scholarly journals Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways

2015 ◽  
Vol 112 (36) ◽  
pp. 11341-11346 ◽  
Author(s):  
Julia L. E. Willett ◽  
Grant C. Gucinski ◽  
Jackson P. Fatherree ◽  
David A. Low ◽  
Christopher S. Hayes
Keyword(s):  
Growth Inhibition ◽  
Antimicrobial Agents ◽  
Membrane Receptors ◽  
Target Cells ◽  
Resistance Mutations ◽  
Inner Membrane ◽  
Terminal Domain ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition ◽  
Terminal Domains

Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal “translocation domains.” These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.

scholarly journals Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Allison M. Jones ◽  
Petra Virtanen ◽  
Disa Hammarlöf ◽  
William J. Allen ◽  
Ian Collinson ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Target Cell ◽  
Bacterial Species ◽  
Genetic Evidence ◽  
Target Cells ◽  
Content Type ◽  
Sec Translocon ◽  
Dependent Growth ◽  
Cell Envelopes ◽  
Contact Dependent Growth Inhibition

ABSTRACT The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secYS281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secYS281F target cells and absent in secYS281F ΔppiD or secYS281F ΔyfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secYG313W) renders ΔppiD or ΔyfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins. IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes.

scholarly journals Delivery of CdiA Nuclease Toxins into Target Cells during Contact-Dependent Growth Inhibition

PLoS ONE ◽  
2013 ◽  
Vol 8 (2) ◽  
pp. e57609 ◽  
Author(s):  
Julia S. Webb ◽  
Kiel C. Nikolakakis ◽  
Julia L. E. Willett ◽  
Stephanie K. Aoki ◽  
Christopher S. Hayes ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Target Cells ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

scholarly journals Receptor Polymorphism Restricts Contact-Dependent Growth Inhibition to Members of the Same Species

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Zachary C. Ruhe ◽  
Adam B. Wallace ◽  
David A. Low ◽  
Christopher S. Hayes
Keyword(s):  
Escherichia Coli ◽  
Growth Inhibition ◽  
Target Cell ◽  
Bacterial Species ◽  
Target Cells ◽  
Content Type ◽  
E Coli ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

ABSTRACT Bacteria that express contact-dependent growth inhibition (CDI) systems outcompete siblings that lack immunity, suggesting that CDI mediates intercellular competition. To further explore the role of CDI in competition, we determined the target cell range of the CDIEC93 system from Escherichia coli EC93. The CdiAEC93 effector protein recognizes the widely conserved BamA protein as a receptor, yet E. coli EC93 does not inhibit other enterobacterial species. The predicted membrane topology of BamA indicates that three of its extracellular loops vary considerably between species, suggesting that loop heterogeneity may control CDI specificity. Consistent with this hypothesis, other enterobacteria are sensitized to CDIEC93 upon the expression of E. coli bamA and E. coli cells become CDIEC93 resistant when bamA is replaced with alleles from other species. Our data indicate that BamA loops 6 and 7 form the CdiAEC93-binding epitope and their variation between species restricts CDIEC93 target cell selection. Although BamA loops 6 and 7 vary dramatically between species, these regions are identical in hundreds of E. coli strains, suggesting that BamAEcoli and CdiAEC93 play a role in self-nonself discrimination. IMPORTANCE Contact-dependent growth inhibition (CDI) systems are widespread among Gram-negative bacteria, enabling them to bind to neighboring bacterial cells and deliver protein toxins that inhibit cell growth. In this study, we tested the role of CDI in interspecies competition using intestinal isolate Escherichia coli EC93 as an inhibitor cell model. Although E. coli EC93 inhibits different E. coli strains, other bacterial species from the intestine are completely resistant to CDI. We show that resistance is due to small variations in the CDI receptor that prevent other species from being recognized as target cells. CDI receptor interactions thus provide a mechanism by which bacteria can distinguish siblings and other close relatives (self) from more distant relatives or other species of bacteria (nonself). Our results provide a possible means by which antimicrobials could be directed to one or only a few related bacterial pathogens by using a specific receptor “zip code.”

scholarly journals Contact-Dependent Growth Inhibition Causes Reversible Metabolic Downregulation in Escherichia coli

2009 ◽  
Vol 191 (6) ◽  
pp. 1777-1786 ◽  
Author(s):  
S. K. Aoki ◽  
J. S. Webb ◽  
B. A. Braaten ◽  
D. A. Low
Keyword(s):  
Escherichia Coli ◽  
Growth Inhibition ◽  
Target Cells ◽  
Immunity Protein ◽  
E Coli ◽  
Outer Membranes ◽  
Altered Cell ◽  
Dependent Growth ◽  
Necessary And Sufficient ◽  
Contact Dependent Growth Inhibition

ABSTRACT Contact-dependent growth inhibition (CDI) is a mechanism identified in Escherichia coli by which bacteria expressing two-partner secretion proteins encoded by cdiA and cdiB bind to BamA in the outer membranes of target cells and inhibit their growth. A third gene in the cluster, cdiI, encodes a small protein that is necessary and sufficient to confer immunity to CDI, thereby preventing cells expressing the cdiBA genes from inhibiting their own growth. In this study, the cdiI gene was placed under araBAD promoter control to modulate levels of the immunity protein and thereby induce CDI by removal of arabinose. This CDI autoinhibition system was used for metabolic analyses of a single population of E. coli cells undergoing CDI. Contact-inhibited cells showed altered cell morphology, including the presence of filaments. Notably, CDI was reversible, as evidenced by resumption of cell growth and normal cellular morphology following induction of the CdiI immunity protein. Recovery of cells from CDI also required an energy source. Cells undergoing CDI showed a significant, reversible downregulation of metabolic parameters, including aerobic respiration, proton motive force (Δp), and steady-state ATP levels. It is unclear whether the decrease in respiration and/or Δp is directly involved in growth inhibition, but a role for ATP in the CDI mechanism was ruled out using an atp mutant. Consistent with the observed decrease in Δp, the phage shock response was induced in cells undergoing CDI but not in recovering cells, based on analysis of levels of pspA mRNA.

scholarly journals Three Distinct Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition by the Cystic Fibrosis Pathogen Burkholderia dolosa

2018 ◽  
Vol 200 (22) ◽  
Author(s):  
Andrew I. Perault ◽  
Peggy A. Cotter
Keyword(s):  
Cystic Fibrosis ◽  
Respiratory Tract ◽  
Growth Inhibition ◽  
Burkholderia Cepacia ◽  
Target Cells ◽  
Content Type ◽  
Dependent Growth ◽  
Polymicrobial Communities ◽  
Contact Dependent Growth Inhibition ◽  
Polymicrobial Infections

ABSTRACT The respiratory tracts of individuals afflicted with cystic fibrosis (CF) harbor complex polymicrobial communities. By an unknown mechanism, species of the Gram-negative Burkholderia cepacia complex, such as Burkholderia dolosa, can displace other bacteria in the CF lung, causing cepacia syndrome, which has a poor prognosis. The genome of B. dolosa strain AU0158 (BdAU0158) contains three loci that are predicted to encode contact-dependent growth inhibition (CDI) systems. CDI systems function by translocating the toxic C terminus of a large exoprotein directly into target cells, resulting in growth inhibition or death unless the target cells produce a cognate immunity protein. We demonstrate here that each of the three bcpAIOB loci in BdAU0158 encodes a distinct CDI system that mediates interbacterial competition in an allele-specific manner. While only two of the three bcpAIOB loci were expressed under the in vitro conditions tested, the third conferred immunity under these conditions due to the presence of an internal promoter driving expression of the bcpI gene. One BdAU0158 bcpAIOB allele is highly similar to bcpAIOB in Burkholderia thailandensis strain E264 (BtE264), and we showed that their BcpI proteins are functionally interchangeable, but contact-dependent signaling (CDS) phenotypes were not observed in BdAU0158. Our findings suggest that the CDI systems of BdAU0158 may provide this pathogen an ecological advantage during polymicrobial infections of the CF respiratory tract. IMPORTANCE Human-associated polymicrobial communities can promote health and disease, and interbacterial interactions influence the microbial ecology of such communities. Polymicrobial infections of the cystic fibrosis respiratory tract impair lung function and lead to the death of individuals suffering from this disorder; therefore, a greater understanding of these microbial communities is necessary for improving treatment strategies. Bacteria utilize contact-dependent growth inhibition systems to kill neighboring competitors and maintain their niche within multicellular communities. Several cystic fibrosis pathogens have the potential to gain an ecological advantage during infection via contact-dependent growth inhibition systems, including Burkholderia dolosa. Our research is significant, as it has identified three functional contact-dependent growth inhibition systems in B. dolosa that may provide this pathogen a competitive advantage during polymicrobial infections.

scholarly journals Escherichia coli EC93 deploys two plasmid-encoded class I contact-dependent growth inhibition systems for antagonistic bacterial interactions

2021 ◽  
Author(s):  
Marcus Wäneskog ◽  
Tiffany Halvorsen ◽  
Klara Filek ◽  
Feifei Xu ◽  
Disa L. Hammarlöf ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Inhibition ◽  
Type Species ◽  
Target Cells ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Immunity Genes ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

The phenomenon of contact-dependent growth inhibition (CDI) and the genes required for CDI (cdiBAI) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAI EC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system (cdiBAIEC93-2 ) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI+ bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media.

scholarly journals Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems

PLoS Genetics ◽  
2011 ◽  
Vol 7 (8) ◽  
pp. e1002217 ◽  
Author(s):  
Stephen J. Poole ◽  
Elie J. Diner ◽  
Stephanie K. Aoki ◽  
Bruce A. Braaten ◽  
Claire t'Kint de Roodenbeke ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Immunity Genes ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

scholarly journals New Players in the Toxin Field: Polymorphic Toxin Systems in Bacteria

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Anne Jamet ◽  
Xavier Nassif
Keyword(s):  
In Silico ◽  
Bacterial Strains ◽  
Secretion Systems ◽  
Toxic Activity ◽  
New Family ◽  
Small Proteins ◽  
Immunity Genes ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition ◽  
Terminal Domains

ABSTRACTBacteria have evolved numerous strategies to increase their competitiveness and fight against each other. Indeed, a large arsenal of antibacterial weapons is available in order to inhibit the proliferation of competitor cells. Polymorphic toxin systems (PTS), recently identified by bioinformatics in all major bacterial lineages, correspond to such a system primarily involved in conflict between related bacterial strains. They are typically composed of a secreted multidomain toxin, a protective immunity protein, and multiple cassettes encoding alternative toxic domains. The C-terminal domains of polymorphic toxins carry the toxic activity, whereas the N-terminal domains are related to the trafficking mode.In silicoanalysis of PTS identified over 150 distinct toxin domains, including putative nuclease, deaminase, or peptidase domains. Immunity genes found immediately downstream of the toxin genes encode small proteins that protect bacteria against their own toxins or against toxins secreted by neighboring cells. PTS encompass well-known colicins and pyocins, contact-dependent growth inhibition systems which include CdiA and Rhs toxins and some effectors of type VI secretion systems. We have recently characterized the MafB toxins, a new family of PTS deployed by pathogenicNeisseriaspp. Many other putative PTS have been identified byin silicopredictions but have yet to be characterized experimentally. However, the high number of these systems suggests that PTS have a fundamental role in bacterial biology that is likely to extend beyond interbacterial competition.

scholarly journals Structural insight into toxin secretion by contact-dependent growth inhibition transporters

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jeremy Guerin ◽  
Istvan Botos ◽  
Zijian Zhang ◽  
Karl Lundquist ◽  
James C Gumbart ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Outer Membrane ◽  
Structural Modifications ◽  
Extracellular Loops ◽  
Toxin Secretion ◽  
Dependent Growth ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Outer Membrane Transporters ◽  
Contact Dependent Growth Inhibition

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane β-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand β1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

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