Research Article: Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator

Date Published: January 23, 2019

Publisher: Public Library of Science

Author(s): Abdallah M. Abdallah, Eveline M. Weerdenburg, Qingtian Guan, Roy Ummels, Stephanie Borggreve, Sabir A. Adroub, Tareq B. Malas, Raeece Naeem, Huoming Zhang, Thomas D. Otto, Wilbert Bitter, Arnab Pain, Riccardo Manganelli.


The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.

Partial Text

Mycobacteria use several different type VII secretion systems (T7S) to transport proteins across their thick and waxy cell envelopes. One of these T7S systems, ESX-1, is responsible for the transport of a number of important virulence factors. Disruption of the esx-1 gene cluster severely reduces the virulence of Mycobacterium tuberculosis [1], whereas restoration of esx-1 in the Mycobacterium bovis-derived vaccine strain BCG, which lacks part of the esx-1 region due to continuous passaging, leads to increased virulence [2]. Many studies have attempted to elucidate the function of ESX-1 substrates in virulence. In the case of pathogenic mycobacteria, such as M. tuberculosis and the fish pathogen Mycobacterium marinum, ESX-1 is responsible for the translocation of the bacteria from the phagolysosomal compartments to the cytosols of macrophages [3–5]. This translocation activity has been attributed to the ESX-1 substrate EsxA (previously also known as ESAT-6) [6, 7]. Interestingly, a closely related homologue of this protein is also secreted by non-pathogenic and non-translocating mycobacteria, such as Mycobacterium smegmatis. A report indicated that, although the EsxA proteins of M. smegmatis and M. tuberculosis are highly homologous, the membrane lysis potentials of these proteins are different [8]. In M. smegmatis, ESX-1 is involved in a completely different process, i.e., conjugative DNA transfer [9]. The direct involvement of EsxA in phagosomal rupture is not undisputed, a recent report indicated that ESX-1–mediated cell lysis occurs through gross disruptions at points of bacterial contact and not through pore formation by EsxA (5). Interestingly, there are more differences between pathogenic and non-pathogenetic mycobacteria, one of which is the presence/absence of the espACD operon, which is exclusively present in mycobacterial species that were shown to be able to induce phagosomal rupture in host‐phagocytes such as M. kansasii Type I, M. bovis, M. tuberculosis [10]. Possibly also other ESX-1 substrates are involved in membrane disruption. However, these other substrates could also be involved in other proposed functions of ESX-1 in pathogenic mycobacterial species, including host cell entry and intercellular spread [11–13].

In this study, we determined the transcriptomes of the M. marinum E11 wild-type and the double-auxotrophic M. tuberculosis mc26020 mutant strains and compared these transcriptomes with those of the corresponding isogenic esx-1 mutants. We found that during the growth of M. marinum in 7H9 culture medium, genes encoding ESX-1 substrates, such as EsxA and other ESX-1-associated proteins, were down-regulated in the mutant strains, whereas the transcription of genes encoding several structural components of the ESX-1 system remained unaffected. This specific decrease in transcription might function as a mechanism to avoid toxic accumulation of ESX-1 substrates. Interestingly, a similar decrease in substrate production has been shown for the ESX-5 secretion system, where the PE_PGRS substrates do not accumulate intracellularly when secretion is blocked [71, 72]. However, for these PE_PGRS substrates, regulation was shown to occur post-transcriptionally [72], implying that a different mechanism is involved.

Sequencing reads have been submitted to the EMBL-EBI European Nucleotide Archive (ENA) Sequence Read Archive (SRA) under the study accession no. PRJEB8560. The expression data have been submitted to the Gene Expression Omnibus (GEO) under the submission no. GSE124341.




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