The Ess/Type VII secretion system of Staphylococcus aureus shows unexpected genetic diversity

SummaryThe Staphylococcus aureus type VII secretion system (T7SS) exports several proteins that are pivotal for bacterial virulence. The mechanisms underlying T7SS-mediated staphylococcal survival during infection nevertheless remain unclear. Here we show that the absence of EsxC, a small secreted effector implicated in bacterial persistence, results in cell membrane defects in S. aureus. Interestingly, isogenic mutants lacking EsxC, other T7SS effectors EsxA and EsxB, or the membrane-bound ATPase EssC, are more sensitive to killing by the host-derived antimicrobial fatty acid, linoleic acid (LA), compared to the wild-type (WT). LA induces more cell membrane damage in the T7SS mutants compared to the WT. Although WT and mutant strains did not differ in their ability to bind labelled LA, membrane lipid profiles show that T7SS mutants are less able to incorporate LA into their membrane phospholipids. Furthermore, proteomic analyses of WT and mutant cell fractions reveal that, in addition to compromising membranes, T7SS defects induce oxidative stress and hamper their response to LA challenge. Thus, our findings indicate that T7SS is crucial for S. aureus membrane integrity and homeostasis, which is critical when bacteria encounter antimicrobial fatty acids.

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EsaB is a core component of the Staphylococcus aureus Type VII secretion system

10.1101/151316 ◽

2017 ◽

Author(s):

M. Guillermina Casabona ◽

Grant Buchanan ◽

Martin Zoltner ◽

Catriona P. Harkins ◽

Matthew T.G. Holden ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Fluorescent Protein ◽

Iron Acquisition ◽

Yellow Fluorescent Protein ◽

Secretion System ◽

Secretion Systems ◽

Core Component ◽

Type Vii Secretion ◽

Bacterial Competition ◽

Type Vii Secretion System

AbstractType VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.

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A membrane-depolarizing toxin substrate of the Staphylococcus aureus Type VII secretion system mediates intra-species competition

10.1101/443630 ◽

2018 ◽

Cited By ~ 1

Author(s):

Fatima R. Ulhuq ◽

Margarida C. Gomes ◽

Gina Duggan ◽

Manman Guo ◽

Chriselle Mendonca ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pathogenic Bacteria ◽

Secretion System ◽

Mucosal Barrier ◽

Infection Model ◽

Species Competition ◽

Type Vii Secretion ◽

Proteomic Approach ◽

Type Vii Secretion System

AbstractThe type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues.Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

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The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria

Nature Microbiology ◽

10.1038/nmicrobiol.2016.183 ◽

2016 ◽

Vol 2 (1) ◽

Cited By ~ 90

Author(s):

Zhenping Cao ◽

M. Guillermina Casabona ◽

Holger Kneuper ◽

James D. Chalmers ◽

Tracy Palmer

Keyword(s):

Staphylococcus Aureus ◽

Secretion System ◽

Type Vii Secretion ◽

Type Vii Secretion System

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Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment

Biochemical Journal ◽

10.1042/bj20121209 ◽

2012 ◽

Vol 449 (2) ◽

pp. 469-477 ◽

Cited By ~ 18

Author(s):

Martin Zoltner ◽

Paul K. Fyfe ◽

Tracy Palmer ◽

William N. Hunter

Keyword(s):

Staphylococcus Aureus ◽

Protein Interactions ◽

Protein Translocation ◽

Secretion System ◽

Full Length ◽

Protein Protein Interactions ◽

Gram Positive Bacteria ◽

Type Vii Secretion ◽

Early Secretory Antigenic Target ◽

Type Vii Secretion System

The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed in Escherichia coli, solubilized from the bacterial membrane, purified to homogeneity and shown to be dimeric. A C-terminal truncation, EssB∆C, and two soluble fragments termed EssB-N and EssB-C, predicted to occur on either side of the cytoplasmic membrane, have been successfully purified in a recombinant form, characterized and, together with the full-length protein, used in crystallization trials. EssB-N, the 25 kDa N-terminal cytoplasmic fragment, gave well-ordered crystals and we report the structure, determined by SAD (single-wavelength anomalous diffraction) targeting an SeMet (selenomethionine) derivative, refined to atomic (1.05 Å; 1 Å=0.1 nm) resolution. EssB-N is dimeric in solution, but crystallizes as a monomer and displays a fold comprised of two globular domains separated by a cleft. The structure is related to that of serine/threonine protein kinases and the present study identifies that the Type VII secretion system exploits and re-uses a stable modular entity and fold that has evolved to participate in protein–protein interactions in a similar fashion to the catalytically inert pseudokinases.

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Functional analysis of the EsaB component of the Staphylococcus aureus Type VII secretion system

Microbiology ◽

10.1099/mic.0.000580 ◽

2017 ◽

Vol 163 (12) ◽

pp. 1851-1863 ◽

Cited By ~ 9

Author(s):

M. Guillermina Casabona ◽

Grant Buchanan ◽

Martin Zoltner ◽

Catriona P. Harkins ◽

Matthew T. G. Holden ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Functional Analysis ◽

Secretion System ◽

Type Vii Secretion ◽

Type Vii Secretion System

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Haem-iron plays a key role in the regulation of the Ess/type VII secretion system of Staphylococcus aureus RN6390

Microbiology ◽

10.1099/mic.0.000579 ◽

2017 ◽

Vol 163 (12) ◽

pp. 1839-1850 ◽

Cited By ~ 7

Author(s):

M. Guillermina Casabona ◽

Holger Kneuper ◽

Daniela Alferes de Lima ◽

Catriona P. Harkins ◽

Martin Zoltner ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Secretion System ◽

Type Vii Secretion ◽

Type Vii Secretion System ◽

Haem Iron

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The type VII secretion system protects Staphylococcus aureus against antimicrobial host fatty acids

Scientific Reports ◽

10.1038/s41598-020-71653-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Arnaud Kengmo Tchoupa ◽

Kate E. Watkins ◽

Rebekah A. Jones ◽

Agnès Kuroki ◽

Mohammad Tauqeer Alam ◽

...

Keyword(s):

Fatty Acids ◽

Staphylococcus Aureus ◽

Unsaturated Fatty Acids ◽

Mutant Cell ◽

Membrane Damage ◽

Membrane Integrity ◽

Secretion System ◽

Membrane Phospholipids ◽

Type Vii Secretion ◽

Type Vii Secretion System

Abstract The Staphylococcus aureus type VII secretion system (T7SS) exports several proteins that are pivotal for bacterial virulence. The mechanisms underlying T7SS-mediated staphylococcal survival during infection nevertheless remain unclear. Here we report that S. aureus lacking T7SS components are more susceptible to host-derived antimicrobial fatty acids. Unsaturated fatty acids such as linoleic acid (LA) elicited an increased inhibition of S. aureus mutants lacking T7SS effectors EsxC, EsxA and EsxB, or the membrane-bound ATPase EssC, compared to the wild-type (WT). T7SS mutants generated in different S. aureus strain backgrounds also displayed an increased sensitivity to LA. Analysis of bacterial membrane lipid profiles revealed that the esxC mutant was less able to incorporate LA into its membrane phospholipids. Although the ability to bind labelled LA did not differ between the WT and mutant strains, LA induced more cell membrane damage in the T7SS mutants compared to the WT. Furthermore, proteomic analyses of WT and mutant cell fractions revealed that, in addition to compromising membranes, T7SS defects induce oxidative stress and hamper their response to LA challenge. Thus, our findings indicate that T7SS contribute to maintaining S. aureus membrane integrity and homeostasis when bacteria encounter antimicrobial fatty acids.

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