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

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD(+) strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness

Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence

Understanding the evolution of virulence is key to appreciating the role specific loci play in pathogenicity. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find homologues of virulence loci. One example of this is the mammalian cell entry (mce) locus, which has been characterised in Mycobacterium tuberculosis. To investigate the role in Streptomyces we deleted the mce locus and studied its impact on cell survival, morphology and interaction with other soil organisms. Disruption of the mce cluster resulted in virulence towards amoebae (Acanthamoeba polyphaga) and reduced colonization of plant (Arabidopsis) models, indicating these genes may play an important role in Streptomyces survival in the environment. Our data suggest that loss of mce in Streptomyces spp. may have profound effects on survival in a competitive soil environment, and provides insight in to the evolution and selection of these genes as virulence factors in related pathogenic organisms

The Enigmatic Esx Proteins:Looking Beyond Mycobacteria

Bacteria export proteins across membranes using a range of transport machineries. Type VII secretion systems (T7SSs), originally described in mycobacteria, are now known to be widespread across diverse bacterial phyla. Recent studies have characterized secretion components and mechanisms of type VII secretion in pathogenic and environmental bacteria. A variety of functions have been attributed to T7SS substrates, including interactions with eukaryotes and with other bacteria. Here, we evaluate the growing body of knowledge on T7SSs, with focus on the nonmycobacterial systems, reviewing their phylogenetic distribution, structure and function in diverse settings

Solution Structure of the Mycobacterium tuberculosis EsxG·EsxH Complex: FUNCTIONAL IMPLICATIONS AND COMPARISONS WITH OTHER M. TUBERCULOSIS Esx FAMILY COMPLEXES*

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn2+ binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn2+-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners

A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism Streptomyces coelicolor A3(2)

A simple and high-throughput transposon mediated mutagenesis system employing in vitro shuttle transposon mutagenesis has been used to systematically mutagenise the Streptomyces coelicolor genome. To achieve the highest coverage, a new ordered cosmid library was also constructed. Individual cosmids from both the existing and new libraries were disrupted using the Tn5-based mini-transposon Tn5062. A total of 35,358 insertions were sequenced resulting in the disruption of 6,482 genes (83% of the predicted open reading frames). Complete information for both the newly generated cosmids as well as all the insertions has been uploaded onto a central database, StrepDB ( http://strepdb.streptomyces.org.uk/ ). All insertions, new cosmids and a range of transposon exchange cassettes are available for study of individual gene function