunopathological concept

“…Бруцеллы проникают в фагоциты через липидный бислой, избегая его бактерицидного действия и фагосомолизосомального слияния. Эта способность бруцелл обеспечивается системой секреции IV типа (T4SS), которая включает 12 структурных генов virB1-virB12, кодирующих белковые компоненты Т4SS и формирующих функциональный оперон, консервативно присутствующий у всех видов бруцелл [19][20][21][22].…”