Mycobacterium tuberculosis SatS is a chaperone for the SecA2 protein export pathway

Miller, Brittany K.; Hughes, Ryan; Ligon, Lauren S.; Rigel, Nathan W.; Malik, Shaukat Iqbal; Anjuwon-Foster, Brandon R.; Sacchettini, James C.; Braunstein, Miriam

doi:10.7554/elife.40063

Cited by 13 publications

(10 citation statements)

References 55 publications

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“…Since there is no SecB protein export chaperone in mycobacteria, one possibility is that the SecA2 pathway is adapted to facilitate export through the SecYEG channel of problematic substrates that can fold in the cytoplasm. Along these lines, a mycobacterial protein export chaperone named SatS was recently identified as playing a role in the SecA2 pathway (124). SatS stabilizes and prevents aggregation of a subset of SecA2 substrates.…”

Section: The Multisubstrates Of the Mycobacterial Seca2 Pathwaymentioning

confidence: 99%

Protein Export into and across the Atypical Diderm Cell Envelope of Mycobacteria

2019

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Section: The Multisubstrates Of the Mycobacterial Seca2 Pathwaymentioning

confidence: 99%

Protein Export into and across the Atypical Diderm Cell Envelope of Mycobacteria

2019

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“…Binding of LepA to the ribosome, facilitated by a series of rare codons, could alter the rate of translation, thus changing the folding of the nascent peptide and affecting its ability to be recognized by the secretory system or other factors (Figure 5). Certainly, a ribosome carrying a nascent signal peptide can interact with a number of distinct components of the Sec system, which enable co-translational secretion of membrane-destined proteins [10, 77, 78]. It is well-established that translation rate is intimately tied to protein folding [79], thus, we do not divorce LepA’s activity from either of these outcomes.…”

Section: Discussionmentioning

confidence: 99%

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

Fishbein

Wolf

Dulberger

et al. 2019

Preprint

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14Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong 15 evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known 16 as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and 17 chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of 18 lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-19 ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we 20 characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-21 type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, 22which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount 23 of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control 24 requires a sequence motif in the 5' region of the porin transcript. Thus, LepA controls the abundance of 25 specific proteins, likely through its activity during translation. 27 Importance 28Our understanding of how ribosomes properly synthesis an entire cellular proteome, in all its 29 complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are 30 not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase 31 conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-32 sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of 33 mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, 34 loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of 35 building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the 36 use of GTPases such as LepA, that have evolved to aid in specific protein synthesis. 37 38 Keywords 39 LepA; ribosome; mycobacteria; porin; drug susceptibility 40 41 45 to adaptive, cellular responses. Indeed, the ribosome machinery and its associated factors/RNA 46 species adapt the cell during periods of environmental transition [2] [3, 4]. Recent advances in 47 techniques such as quantitative proteomics, ribosome profiling, and cryo-electron microscopy (cryo-48 EM) have revealed an intricate balance of mRNA sequences and associating proteins at the ribosome 49 that are necessary for the homeostatic cellular proteome [5-7]. Regardless of growth environment,50bacterial cells are likely composed of a heterogenous population of ribosomes [8, 9], with some inactive 51 and others actively translating parts of the proteome [10, 11]. Associating factors and RNA species tune 52 each ribosome, creating a cell with a spatially-and temporally-regulated proteome [8, 12]. 53The successful synthesis of a given protein at the ribosome is ...

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“…Secreted SapM phosphatase activity was measured as previously described(5, 34). Briefly, Mtb and BCG cultures were grown to mid-log phase and supernatants were normalized by culture OD 600 .…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial whole cell lysates, normalized by OD 600 to load equal protein, were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked and subsequently proteins were detected using anti-rabbit primary antibodies specific for SapM (1:10,000) or SatS (1:10,000)(34). An α-Rabbit IgG conjugated horseradish peroxidase secondary antibody (Bio-Rad) was used and the signal was detected using Western Lightning Plus-ECL chemiluminescent detection reagent (Perkin-Elmer).…”

Section: Methodsmentioning

confidence: 99%

The SapM phosphatase arrests phagosome maturation in an ESX-1 independent manner inMycobacterium tuberculosisand BCG

Xander

Saranathan

Jacobs

et al. 2023

Preprint

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Mycobacterium tuberculosis(Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used byMtbto achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA),Mtbremains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector ofMtbis the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to be both released by the mycobacteria and escape the phagosome to carry out its function. To date, the only mechanism known forMtbmolecules to escape the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-framesapMmutants in both the attenuatedMycobacterium bovisbacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, andMtb. Characterization of these mutants demonstrated that SapM is required for PMA in both BCG andMtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in aMtbmutant lacking the ESX-1 system, we demonstrated that the role of SapM is ESX-1-independent. We further determined that ESX-2 or ESX-4 are also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG andMtband that it functions independent of the known mechanism forMtbmolecules to escape the phagosome.

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Mycobacterium tuberculosis SatS is a chaperone for the SecA2 protein export pathway

Cited by 13 publications

References 55 publications

Protein Export into and across the Atypical Diderm Cell Envelope of Mycobacteria

Protein Export into and across the Atypical Diderm Cell Envelope of Mycobacteria

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

The SapM phosphatase arrests phagosome maturation in an ESX-1 independent manner inMycobacterium tuberculosisand BCG

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