A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria

Abstract

The ESX-5 secretion system of Mycobacterium tuberculosis is important for bacterial virulence and for the secretion of the large PE/PPE protein family, whose genes constitute 10% of the M. tuberculosis genome. A four-gene region of the ESX-5 system is duplicated three times in the M. tuberculosis genome, but the functions of these duplicates are unknown. Here we investigated one of these duplicates: the region carrying the esxI, esxJ, ppe15, and pe8 genes (ESX-5a). An ESX-5a deletion mutant in the model system M. marinum background was deficient in the secretion of some members of the PE/PPE family of proteins. Surprisingly, we also identified other proteins that are not members of this family, thus expanding the range of ESX-5 secretion substrates. In addition, we demonstrated that ESX-5a is important for the virulence of M. marinum in the zebrafish model. Furthermore, we showed the role of the M. tuberculosis ESX-5a region in inflammasome activation but not host cell death induction, which is different from the case for the M. tuberculosis ESX-5 system. In conclusion, the ESX-5a region is nonredundant with its ESX-5 paralog and is necessary for secretion of a specific subset of proteins in M. tuberculosis and M. marinum that are important for bacterial virulence of M. marinum. Our findings point to a role for the three ESX-5 duplicate regions in the selection of substrates for secretion via ESX-5, and hence, they provide the basis for a refined model of the molecular mechanism of this type VII secretion system.

FIG 1

Confirmation of M. marinum ΔesxIJ mutant (Δesx-i/j) and M. tuberculosis ΔESX-5a mutant. (A) Genome organization of the ESX-5a region in M. tuberculosis (Mtb) and M. marinum (Mm) compared to the parent ESX-5 region. (B) Schematic diagram of the ESX-5a gene region deleted in M. marinum (left) and a Southern blot to confirm the deletion of the esxI and esxJ genes (right). (C) Schematic diagram of the ESX-5a gene region deleted in M. tuberculosis (left) and a Southern blot to confirm the deletion of the esxI, esxJ, ppe15, and pe8 genes (right).

FIG 2

No in vitro growth defects due to ESX-5 deletion were seen. OD₆₀₀ values for M. marinum WT, ΔesxIJ mutant (Δesx-i/j), and complemented mutant (Δesx-i/jC) cultures grown in 7H9 growth medium (A) and Sauton's minimal medium (B) were measured over time. OD₆₀₀ values for M. tuberculosis WT, ΔESX-5a mutant, and complemented mutant (ΔESX-5aC) cultures grown in 7H9 growth medium (C) and Sauton's minimal medium (D) were measured over time. Data shown are for one representative experiment out of three.

FIG 3

Secretion defects in M. marinum ΔesxIJ and M. tuberculosis ΔESX-5a strains. (A) 2D gel electrophoresis was performed on secreted proteins from M. marinum (top) and the ESX-5a-deficient ΔesxIJ mutant (bottom). The encircled protein spot was consistently absent in the mutant proteome. It was collected from the M. marinum gel and identified as alanine-l-dehydrogenase (ALD) via mass spectroscopy analysis. The 2D gel image is one representative of two independent experiments. (B) Western blot of WT M. marinum and the ESX-5a mutant (ΔesxIJ). Culture filtrates (CFs) and bacterial cell lysates (CLs) were probed for ALD. (C) WT M. tuberculosis and ΔESX-5a mutant cultures were exposed to superoxide for different times, as indicated, and the corresponding CLs were probed for ALD (top) and FAP (bottom) by Western blotting. For this particular image, an empty lane between the 1st and 2nd lanes was cropped. (D) The secreted proteins (CFs) and CLs from WT M. tuberculosis and the ΔESX-5a strain, constitutively expressing ALD, were blotted for ALD, FAP, and GroEL. Detection of GroEL was used as a quality control for the CFs, while the FAP signal served as a loading control. Data shown are for one representative experiment out of three.

FIG 4

ESX-5a contributes to the secretion of PE_PGRS proteins and SodA. Western blots of the CFs and CLs of the M. marinum WT, ΔesxIJ, and complemented strains were probed for PGRS-containing proteins, which are a subgroup of the PE family, and for SodA. Detection of GroEL was used as a quality control for the CFs, while the FAP signal served as a loading control. Images are for samples run on the same gel and developed for the same length of time, but the lane between the ΔesxIJ and ΔesxIJC lanes was cropped. Data shown are for one representative experiment out of three.

FIG 5

ALD is secreted via the ESX-5 system. (A) CLs and CFs were collected from WT M. marinum and a transposon insertion mutant of the ESX-5 ATPase (eccA₅::Tn). The Western blots were probed for ALD, GroEL, FAP, and PE_PGRS proteins. Images are for samples run on the same gel and developed for the same length of time, but the lane between the WT and eccA₅::Tn lanes was cropped. (B) CFs and CLs were collected from the M. marinum WT, eccA₅::Tn, and ΔsecA2 strains and run on a gel. The Western blot was then probed for SodA, GroEL, and FAP. Data shown are for one representative experiment out of three.

FIG 6

ESX-5a deletion has no effect on host cell death induction. Apoptotic cell death (blue bars; left y axis) induction by the M. tuberculosis WT, ΔESX-5a, and complemented (ΔESX-5aC) strains was measured in THP-1 macrophages by TUNEL assay at day 3 (A), in BMDMs by hypodiploid staining at day 1 (B), and in BMDCs by hypodiploid staining at day 1 (C), while the release of adenylate kinase into the supernatant was used as a measure of necrosis (red bars; right y axis). The data are shown as fold changes over the WT levels. Data shown are the means ± standard deviations (SD) for triplicate measurements from one representative experiment out of three. ns, not significant.

FIG 7

ESX-5a deletion in M. tuberculosis reduces host cell inflammasome activation and secretion of IL-1β. IL-1β ELISAs were performed on supernatants from cells infected with WT M. tuberculosis, the ΔESX-5a mutant, or the complemented strain (ΔESX-5aC) in THP-1 cells at day 3 postinfection (A) or in mouse BMDMs (B) or mouse BMDCs (C) at 24 h. (D) The mRNA levels of pro-IL-1β were measured in BMDCs infected with M. tuberculosis, the ESX-5a mutant (ΔESX-5a), or the complemented strain right after the 4-h infection period by using quantitative reverse transcription-PCR (RT-PCR). (E) Western blots of supernatants (SN) collected at 6 h postinfection, showing the active fragments of caspase-1 (10 kDa) and IL-1β (17 kDa). The corresponding CLs were probed for pro-caspase-1 (45 kDa) and pro-IL-1β (35 kDa). Tubulin (55 kDa) served as the loading control. Data are shown as means ± SD for triplicate measurements from one representative experiment out of three.

FIG 8

Virulence defect of the M. marinum ESX-5a mutant. Adult zebrafish were injected i.p. with 10⁴ bacteria per fish of WT M. marinum (WT Mm), the M. marinum ESX-5a-deficient ΔesxIJ mutant (Δesx I/J), or the complemented mutant strain (Δesx I/J C) and monitored for survival. There were 10 fish per group (n = 10), with saline (PBS)-injected fish as a control group. Data are shown as means ± SD for triplicate measurements from one representative experiment out of three. Statistical analysis was performed using the log rank test.