Dataset Information

Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

ABSTRACT: Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for an efficient conjugative transfer, 'extracellular complementation' potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the ?-barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.

SUBMITTER: Gonzalez-Rivera C

PROVIDER: S-EPMC6351158 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

González-Rivera Christian C Khara Pratick P Awad Dominik D Patel Roosheel R Li Yang Grace YG Bogisch Maxim M Christie Peter J PJ

Molecular microbiology 20181021 1

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment me ...[more]

PMID: 30264928

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Project description:UnlabelledBacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains.ImportanceBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.

Project description:A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.

Dataset Information

Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

Publications

Two pKM101-encoded proteins, the pilus-tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer.

Similar Datasets

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