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 Cytotoxicity in Macrophages Infected with Rough Brucella Mutants Is Type IV Secretion System Dependent

2007 ◽  
Vol 76 (1) ◽  
pp. 30-37 ◽  
Author(s):  
Jianwu Pei ◽  
Qingmin Wu ◽  
Melissa Kahl-McDonagh ◽  
Thomas A. Ficht
Keyword(s):  
Cell Death ◽  
Brucella Melitensis ◽  
Regulatory Element ◽  
Random Mutagenesis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Mutant Bank

ABSTRACT Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16MΔmanBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (ΔmanBA) and the T4SS (ΔvirB). As predicted, deletion of virB from 16MΔmanBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.

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 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 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 Quorum-Sensing and BvrR/BvrS Regulation, the Type IV Secretion System, Cyclic Glucans, and BacA in the Virulence of Brucella ovis: Similarities to and Differences from Smooth Brucellae

2012 ◽  
Vol 80 (5) ◽  
pp. 1783-1793 ◽  
Author(s):  
Ana I. Martín-Martín ◽  
Pilar Sancho ◽  
María Jesús de Miguel ◽  
Luis Fernández-Lago ◽  
Nieves Vizcaíno
Keyword(s):  
Quorum Sensing ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Multiplication Rate ◽  
Content Type ◽  
Murine Macrophages ◽  
Brucella Ovis ◽  
Type Iv

ABSTRACTBrucella ovisis a rough bacterium—lacking O-polysaccharide chains in the lipopolysaccharide—that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguishB. ovisfrom smoothBrucella, which require O-polysaccharide chains for virulence, we have analyzed the significance inB. ovisof the main virulence factors described for smoothBrucella. Attempts to obtain strains of virulentB. ovisstrain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system forin vitrosurvival, while the inactivation ofbacA—in contrast to the results seen with smoothBrucella—did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake ofB. ovisPA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by thevirBoperon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described forB. melitensis, VjbR regulates the transcription of thevirBoperon positively, and theN-dodecanoyl-dl-homoserine lactone (C12-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of thefliFflagellar gene was observed inB. ovis, probably due to the two deletions detected upstream offliF. These results, together with others reported in the text, point to similarities between rough virulentB. ovisand smoothBrucellaspecies as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics ofB. ovis.

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 Implication of the Type IV Secretion System in the Pathogenicity of Vibrio tapetis, the Etiological Agent of Brown Ring Disease Affecting the Manila Clam Ruditapes philippinarum

2021 ◽  
Vol 11 ◽  
Author(s):  
Alexandra Rahmani ◽  
François Delavat ◽  
Christophe Lambert ◽  
Nelly Le Goic ◽  
Eric Dabas ◽  
...  
Keyword(s):  
Ruditapes Philippinarum ◽  
Secretion System ◽  
Infection Process ◽  
Type Iv Secretion ◽  
Manila Clam ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
T4ss Gene ◽  
Brown Ring Disease

Vibrio tapetis is a Gram-negative bacterium that causes infections of mollusk bivalves and fish. The Brown Ring Disease (BRD) is an infection caused by V. tapetis that primarily affects the Manila clam Ruditapes philippinarum. Recent studies have shown that a type IV secretion system (T4SS) gene cluster is exclusively found in strains of V. tapetis pathogenic to clams. However, whether the T4SS is implicated or not during the infection process remains unknown. The aim of this study was to create and characterize a V. tapetis T4SS null mutant, obtained by a near-complete deletion of the virB4 gene, in order to determine the role of T4SS in the development of BRD. This study demonstrated that the T4SS is neither responsible for the loss of hemocyte adhesion capacities, nor for the decrease of the lysosomal activity during BRD. Nevertheless, we observed a 50% decrease of the BRD prevalence and a decrease of mortality dynamics with the ΔvirB4 mutant. This work demonstrates that the T4SS of V. tapetis plays an important role in the development of BRD in the Manila clam.

scholarly journals Brucella abortus VirB12 Is Expressed during Infection but Is Not an Essential Component of the Type IV Secretion System

2005 ◽  
Vol 73 (9) ◽  
pp. 6048-6054 ◽  
Author(s):  
Yao-Hui Sun ◽  
Hortensia G. Rolán ◽  
Andreas B. den Hartigh ◽  
David Sondervan ◽  
Renée M. Tsolis
Keyword(s):  
Brucella Abortus ◽  
Secretion System ◽  
Splenic Tissue ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Deletion Mutants ◽  
Wild Type ◽  
Intracellular Replication ◽  
Type Iv

ABSTRACT The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.

scholarly journals The Brucella suis Type IV Secretion System Assembles in the Cell Envelope of the Heterologous Host Agrobacterium tumefaciens and Increases IncQ Plasmid pLS1 Recipient Competence

2006 ◽  
Vol 74 (1) ◽  
pp. 108-117 ◽  
Author(s):  
Anna Carle ◽  
Christoph Höppner ◽  
Khaled Ahmed Aly ◽  
Qing Yuan ◽  
Amke den Dulk-Ras ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Mammalian Cells ◽  
Cell Envelope ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Heterologous Host ◽  
Type Iv ◽  
Brucella Suis

ABSTRACT Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related α2-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.

scholarly journals Cytotoxicity of Brucella smooth strains for macrophages is mediated by increased secretion of the type IV secretion system

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3392-3402 ◽  
Author(s):  
Zhijun Zhong ◽  
Yufei Wang ◽  
Feng Qiao ◽  
Zhoujia Wang ◽  
Xinying Du ◽  
...  
Keyword(s):  
Cell Density ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Dependent Manner ◽  
Density Dependent ◽  
Type Iv ◽  
A Cell

Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BMΔvirB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMΔvirB. The quorum sensing signal molecule N-dodecanoyl-dl-homoserine lactone (C12-HSL), a molecule that can inhibit expression of virB, inhibited the cytotoxicity of BM and BM-VIR, but not of BMΔvirB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner.

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