Mycobacterial Esx-3 requires multiple components for iron acquisition

Abstract

ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ΔmycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ΔmycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. IMPORTANCE Mycobacteria have several paralogous type VII secretion systems, Esx-1 through Esx-5. Whereas Esx-1 is required for pathogenic mycobacteria to grow within an infected host, Esx-3 is essential for growth in vitro. We and others have shown that Esx-3 is required for siderophore-mediated iron acquisition. In this work, we identify individual Esx-3 components that contribute to this process. As in the Esx-1 system, most mutations that abolish Esx-3 protein export also disrupt its function. Unexpectedly, however, ultrasensitive quantitation of Esx-3 secretion by multiple-reaction-monitoring mass spectrometry (MRM-MS) revealed that very low levels of export were sufficient for iron acquisition under similar conditions. Although protein export clearly contributes to type VII function, the relationship is not absolute.

FIG 1

Schematic diagram of gene conservation in mycobacterial esx-1 and esx-3 loci. ∗, the M. smegmatis esx-1 locus has a slightly different organization than the M. tuberculosis region from espJ to espB (5, 8); ∗∗, there is no MSMEG_0625, mycP_3ms is MSMEG_0624, and eccE_3ms is MSMEG_0626. We use the nomenclature proposed by Bitter et al. (29). Briefly, the terms ecc and esp, respectively, stand for esx conserved component and Esx-1 secretion-associated protein. The alphabetic suffix of conserved esx genes follows the gene order in the esx-1 locus. The numerical subscript at the end of the gene name refers to the esx cluster to which the gene belongs. We also provide the standard M. tuberculosis and M. smegmatis gene numbers at the beginning and end of each locus for comparison. Although not listed in the NCBI or SmegmaList databases, MSMEG_0620 is esxG_ms and MSMEG_0621 is esxH_ms.

FIG 2

Esx-3 components contribute to low-iron growth. The growth of the fxbA and fxbA esx-3 M. smegmatis mutants in low-iron medium was monitored by determining their optical densities at 600 nm. pJ, pJEB402 vector; pJ-[gene name], pJEB402 containing the indicated M. smegmatis gene. The experiments were performed 2 to 6 times in triplicate. Representative data are shown, and error bars represent the standard deviations from the replicates.

FIG 3

Loss of Esx-3 components is not rescued by exogenous mycobactin. Growth of fxbA, fxbA mbtD, and fxbA esx-3 mutants in unsupplemented, low-iron medium or in low-iron medium containing 20 µg/ml mycobactin S at 48 h. The experiment was performed at least three times in triplicate. Representative data are shown and are expressed as percentages of the fxbA mutant’s growth in low-iron medium. Error bars indicate the standard errors of the proportions.

FIG 4

EsxG_ms (A) and EsxH_ms (B) abundances in wild-type and esx-3 mutant supernatants. Protein concentrations for EsxG_ms and EsxH_ms were approximated, respectively, by measuring the concentration of the FVEVSAK peptide alone and by summing the individual concentrations above the LOD of three of the methionine forms of AMATTHEQNTMAMSAR peptides (Fig. S4). Dotted lines show the LOQ (Fig. S4). The experiments were performed in technical replicate across two biological replicates. The protein abundance data from one of the biological replicates are shown here in graphical and table format, and the data from the other replicate are reported in the Fig. S5 table. comp, complemented; ND, none detected.

FIG 5

Export of epitope-tagged EsxH_ms in the presence or absence of Esx-3 components. Anti-FLAG immunoblotting of whole-cell lysates (wcl) and culture supernatants (sup) from wild-type and esx-3 M. smegmatis containing pSYMP-esxGH_ms-FLAG in low-iron medium. All strains contain either the empty pJEB402 vector or pJEB402 containing the complementing gene. The antibody against the intracellular protein RNAP is a loading and lysis control. The experiment was performed twice with similar results.

FIG 6

One model for Esx-3 function in which EsxG and EsxH are both substrates and chaperones or structural components of the secretion apparatus. IrtA and IrtB are components of a mycobactin transporter system (63–65). We hypothesize that at least one of the functions of EsxG and EsxH is to ferry effectors of iron-loaded mycobactin uptake within or across the mycobacterial cell wall. In the absence of an Esx-3 core component, such as EccD₃, we do not detect EsxG or EsxH in culture supernatants. We hypothesize that the proteins are not exported across the cytoplasmic membrane under this condition and therefore are completely unable to contribute to iron acquisition. In contrast, although supernatants from fxbA ΔmycP_3ms cultures do not contain detectable EsxG or EsxH, the strain itself retains intermediate growth under low-iron conditions. We suggest the MycP₃ protease may be an accessory Esx-3 component that is required either for a stable EsxG-EsxH structure or for optimal chaperone activity. Thus, in the absence of this accessory factor, EsxG and EsxH may still traverse the cytoplasmic membrane to the periplasm but be only partially functional.