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Mycobacteria, such as the etiological agent of human tuberculosis, Mycobacterium tuberculosis, are protected by an impermeable cell envelope composed of an inner cytoplasmic membrane, a peptidoglycan layer, an arabinogalactan layer, and an outer membrane. This second membrane consists of covalently linked, tightly packed long-chain mycolic acids , and non-covalently bound shorter lipids involved in pathogenicity –. To ensure protein transport across this complex cell envelope, mycobacteria use various secretion pathways, such as the SecA1-mediated general secretory pathway ,, an alternative SecA2-operated pathway , a twin-arginine translocation system ,, and a specialized secretion pathway variously named ESAT-6-, SNM-, ESX-, or type VII secretion –. The latter pathway, hereafter referred to as type VII secretion (T7S), has recently become a large and competitive research topic that is closely linked to studies of host–pathogen interactions of M. tuberculosis  and other pathogenic mycobacteria . Molecular details are just beginning to be revealed – showing that T7S systems are complex machineries with multiple components and multiple substrates. Despite their biological importance, there has been a lack of a clear naming policy for the components and substrates of these systems. As there are multiple paralogous T7S systems within the Mycobacteria and orthologous systems in related bacteria, we are concerned that, without a unified nomenclature system, a multitude of redundant and obscure gene names will be used that will inevitably lead to confusion and hinder future progress. In this opinion piece we will therefore propose and introduce a systematic nomenclature with guidelines for name selection of new components that will greatly facilitate communication and understanding in this rapidly developing field of research.
The first T7S-associated protein to be identified was the 6-kD early secreted antigenic target ESAT-6 . This small, highly immunogenic protein lacks a classical N-terminal signal sequence and is present in large amounts in the culture filtrate of M. tuberculosis , but is missing from the closely related attenuated live vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG)  due to the deletion of region of difference 1 (RD1) . ESAT-6 and its protein partner, the 10-kD culture filtrate protein CFP-10 , form a 11 protein complex  that involves hydrophobic interaction ,. Secretion of ESAT-6 and CFP-10 is required for the pathogenicity of M. tuberculosis –. The absence of ESAT-6 secretion is responsible in part for the attenuation of the BCG and Mycobacterium microti vaccines ,,, as well as for the decrease in virulence of the attenuated M. tuberculosis H37Ra strain .
In M. tuberculosis, ESAT-6 and CFP-10 belong to the WXG100 family of 23 small secreted proteins that share a size of approximately 100 amino acids, a helical structure, and a characteristic hairpin bend formed by the conserved Trp-Xaa-Gly (W-X-G) motif . The genes encoding these proteins, many of which represent immunodominant T cell antigens , are called esx genes in M. tuberculosis (esxA-W, Table 1) and are arranged in tandem pairs at 11 genomic loci . In five of these genomic loci (ESX-1–ESX-5), the esx genes are flanked by genes coding for components of secretion machineries involved in the export of the corresponding ESX proteins (Figure 1). These proteins constitute the major building blocks of the T7S systems ,,,,. Four of these regions are also characterized by the presence of genes encoding PE and/or PPE proteins (Figure 1, Table 2), named after their characteristic N-terminal motifs proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) . Apart from genes localized in these core ESX regions, additional genes situated elsewhere on the chromosome may be required for the function of T7S systems. For example, the rv3616c-rv3614c genes are required for secretion of ESAT-6 and CFP-10 by ESX-1 –.
Apart from members of the M. tuberculosis complex, the ESX-1 cluster is also present in a range of mycobacteria, including Mycobacterium kansasii  and Mycobacterium leprae . However, experimental work has mainly focused on the ESX-1 system of Mycobacterium marinum , , –, a fish pathogen that shows high homology in its ESX loci with M. tuberculosis , and the fast grower Mycobacterium smegmatis –. M. marinum has also been used to define a role for the paralogous system ESX-5, which is required for the secretion of PE and PPE proteins ,,. For the remaining ESX-2, ESX-3, and ESX-4 systems, only very limited predictions of their putative functions can be made. ESX-3 transcriptome data suggest that this system is involved in iron/zinc homeostasis ,, which would be consistent with the essential role of ESX-3 in M. tuberculosis . The putative functions of ESX-2 and ESX-4 remain unknown. ESX-4, which harbors a smaller number of genes than other ESX loci (Table 2), appears to represent the most ancestral T7S system in mycobacteria . This hypothesis is based on the observation that ESX-4-like loci are the only ESX clusters that are found in other high GC Gram-positive bacteria, suggesting that the last common ancestor of mycobacteria already harbored an ESX-4 T7S system. Other ESX clusters may have evolved later by gene duplication and gene diversification events. However, the finding that Nocardia farcinica (http://nocardia.nih.go.jp/) contains two T7S systems, one orthologous to ESX-4 and one locus that shows some similarity to all the conserved components of larger T7S systems, suggests that evolution of T7S systems is more complex than previously anticipated. This second T7S locus in N. farcinica even contains two PPE-like genes that were originally thought to be specific for the mycobacteria .
T7S-like systems are also found outside the high GC Gram-positive bacteria, since a number of Firmicutes have WXG100 members . However, the loci containing these WXG100 genes are only weakly similar to the mycobacterial T7S systems: in fact, only the gene encoding the FtsK/SpoIIIE-like protein is present. Therefore, these systems should be called WXG100 systems to differentiate them from true T7S systems. Both Staphylococcus aureus and Bacillus anthracis have an active WXG100 system, and the WXG100 system encoded by S. aureus is important for virulence ,.
Research in the T7S/ESX field is relatively new, but is now rapidly expanding and we therefore would like to propose a systematic nomenclature for all components involved. Until now a small number of genes within the different ESX loci of mycobacteria have been named, but for most genes the original genome annotation numbers are used. These gene numbers vary between different species and even between different strains of the same species, and therefore make comparative studies confusing. Our nomenclature is appropriate for all T7S systems in high GC-Gram-positive species. Extending this nomenclature to the T7S-like systems of Firmicutes is not recommended, since there are only a very few conserved components.
As a starting point for the new nomenclature, we focus on the most studied system, the ESX-1 system of M. tuberculosis, which is the paradigm T7S system. The new nomenclature is given for ESX-1 in M. tuberculosis (Figure 1 and Table 2) and for all ESX systems in various Mycobacteria (Table S1). The proposed rules for the nomenclature are as follows:
In order to ensure wide visibility for this new nomenclature it will be included in the most extensively used mycobacterial genome databases. As a first step, selected genome browsers available at the Institut Pasteur (http://genolist.pasteur.fr/) and/or the Ecole Polytechnique Federale de Lausanne (http://tuberculist.epfl.ch/) will adopt these new rules; other databases could follow this example.
In conclusion, we would like to emphasize that the introduction of a uniform gene nomenclature for other secretion systems in Gram-negative bacteria (type II, type III) has facilitated comparative analysis of these systems. We anticipate that the acceptance/implementation of this proposal will provide similar advantages for the T7S systems.
New and old nomenclature of the different conserved components of the T7S systems in selected mycobacteria (M. tuberculosis H37Rv, M. marinum M, M. smegmatis mc2155, M. leprae TN, M. avium paratuberculosis K10). The numeral suffices to indicate the ESX clusters to which the genes belong are omitted. Note that the ESX-2 genes of M. avium paratuberculosis are located in two separate genomic loci. TM, transmembrane domain.
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Comparison of the transmembrane topologies and signal sequence predictions of the M. tuberculosis H37Rv Ecc membrane proteins. Amongst the different topology prediction programs that were used (TMHMM Server v. 2.0, MEMSAT3, Philius, SCAMPI, HMMTOP and Phobius) MEMSAT3 gave the correct prediction for the highest number of Ecc membrane proteins. Therefore, only the topology prediction results of TMHMM (used on the TubercuList server) and MEMSAT3 are shown. The clearly incorrect predictions are depicted in gray. TM, transmembrane domain; in, cytoplasmic location; out, periplasmic location; C, C-terminus; N, N-terminus; ss, signal sequence.
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We are grateful to our many collaborators and colleagues, who have contributed to the rapid progress in this field.
The authors have declared that no competing interests exist.
This work has received funding from the European Community's Seventh Framework Programme ([FP7/2007–2013]) under grant agreement n°201762. In addition, the authors acknowledge their individual external funding for T7S/ESX related research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.