scholarly journals The β-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion

2020 ◽  
Vol 117 (52) ◽  
pp. 33540-33548
Author(s):  
Sonya L. Donato ◽  
Christina M. Beck ◽  
Fernando Garza-Sánchez ◽  
Steven J. Jensen ◽  
Zachary C. Ruhe ◽  
...  
Keyword(s):  
Enterobacter Cloacae ◽  
Time Lapse ◽  
Secretion System ◽  
Target Cells ◽  
Transmembrane Helices ◽  
Type Vi Secretion System ◽  
Cage Structure ◽  
Time Lapse Microscopy ◽  
Type Vi Secretion ◽  
Secretion Efficiency

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs− mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro−Ala−Ala−Arg (PAAR) repeat domains, which bind the β-spike of trimeric valine−glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact β-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs+ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the β-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact β-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

2020 ◽  
Vol 202 (10) ◽  
Author(s):  
Yannick R. Brunet ◽  
Christophe S. Bernard ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Internal Promoter ◽  
E Coli ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

scholarly journals VipA N-terminal linker and VipB-VipB interaction modulate the contraction of Type VI secretion system sheath

2017 ◽  
Author(s):  
Maximilian Brackmann ◽  
Jing Wang ◽  
Marek Basler
Keyword(s):  
Protein Translocation ◽  
Secretion System ◽  
Mass Spectrometry Analysis ◽  
Target Cells ◽  
Extended State ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Spectrometry Analysis ◽  
Type Vi Secretion ◽  
Bacterial Type

AbstractSecretion systems are essential for bacteria to survive and manipulate their environment. The bacterial Type VI Secretion System (T6SS) generates the force needed for protein translocation by the contraction of a long polymer called sheath, which is composed of interconnected VipA/VipB subunits forming a six-start helix. The mechanism of T6SS sheath contraction and the structure of its extended state are unknown. Here we show that elongating the N-terminal VipA linker or eliminating charge of a specific VipB residue abolished sheath contraction and delivery of effectors into target cells. The assembly of the non-contractile sheaths was dependent on the baseplate component TssE and mass-spectrometry analysis identified Hcp, VgrG and other components of the T6SS baseplate specifically associated with stable non-contractile sheaths. The ability to lock T6SS in the pre-firing state opens new possibilities for understanding its mode of action.

scholarly journals Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin

2007 ◽  
Vol 104 (39) ◽  
pp. 15508-15513 ◽  
Author(s):  
S. Pukatzki ◽  
A. T. Ma ◽  
A. T. Revel ◽  
D. Sturtevant ◽  
J. J. Mekalanos
Keyword(s):  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Type Vi Secretion ◽  
Phage Tail ◽  
Cross Links ◽  
Tail Spike

scholarly journals TheVibrio choleraeType VI Secretion System Can Modulate Host Intestinal Mechanics to Displace Commensal Gut Bacteria

2017 ◽  
Author(s):  
Savannah L. Logan ◽  
Jacob Thomas ◽  
Jinyuan Yan ◽  
Ryan P. Baker ◽  
Drew S. Shields ◽  
...  
Keyword(s):  
Secretion System ◽  
Effector Proteins ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Zebrafish Model ◽  
Microbial Competition ◽  
Aeromonas Veronii ◽  
Type Vi Secretion ◽  
Commensal Strain

AbstractHost-associated microbiota help defend against bacterial pathogens; the mechanisms that pathogens possess to overcome this defense, however, remain largely unknown. We developed a zebrafish model and used live imaging to directly study how the human pathogenVibrio choleraeinvades the intestine. The gut microbiota of fish mono-colonized by commensal strainAeromonas veroniiwas displaced byV. choleraeexpressing its Type VI Secretion System (T6SS), a syringe-like apparatus that deploys effector proteins into target cells. Surprisingly, displacement was independent of T6SS-mediated killing ofAeromonas, driven instead by T6SS-induced enhancement of zebrafish intestinal movements that led to expulsion of the resident commensal by the host. Deleting an actin crosslinking domain from the T6SS apparatus returned intestinal motility to normal and thwarted expulsion, without weakeningV. cholerae′sability to killAeromonas in vitro. Our finding that bacteria can manipulate host physiology to influence inter-microbial competition has implications for both pathogenesis and microbiome engineering.

scholarly journals Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis

2017 ◽  
Vol 114 (11) ◽  
pp. E2233-E2242 ◽  
Author(s):  
Meiru Si ◽  
Chao Zhao ◽  
Brianne Burkinshaw ◽  
Bing Zhang ◽  
Dawei Wei ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Outer Membrane ◽  
Secretion System ◽  
Cellular Damage ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Burkholderia Thailandensis ◽  
Type Vi Secretion ◽  
Independent Functions ◽  
And Oxidative Stress

Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe–host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn2+) under oxidative stress. Next, we identified a T6SS-4–dependent Mn2+-binding effector TseM, and its interacting partner MnoT, a Mn2+-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn2+ conditions. Both TseM and MnoT are required for efficient uptake of Mn2+ across the outer membrane under Mn2+-limited and -oxidative stress conditions. The TseM–MnoT-mediated active Mn2+ transport system is also involved in contact-independent bacteria–bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria–bacteria competition.

scholarly journals Role and Recruitment of the TagL Peptidoglycan-Binding Protein during Type VI Secretion System Biogenesis

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Yoann G. Santin ◽  
Claire E. Camy ◽  
Abdelrahim Zoued ◽  
Thierry Doan ◽  
Marie-Stéphanie Aschtgen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Secretion System ◽  
Target Cells ◽  
Functional Domain ◽  
Type Vi Secretion System ◽  
Inner Membrane ◽  
Content Type ◽  
Membrane Complex ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, called the type VI secretion system (T6SS), could be compared to a nano-spear gun using a springlike mechanism for effector injection. By targeting bacteria and eukaryotic cells, the T6SS reshapes bacterial communities and hijacks host cell defenses. In enteroaggregative Escherichia coli, the T6SS is a multiprotein machine that comprises a cytoplasmic tail and a peptidoglycan-anchored trans-envelope channel. In this work, we show that TagL comprises an N-terminal domain that mediates contact with the channel and a peptidoglycan-binding domain that binds the cell wall. We then determine at which stage of T6SS biogenesis TagL is recruited and how TagL absence impacts the assembly pathway.

scholarly journals Characterization of a lysozyme-like effector TseP and secretion co-dependence of the type VI secretion system in Aeromonas dhakensis SSU

2021 ◽  
Author(s):  
Xiaoye Liang ◽  
Tong-Tong Pei ◽  
Zeng-Hang Wang ◽  
Weiliang Xiong ◽  
Li-Li Wu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Delivery ◽  
Systemic Infection ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Secretion Systems ◽  
Bacterial Killing ◽  
Promising Tool ◽  
Type Vi Secretion

The type VI secretion system (T6SS) is a widespread weapon employed by gram-negative bacteria for interspecies interaction in complex communities. Analogous to a contractile phage tail, the double-tubular T6SS injects toxic effectors into prokaryotic and eukaryotic neighboring cells. Although effectors dictate T6SS functions, their identities remain elusive in many pathogens. Here we report a lysozyme-like effector TseP in Aeromonas dhakensis, a waterborne pathogen that can cause severe gastroenteritis and systemic infection. Using secretion, competition and enzymatic assays, we demonstrate that TseP is a T6SS-dependent effector with cell wall-lysing activities and TsiP is its cognate immunity protein. Triple deletion of tseP and two known effector genes tseI and tseC abolished T6SS-mediated secretion, while complementation with any single effector gene partially restores bacterial killing and Hcp secretion. By contrast to whole-gene deletions, a triple-effector-inactivated mutant 3effc showed abolished antibacterial killing but retained T6SS secretion. We further demonstrate that the 3effc mutation abolished T6SS-mediated toxicity of SSU to Dictyostelium discoideum amoeba, suggesting that the T6SS physical puncture is nontoxic to eukaryotic cells. These data highlight not only the necessity of possessing functionally diverse effectors for survival in multispecies communities but also that effector inactivation would be an efficient strategy to detoxify the T6SS while preserving its delivery efficiency, converting the T6SS to a protein delivery platform to a variety of recipient cells. Importance Delivery of cargo proteins via protein secretion systems has been shown as a promising tool in various applications. However, secretion systems are often used by pathogens to cause disease. Thus, strategies are needed to detoxify secretion systems while preserving their efficiency. The T6SS can translocate proteins through physical puncture of target cells without specific surface receptorsand can target a broad range of recipients. In this study, we identified a cell-wall lysing effector and by inactivating it and the other two known effectors, we have built a detoxified T6SS-active strain that may be used for protein delivery towards prokaryotic and eukaryotic recipient cells.

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