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 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 Diversity of Contact-Dependent Growth Inhibition Systems ofPseudomonas aeruginosa

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Jonathan P. Allen ◽  
Alan R. Hauser
Keyword(s):  
Growth Inhibition ◽  
Target Cell ◽  
Affinity Binding ◽  
Immunity Protein ◽  
High Affinity Binding ◽  
Content Type ◽  
High Affinity ◽  
Bacterial Competition ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

ABSTRACTContact-dependent growth inhibition (CDI) systems are used in bacterial competition to hinder the growth of neighboring microbes. These systems utilize a two-partner secretion mechanism to display the CdiA exoprotein at the bacterial cell surface. CdiA forms a long filamentous stalk that facilitates binding to a target cell and delivery of a C-terminal toxin (CT) domain. This CT domain is processed and delivered into the cytoplasm of a target cell upon contact. CDI systems also encode a cognate immunity protein (CdiI) that protects siblings and resistant targeted cells from intoxication by high-affinity binding to the CT. CdiA CT domains vary among strains within a species, and many alleles encode enzymatic functions that target nucleic acids. This variation is thought to help drive diversity and adaptation within a species. CdiA diversity is well studied inEscherichia coliand several other bacteria, but little is known about the extent of this diversity inPseudomonas aeruginosa. The purpose of this review is to highlight the variability that exists in CDI systems ofP. aeruginosa. We show that this diversity is apparent even among strains isolated from a single geographical region, suggesting that CDI systems play an important role in the ecology ofP. aeruginosa.

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 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 Lipidation of Class IV CdiA Effector Proteins Promotes Target Cell Recognition during Contact-Dependent Growth Inhibition

mBio ◽  
2021 ◽  
Author(s):  
Tiffany M. Halvorsen ◽  
Fernando Garza-Sánchez ◽  
Zachary C. Ruhe ◽  
Nicholas L. Bartelli ◽  
Nicole A. Chan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Growth Inhibition ◽  
Target Cell ◽  
Effector Proteins ◽  
Cell Recognition ◽  
Specific Receptor ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition ◽  
Class Iv ◽  
Toxic Proteins

Contact-dependent growth inhibition (CDI) is a common form of interbacterial competition in which cells use CdiA effectors to deliver toxic proteins into their neighbors. CdiA recognizes target bacteria through specific receptor molecules on the cell surface.

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 Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Tanya Myers-Morales ◽  
A. Elizabeth Oates ◽  
Matthew S. Byrd ◽  
Erin C. Garcia
Keyword(s):  
Cystic Fibrosis ◽  
Growth Inhibition ◽  
Burkholderia Cepacia ◽  
Gram Negative Bacteria ◽  
Immunity Protein ◽  
Gram Negative ◽  
Content Type ◽  
Burkholderia Multivorans ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

ABSTRACT Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens. IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

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.

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