Research Article: EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF

Date Published: August 13, 2018

Publisher: Public Library of Science

Author(s): Trang H. Phan, Lisanne M. van Leeuwen, Coen Kuijl, Roy Ummels, Gunny van Stempvoort, Alba Rubio-Canalejas, Sander R. Piersma, Connie R. Jiménez, Astrid M. van der Sar, Edith N. G. Houben, Wilbert Bitter, Christopher M. Sassetti.


The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.

Partial Text

Mycobacterium tuberculosis, the etiological agent for the disease tuberculosis (TB), is still one of the most dangerous pathogens for global health [1]. Successful infection requires secretion of multiple virulence factors, facilitated by type VII secretion systems (T7SS). Pathogenic mycobacteria have up to five T7SS, called ESX-1 to ESX-5 [2], of which at least three are essential for growth and/or virulence [3,4]. The ESX-1 locus was the first T7SS to be identified. The loss of ESX-1 function in Mycobacterium bovis BCG is considered a decisive factor of attenuation of this vaccine strain [5]. Mouse infection experiments utilizing M. tuberculosis with a partial deletion in ESX-1 showed reduced granuloma formation, the characteristic pathological hallmark of mycobacterial disease [6,7]. Similarly, efficient granuloma formation, dissemination of disease and invasion of endothelial cells in the fish-pathogen Mycobacterium marinum is dependent on a functional ESX-1 secretion system [8–10]. More detailed analysis showed that ESX-1 substrates are required for phagosomal membrane rupture [11,12].

A number of studies have shown that the mycobacterial ESX-1 system plays a pivotal role in mycobacterial pathogenesis [6,21,27,33]. The system affects virulence through secretion of protein effectors with host-modulatory effects. Here, we show that EccA1 is not strictly required for the secretion of ESX-1 substrates. The finding that EccA1 is important for secretion is in line with previous reports [8,34], but the fact that the role of EccA1 is depending on the growth medium is entirely surprising. This difference could also explain the variable results described for the role of EccA1 in EsxA secretion by M. tuberculosis [49]. Of all ESX-1 substrates, EspE, EspF, EspJ and EspK secretion was mostly affected in our eccA1 mutant strain, while secretion of EspB, EsxA/EsxB and PE/PPE was hardly altered. An interesting observation here is the discrepancy between the active secretion of EsxA in the ΔeccA1 strain and at the same time loss of hemolytic activity. Although this observation has been described before, this was always linked to a reduced secretion of EsxA in these strains [8,34]. In a recent study, the importance of EsxA in lysing membranes was questioned [50]. Our results also supports an alternative mechanism: we find a strong correlation between ESX-1 functionality and hemolysis, but this correlation is not seen for EsxA secretion. Our finding is in line with several other recent studies, who showed that both EsxA and the cell-surface lipid PDIM are important for phagosomal rupture and escape by M. tuberculosis [51–53]. We propose that it is not the loss of secreted EsxA, but the loss of (multiple) surface-exposed Esp proteins that results in hemolytic deficiency.