Homologous recombination between tandem paralogues drives evolution of Type VII secretion system immunity genes in firmicute bacteria

The Type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified in Staphylococcus aureus, EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identical esaG and tsaI immunity genes are encoded in all S. aureus strains. During genome sequence analysis of closely related S. aureus strains we noted that there had been a deletion of six consecutive esaG copies in one lineage. To investigate this further, we analysed the sequences of the tandem esaG genes and their encoded proteins. We identified three blocks of high sequence homology shared by all esaG genes, and identified evidence of extensive recombination events between esaG paralogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss of esaG genes from S. aureus genomes. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence homology locate to the 5-prime and 3-prime end of tipC genes, and we found strong evidence for recombination between tipC paralogues encoded by Streptococcus mitis BCC08. By contrast, we found only a single block of homology across tsaI genes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.

A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence

Type VII secretion systems (T7SS) have been identified in Actinobacteria and Firmicutes and have been shown to secrete effector proteins with functions in virulence, host toxicity, and/or interbacterial killing in a few genera. Bioinformatic analysis indicates that isolates of Group B Streptococcus (GBS) encode at least four distinct subtypes of T7SS machinery, three of which encode adjacent putative T7SS effectors with WXG and LXG motifs. However, the function of T7SS in GBS pathogenesis is unknown. Here we assessed the role of the most abundant GBS T7SS subtype during GBS pathogenesis. In a murine model of hematogenous meningitis, mice infected with GBS lacking a functional T7SS or lacking the secreted WXG100 effector EsxA exhibited less mortality, lower bacterial burdens in tissues, and decreased inflammation in the brain compared to mice infected with the parental GBS strain. We further showed that this T7SS induces cytotoxicity in brain endothelium and that EsxA contributes to these cytotoxicity phenotypes in a WXG motif-dependent manner. Finally, we determined that EsxA is a pore-forming protein, thus demonstrating the first role for a non-mycobacterial EsxA homolog in pore formation. This work reveals the importance of a T7SS in host–GBS interactions and has implications for T7SS effector function in other Gram-positive bacteria.

Toxin secretion and trafficking by Mycobacterium tuberculosis

The tuberculosis necrotizing toxin (TNT) is the major cytotoxicity factor of Mycobacterium tuberculosis (Mtb) in macrophages. TNT is the C-terminal domain of the outer membrane protein CpnT and gains access to the cytosol to kill macrophages infected with Mtb. However, molecular mechanisms of TNT secretion and trafficking are largely unknown. A comprehensive analysis of the five type VII secretion systems of Mtb revealed that the ESX-4 system is required for export of CpnT and surface accessibility of TNT. Furthermore, the ESX-2 and ESX-4 systems are required for permeabilization of the phagosomal membrane in addition to the ESX-1 system. Thus, these three ESX systems need to act in concert to enable trafficking of TNT into the cytosol of Mtb-infected macrophages. These discoveries establish new molecular roles for the two previously uncharacterized type VII secretion systems ESX-2 and ESX-4 and reveal an intricate link between toxin secretion and phagosomal permeabilization by Mtb.

Extensive Horizontal Gene Transfer within and between Species of Coagulase-Negative Staphylococcus

Members of the gram-positive bacterial genus Staphylococcus have historically been classified into coagulase-positive Staphylococcus (CoPS) and coagulase-negative Staphylococcus (CoNS) based on the diagnostic presentation of the coagulase protein. Previous studies have noted the importance of horizontal gene transfer (HGT) and recombination in the more well-known CoPS species Staphylococcus aureus, yet little is known of the contributions of these processes in CoNS evolution. In this study, we aimed to elucidate the phylogenetic relationships, genomic characteristics, and frequencies of HGT in CoNS, which are now being recognized as major opportunistic pathogens of humans. We compiled a data set of 1,876 publicly available named CoNS genomes. These can be delineated into 55 species based on allele differences in 462 core genes and variation in accessory gene content. CoNS species are a reservoir of transferrable genes associated with resistance to diverse classes of antimicrobials. We also identified nine types of the mobile genetic element SCCmec, which carries the methicillin resistance determinant mecA. Other frequently transferred genes included those associated with resistance to heavy metals, surface-associated proteins related to virulence and biofilm formation, type VII secretion system, iron capture, recombination, and metabolic enzymes. The highest frequencies of receipt and donation of recombined DNA fragments were observed in Staphylococcus capitis, Staphylococcus caprae, Staphylococcus hominis, Staphylococcus haemolyticus, and members of the Saprophyticus species group. The variable rates of recombination and biases in transfer partners imply that certain CoNS species function as hubs of gene flow and major reservoir of genetic diversity for the entire genus.

The Type VII Secretion System of Staphylococcus

The type VII protein secretion system (T7SS) of Staphylococcus aureus is encoded at the ess locus. T7 substrate recognition and protein transport are mediated by EssC, a membrane-bound multidomain ATPase. Four EssC sequence variants have been identified across S. aureus strains, each accompanied by a specific suite of substrate proteins. The ess genes are upregulated during persistent infection, and the secretion system contributes to virulence in disease models. It also plays a key role in intraspecies competition, secreting nuclease and membrane-depolarizing toxins that inhibit the growth of strains lacking neutralizing immunity proteins. A genomic survey indicates that the T7SS is widely conserved across staphylococci and is encoded in clusters that contain diverse arrays of toxin and immunity genes. The presence of genomic islands encoding multiple immunity proteins in species such as Staphylococcus warneri that lack the T7SS points to a major role for the secretion system in bacterial antagonism. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The beneficial rhizobacterium Bacillus velezensis acquires iron from roots via a type VII secretion system for colonization

Niche colonization is the key for bacterial adaptation to the environment, and competition for iron largely determines root colonization by rhizosphere microbes. Pathogenic and beneficial symbiotic bacteria use various unique secretion systems to support plant colonization or acquire limited resources from the environment. However, ubiquitous nonsymbiotic beneficial rhizobacteria have never been reported to use a unique secretion system to facilitate colonization. Here, we show that the type VII secretion system (T7SS) of the beneficial rhizobacterium Bacillus velezensis SQR9 contributes to root colonization. Knocking out T7SS and the major secreted protein YukE in SQR9 caused a significant decrease in root colonization. Moreover, the T7SS and YukE caused iron loss in plant roots in the early stage after inoculation, which contributed to root colonization by SQR9. Interestingly, purified YukE, but not inactivated YukE, could change the permeability of root cells. We speculated that secreted YukE might be directly inserted into the root cell membrane to cause iron leakage, indicating that the bacterial protein and root cell membrane interact directly. Moreover, a bacterial siderophore and the T7SS may be coordinately involved in iron acquisition by B. velezensis SQR9 for efficient root colonization. We showed that the beneficial rhizobacterium B. velezensis SQR9 could acquire iron from roots via the T7SS for rapid colonization. These findings provide the first insight into the function of the unique secretion system in nonsymbiotic beneficial rhizobacteria and reveal a novel mutualism in which plants and bacteria might share iron in a sequential manner.

Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis , to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis . In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus ; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.