Laboratório Nacional
de Luz Síncrotron



First Demonstration of the Involvement of T4ss in Bacterial Killing


This immediately suggests the hypothesis that, since very few T4SS have been characterized to date, T4SS-mediated bacterial killing may not be restricted to the Xanthomonadaceae family, and may in fact be a more widespread phenomenon.

Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002 [1]. Bacterial cells are continuously interacting with other bacterial and eukaryotic cells in a battle for survival. These interactions have driven the evolution of several mechanisms by which they quickly deploy proteinaceous and nucleic acid effectors that manipulate the behaviour of the target organism, often resulting in growth inhibition or death. Distinct among these mechanisms are the type III, type IV and type VI secretion systems (T3SS, T4SS and T6SS, respectively) that are all capable of transferring proteins, and in the case of the T4SS, protein–DNA complexes, directly into neighbouring cells in a contact-dependent manner. All three of these systems have been shown to be able to inject virulence factors into eukaryotic hosts, but only the T6SSs have been shown to deliver lethal toxins into bacterial cells.

T4SSs are perhaps the most versatile of the macromolecular transport systems, being essential for host colonization by many medically important microbes, the horizontal transfer of genetic material between bacteria and from bacteria to plants as well as the special cases of the Helicobacter pylori DNA uptake and Neisseria gonorrhoeae DNA release systems. The prototypical T4SS is that of the plant pathogen Agrobacterium tumefaciens that uses its Vir system to cause tumours in most dicotyledonous species. T4SSs are generally made up of a core set of 12 proteins (see Figure 1), named VirB1–VirB11 plus VirD4 with a structural organization that is only now being elucidated:

  • a set of three cytoplasmic ATPases (VirB4, VirB11 and VirD4) that energize secretion
  • a periplasmic core complex made up of 14 repeats of a VirB7–VirB9–VirB10 trimer, in which VirB10 inserts into both inner and outer membranes and VirB7 is an outer membrane lipoprotein
  • an inner membrane complex that includes VirB3, VirB6 and VirB8
  • an extracellular pilus formed by VirB2 and VirB5 the periplasmic transglycosylase VirB1


Figure 1: The chromosomal T4SS gene cluster in XAC.

(A) This T4SS cluster is located between regions XACSR12 and XACSR13. Genes shared with XauB and XauC are shown with a black asterisk. Two of the genes are shared only by XAC and XauB (red asterisk).

(B) Interactions among T4SS proteins, based on data presented by Alegria et al. [2]. Proteins specific to XAC are represented in yellow, and proteins shared by XAC, XauB and XauC are represented in orange. Six of the proteins colored in yellow are in XAC-specific regions. Genes XAC0095 and XAC0096, although not part of XAC-specific regions, seem to play an especially important role in pathogenicity. Under this model the protein coded for by XAC0095 interacts with HrpG, a protein that participates in the T3SS apparatus, and that causes phenotypic changes when its gene is mutated. The product of gene XAC0096, which is next to XAC0095, interacts wirth VirD4, a component of the T4SS [2].

Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. One of these distinguishing features is that the Xanthomonas citri (XAC) VirD4 protein (VirD4XAC2623), a homologue of the coupling protein that recruits effectors to the T4SS for secretion, interacts with a set of uncharacterized Xanthomonas VirD4-interacting proteins (XVIPs) that all contain a conserved C-terminal domain termed XVIPCD (XVIP- conserved domain).

Recently, D. P. Souza et al. [3], using various biochemical, spectroscopic, structural and genetic techniques, in combination with the production of recombinant proteins to study the type III (T3SS) and type IV (T4SS) secretion systems and quorum sensing signal transduction pathway in XAC, have made important findings:

On the basis of various observations, they emitted the hypothesis that the XAC T4SS could secrete toxins participating in toxin-immunity protein systems.

To test this, they performed a set of in vitro experiments to determine whether XAC2609 has the predicted lysozyme-like activity. They showed that XAC2609 lyses PG (a rigid, but flexible, macromolecule that surrounds and protects individual bacterial cells) and XAC2610 is its inhibitor.

The X-TfiXAC2610 protein (Xanthomonadaceae-4TSS immunity protein) does not share sequency similarity with any proteins of known function. So, crystals of X-TfiXAC2610 were obtained and transferred to reservoir solution supplemented with 20% (v/v) glycerol and flash frozen at 100 K. X-ray diffraction data were collected using an in-house Rigaku MicroMax-007 HF microfocus rotating Cu anode generator (Institute of Chemistry, University of São Paulo) and the beamline W01B-MX2 of the Brazilian Synchrotron Light Laboratory (LNLS). The crystal structure was determined by SIRAS (single isomorphous replacement with anomalous scattering). This was complemented by small-angle X-ray scattering (SAXS) experiments collected at the D11A-SAXS1 beamline of the LNLS. The structure is similar to PG hydrolase inhibitors.

They also showed that X-TfeXAC2609 (Xsanthomonadaceae-4TSS effector) was secreted in a manner dependent of the XAC T4SS and the secretion requires a XVIPCD.

XAC4TSS mediates bacterial killing: this is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of 4TSS as a mediator of both antagonistic and cooperative interbacterial interactions.

However, since Diorge P. Souza et al. have now demonstrated that some T4SSs pose a more immediate lethal threat to neighbouring bacteria, many bacterial species may have been under much stronger evolutionary pressure to have evolved an automatic T6SS counterattack response. This immediately suggests the hypothesis that, since very few T4SS have been characterized to date, T4SS-mediated bacterial killing may not be restricted to the Xanthomonadaceae family, and may in fact be a more widespread phenomenon.


[1]  Da Silva, A. C. et al. “Comparison of the genomes of two Xanthomonas pathogens with differing host specificities”. Nature 417, 459, 2002.

[2] Alegria, M. C. et al.  “Identification of new protein-protein interactions involving the products of the chromosome-and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonasaxonopodis pv. citri”. Journal of Bacteriology 187, 2315, 2005.

[3] Diorge P. Souza, Gabriel U. Oka, Cristina E. Alvarez-Martinez, Alexandre W. Bisson-Filho, German Dunger, Lise Hobeika, Nayara S. Cavalcante3, Marcos C. Alegria, Leandro R.S. Barbosa, Roberto K. Salinas, Cristiane R. Guzzo & Chuck S. Farah. “Bacterial killing via a type IV secretion system” Nature Communications 6, 6453, 2015. doi:10.1038/ncomms7453