ESX‐1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria

Abstract

SummaryThe ESX-1 secretion system of Mycobacterium tuberculosis delivers bacterial virulence factors to host cells during infection. The most abundant factor, the ESAT-6/CFP-10 dimer, is targeted for secretion via a C-terminal signal sequence on CFP-10 that is recognized by the cytosolic ATPase, Rv3871. However, the selection determinants for other ESX-1 substrates appear to be more complex. Some substrates, such as ESAT-6, are secreted despite lacking signal sequences. Furthermore, all substrates require targeting of the other ESX-1 secreted proteins, a distinguishing feature of this system. How ESX-1 substrates are selected and the basis for co-dependent secretion is unknown. Here we show that the EspC substrate interacts with Rv3868, a cytosolic AAA ATPase, through its C-terminus. Swapping the C-termini of EspC and CFP-10 revealed that these signals are functionally distinct, suggesting that the proteins are targeted via interactions with different ATPases. Surprisingly, biochemical purification experiments demonstrate that these substrates and ATPases form multi-protein complexes inside the cell and identified a new secreted substrate. By interfering with this protein interaction network, we have partially uncoupled co-dependent substrate secretion. Our results suggest that proper functioning of the ESX-1 pathway requires the interaction of multiple ESX-1 substrates and components prior to their secretion. Ultimately, understanding the details of ESX-1 targeting may allow for engineering of better vaccines to prevent tuberculosis.

Activation of BlaR1 Protein of Methicillin-resistant Staphylococcus aureus, Its Proteolytic Processing, and Recovery from Induction of Resistance

“…N-terminal blocking of bacterial proteins can be due to N-acetylation or N-formylation of methionine. N-Acetylation is an N-terminal modification in prokaryotes, which has so far been reported for proteins associated with translation (23) and secreted proteins (11). N-Formylmethionine is used to initiate protein synthesis in eubacteria (24), and although deformylation is generally highly efficient, high levels of expression in E. coli can result in partial retention of the formyl group (25,26).…”

“…Analysis by MS/MS was performed on a QTrap 5500 mass spectrometry system (ABSciex) running in ion trap IDA mode coupled to a two-dimensional Eksignet Ultra Nano ultrahigh performance liquid chromatograph. Acquisition parameters were essentially identical to those reported earlier (11). Two summed survey scans (EMS) were followed by six data-dependent full-scan MS/MS scans (enhanced product ion) by the TOP6 method.…”

“…Targeted detection and sequencing of specific peptides from BlaR1 for enhanced sensitivity and quantification were performed on samples prepared identically. Generation of the MRM transitions was determined using MRM-initiated detection and sequencing as well as the empirical MS/MS data from the LC/MS/MS of the gel bands (11,14,15). In-gel-digested E. coliexpressed rBlaR1 was used to determine appropriate peptide transitions for analysis.…”

A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

“…Mycobacterium marinum strains were grown at 30°C in Middlebrook 7H9 liquid broth (Sigma-Aldrich, St. Louis, MO) with 0.1% Tween 80 (Fisher Scientific, Pittsburgh, PA) or on Middlebrook 7H11 agar as described previously (19), supplemented with kanamycin (20 g/ml; IBI, Poesta, IL) or hygromycin (50 g/ml; EMD Millipore, Billerica, CA) as necessary. The mycobacterial strains used in this study are listed in Table 1.…”

“…Several genes are required for protein translocation, disruption of which results in loss of substrate secretion into the bacteriological medium (culture supernatant) in vitro (3)(4)(5)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). It is unknown how the protein substrates are secreted across the mycolate outer membrane (MOM) out of the mycobacterial cell or if the known ESX genes promote MOM translocation.…”