scholarly journals In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance

2021 ◽  
Author(s):  
Pratick Khara ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Cell Envelope ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Inner Membrane Complex ◽  
Cytoplasmic Complex

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCCexhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMCclosely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers.  Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as ‘minimized’ or ‘expanded’: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.

scholarly journals In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center

2021 ◽  
Author(s):  
Pratick Khara ◽  
Liqiang Song ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Electron Microscopy ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Inner Membrane Complex ◽  
Cytoplasmic Complex

ABSTRACTBacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1-VirB11 and VirD4. We visualized the pKM101-encoded T4SS in the native cellular context by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but differs strikingly from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers-of-dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially-dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.SIGNIFICANCEBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for development of intervention strategies aimed at suppressing the action of T4SS nanomachines.

scholarly journals Polar targeting and assembly of theLegionellaDot/Icm type IV secretion system (T4SS) by T6SS-related components

2018 ◽  
Author(s):  
KwangCheol C. Jeong ◽  
Jacob Gyore ◽  
Lin Teng ◽  
Debnath Ghosal ◽  
Grant J. Jensen ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Legionnaires Disease ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

SummaryLegionella pneumophila, the causative agent of Legionnaires’ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells’ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of theLegionellacontaining vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles ofL. pneumophilaby themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose thatLegionellaco-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of theLegionellaT4BSS apparatus is mediated by an innovative “outside-inside” mechanism.

scholarly journals Bactericidal Type IV Secretion System Homeostasis in Xanthomonas citri

2019 ◽  
Author(s):  
William Cenens ◽  
Maxuel O. Andrade ◽  
Chuck S. Farah
Keyword(s):  
Cell Envelope ◽  
Growth Conditions ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Gram Negative Bacteria ◽  
Global Regulator ◽  
Xanthomonas Citri ◽  
Gram Negative ◽  
Type Iv

AbstractSeveral Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized T4SS, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the entirety of structural genes of the T4SS in X. citri, is regulated by the global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citri’s aggressive posture when confronted by competitors.Author SummaryXanthomonas citri is a member of a family of phytopathogenic bacteria that can cause substantial losses in crops. At different stages of the infection cycle, these cells will encounter other bacterial species with whom they will have to compete for space and nutrients. One mechanism which improves a cell’s chance to survive these encounters is a type IV secretion system that transfers a cocktail of antimicrobial effector proteins into other Gram-negative bacteria in a contact-dependent manner. Here, we show that this system is constitutively produced at a low basal level, even during low nutrient conditions, despite representing a significant metabolic burden to the cell. The conserved global regulator, CsrA, provides a constant, nutrient-independent, repression on the production T4SS components, thereby holding production costs to a minimum while at the same time ensuring X. citri’s competitiveness during encounters with bacterial rivals.

scholarly journals In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System

Cell Reports ◽  
2018 ◽  
Vol 23 (3) ◽  
pp. 673-681 ◽  
Author(s):  
Yi-Wei Chang ◽  
Carrie L. Shaffer ◽  
Lee A. Rettberg ◽  
Debnath Ghosal ◽  
Grant J. Jensen
Keyword(s):  
Helicobacter Pylori ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv

scholarly journals Type IV Secretion System Is Not Involved in Infection Process in Citrus

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Tiago Rinaldi Jacob ◽  
Marcelo Luiz de Laia ◽  
Leandro Marcio Moreira ◽  
Janaína Fernandes Gonçalves ◽  
Flavia Maria de Souza Carvalho ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Positive Control ◽  
Secretion System ◽  
Infection Process ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Type Iv Secretion System ◽  
In Planta ◽  
Type Iv

The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells.Xanthomonas citrisubsp.citricontains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS inXcc, we analyzed,in vitroandin planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, onlyhrpAwas downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated onCitrusleaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.

scholarly journals Brucella melitensis Invades Murine Erythrocytes during Infection

2014 ◽  
Vol 82 (9) ◽  
pp. 3927-3938 ◽  
Author(s):  
Marie-Alice Vitry ◽  
Delphine Hanot Mambres ◽  
Michaël Deghelt ◽  
Katrin Hack ◽  
Arnaud Machelart ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Bacterial Cells ◽  
Content Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Time Point

ABSTRACTBrucellaspp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed thatBrucella melitensisis able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced followingBrucellavaccination. In contrast toBartonella, the type IV secretion system and flagellar expression are not critically required for the persistence ofBrucellain blood. ImageStream analysis of blood cells showed that following a brief extracellular phase,Brucellais associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated thatB. melitensisis able to invade erythrocytesin vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possibleBrucellatransmission by bloodsucking insects in nature.

scholarly journals Phylogenomics Reveals a Diverse Rickettsiales Type IV Secretion System

2010 ◽  
Vol 78 (5) ◽  
pp. 1809-1823 ◽  
Author(s):  
Joseph J. Gillespie ◽  
Kelly A. Brayton ◽  
Kelly P. Williams ◽  
Marco A. Quevedo Diaz ◽  
Wendy C. Brown ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Cell Envelope ◽  
Secretion System ◽  
Current Data ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Dna Uptake ◽  
Type Iv ◽  
Conserved Core

ABSTRACT With an obligate intracellular lifestyle, Alphaproteobacteria of the order Rickettsiales have inextricably coevolved with their various eukaryotic hosts, resulting in small, reductive genomes and strict dependency on host resources. Unsurprisingly, large portions of Rickettsiales genomes encode proteins involved in transport and secretion. One particular transporter that has garnered recent attention from researchers is the type IV secretion system (T4SS). Homologous to the well-studied archetypal vir T4SS of Agrobacterium tumefaciens, the R ickettsiales v ir homolog (rvh) T4SS is characterized primarily by duplication of several of its genes and scattered genomic distribution of all components in several conserved islets. Phylogeny estimation suggests a single event of ancestral acquirement of the rvh T4SS, likely from a nonalphaproteobacterial origin. Bioinformatics analysis of over 30 Rickettsiales genome sequences illustrates a conserved core rvh scaffold (lacking only a virB5 homolog), with lineage-specific diversification of several components (rvhB1, rvhB2, and rvhB9b), likely a result of modifications to cell envelope structure. This coevolution of the rvh T4SS and cell envelope morphology is probably driven by adaptations to various host cells, identifying the transporter as an important target for vaccine development. Despite the genetic intractability of Rickettsiales, recent advancements have been made in the characterization of several components of the rvh T4SS, as well as its putative regulators and substrates. While current data favor a role in effector translocation, functions in DNA uptake and release and/or conjugation cannot at present be ruled out, especially considering that a mechanism for plasmid transfer in Rickettsia spp. has yet to be proposed.

scholarly journals Structure of the outer membrane complex of a type IV secretion system

Nature ◽  
2009 ◽  
Vol 462 (7276) ◽  
pp. 1011-1015 ◽  
Author(s):  
Vidya Chandran ◽  
Rémi Fronzes ◽  
Stéphane Duquerroy ◽  
Nora Cronin ◽  
Jorge Navaza ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv

scholarly journals Brucella suppress STING expression via miR-24 to enhance infection

2020 ◽  
Vol 16 (10) ◽  
pp. e1009020 ◽  
Author(s):  
Mike Khan ◽  
Jerome S. Harms ◽  
Yiping Liu ◽  
Jens Eickhoff ◽  
Jin Wen Tan ◽  
...  
Keyword(s):  
Damage Control ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Dependent Manner ◽  
Chronic Infections ◽  
Type Iv ◽  
Live Bacteria ◽  
The Impact

Brucellosis, caused by a number of Brucella species, remains the most prevalent zoonotic disease worldwide. Brucella establish chronic infections within host macrophages despite triggering cytosolic innate immune sensors, including Stimulator of Interferon Genes (STING), which potentially limit infection. In this study, STING was required for control of chronic Brucella infection in vivo. However, early during infection, Brucella down-regulated STING mRNA and protein. Down-regulation occurred post-transcriptionally, required live bacteria, the Brucella type IV secretion system, and was independent of host IRE1-RNase activity. STING suppression occurred in MyD88-/- macrophages and was not induced by Toll-like receptor agonists or purified Brucella lipopolysaccharide (LPS). Rather, Brucella induced a STING-targeting microRNA, miR-24-2, in a type IV secretion system-dependent manner. Furthermore, STING downregulation was inhibited by miR-24 anti-miRs and in Mirn23a locus-deficient macrophages. Failure to suppress STING expression in Mirn23a-/- macrophages correlated with diminished Brucella replication, and was rescued by exogenous miR-24. Mirn23a-/- mice were also more resistant to splenic colonization one week post infection. Anti-miR-24 potently suppressed replication in wild type, but much less in STING-/- macrophages, suggesting most of the impact of miR-24 induction on replication occurred via STING suppression. In summary, Brucella sabotages cytosolic surveillance by miR-24-dependent suppression of STING expression; post-STING activation “damage control” via targeted STING destruction may enable establishment of chronic infection.

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