Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri

Sgro, Germán G.; Costa, Tiago; Cenens, William; Souza, Diorge P.; Cassago, Alexandre; Oliveira, Luciana Coutinho de; Salinas, Roberto Köpke; Portugal, Rodrigo Villares; Farah, Chuck S.; Waksman, Gabriel

doi:10.1038/s41564-018-0262-z

Cited by 69 publications

(157 citation statements)

References 40 publications

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“…Although different in size and shape, both T4SS showed an outer membrane complex (OMC) with a 14‐fold symmetry. The atomic structure of the OMC of the VirB T4SS from Xanthomonas citrii, which mediates bacterial killing, confirmed this 14‐fold symmetry, as reported Tiago R. Costa from Gabriel Waksman lab (Imperial College, London, UK; Sgro et al, ). Intriguingly, the fine structure of two different T4SS, solved in situ by cryo‐ET, shed different results.…”

Section: Structure and Function Of T3 T4 And T6sssupporting

confidence: 76%

Bacterial injection machines: Evolutionary diverse but functionally convergent

Bleves

Galán

Llosa

2020

Cellular Microbiology

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Many human pathogens use Type III, Type IV, and Type VI secretion systems to deliver effectors into their target cells. The contribution of these secretion systems to microbial virulence was the main focus of a workshop organised by the International University of Andalusia in Spain. The meeting addressed structure–function, substrate recruitment, and translocation processes, which differ widely on the different secretion machineries, as well as the nature of the translocated effectors and their roles in subverting the host cell. An excellent panel of worldwide speakers presented the state of the art of the field, highlighting the involvement of bacterial secretion in human disease and discussing mechanistic aspects of bacterial pathogenicity, which can provide the bases for the development of novel antivirulence strategies.

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Section: Structure and Function Of T3 T4 And T6sssupporting

confidence: 76%

Bacterial injection machines: Evolutionary diverse but functionally convergent

Bleves

Galán

Llosa

2020

Cellular Microbiology

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“…Structural details for several IVA machines have furthered our understanding of how these systems function. Xanthomonas citri VirB/D-, Helicobacter pylori Cag-, and E. coli R388-encoded IVA secretion machine structures resolved by single-particle cryoelectron microscopy (cryo-EM) and X-ray crystallography revealed that the IVA secretion machines are composed of an outer membrane-associated core complex (OMCC) that includes a large periplasmic assembly of proteins that connects to the secretion channel in the outer membrane (9)(10)(11)(12)(13). The OMCC recruits integral inner membrane (IM) proteins that connect to a cytoplasmic complex.…”

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confidence: 99%

Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding

Park

Chetrit

et al. 2020

mBio

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Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

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“…Having established the direct role of CsrA in the repression of the virB operon, we asked 171 whether the interbacterial killing efficiency of X. citri would be enhanced in a strain lacking 172 the 5'UTR B7 . We used a X. citri virB10-msfgfp TL translational fusion strain (Sgro et al 2018) in 173 order to assess the number of T4SSs that are present per cell (see Materials and Methods 174 and Figure 2b). In this strain, the periplasmic VirB10 component has been replaced by a 175…”

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confidence: 99%

“…VirB10-msfGFP chimera. Since each T4SS contains 14 copies of VirB10, assembled T4SSs can 176 be observed as fluorescent periplasmic foci and counted (Sgro et al 2018). Deleting the 177 5'UTR B7 in the X. citri virB10-msfgfp TL genetic background resulted in a 2.6-fold increase in 178 the number of fluorescent T4SS foci that were counted per cell ( Figure 2b).…”

mentioning

confidence: 99%

“…Basler 2016;Sgro et al 2018).Briefly, 443 to each well of a clear U-shaped bottom 96-well plate, 100 µL of a mixture of 0.5 X buffer A 444 (7.5mM (NH 4 ) 2 SO 4 , 8.5mM Na 2 HPO 4 , 11mM KH 2 PO 4 , 25mM NaCl, pH 7.0), 1.5% agarose and 445 40 µg/mL CPRG (Sigma-Aldrich) was added, and plates were thoroughly dried under a 446 laminar flow. X. citri cells grown in the appropriate media, were mixed in a 1:1 volume ratio 447 with a concentrated E. coli culture.…”

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confidence: 99%

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Bactericidal Type IV Secretion System Homeostasis in Xanthomonas citri

Cenens

Andrade

Farah

2019

Preprint

Self Cite

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11Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of 12 antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis 13 upon cell contact. This capability represents an obvious fitness benefit since it can eliminate 14 competition while the liberated contents of the lysed bacteria could provide an increase in 15 the local availability of nutrients. However, the production of this Mega Dalton-sized T4SS, 16with over a hundred subunits, also imposes a significant metabolic cost. Here we show that 17 the chromosomal virB operon, which encodes the entirety of structural genes of the T4SS in 18 X. citri, is regulated by the global regulator CsrA. Relieving CsrA repression from the virB 19 operon produced a greater number of T4SSs in the cell envelope and an increased efficiency 20 in contact dependent lysis of target cells. However, this was also accompanied by a 21 physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We 22show that T4SS production is constitutive despite being downregulated by CsrA. Cells 23 subjected to a wide range of rich and poor growth conditions maintain a constant density of 24 T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results 25show that CsrA provides a constant though partial repression on the virB operon, 26 independent of the tested growth conditions, in this way controlling T4SS-related costs while 27 at the same time maintaining X. citri's aggressive posture when confronted by competitors. 28 29 2

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Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri

Cited by 69 publications

References 40 publications

Bacterial injection machines: Evolutionary diverse but functionally convergent

Bacterial injection machines: Evolutionary diverse but functionally convergent

Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding

Bactericidal Type IV Secretion System Homeostasis in Xanthomonas citri

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