Streptococcus agalactiae causes sepsis and meningitis in neonates, presenting substantial clinical challenges. Type VII secretion system (T7SS), an important secretion system identified in Mycobacterium sp. and Gram-positive bacteria, was recently characterized in S. agalactiae and considered to contribute to its virulence and pathogenesis. In the present study, 128 complete genomic sequences of S. agalactiae were retrieved from GenBank to build a public dataset, and their sequences, capsular types, and clonal complexes were determined. Polymerase chain reaction (PCR) screening and genomic sequencing were conducted in an additional clinical dataset. STs and capsular types were determined using PCR. Eleven different types of T7SS were detected with similarities in gene order but differences in gene content. Strains with incomplete T7SS or lack of T7SS were also identified. Deletion, insertion, and segmentation of T7SS might be related to insertion sequences. The genetic environment of T7SS in S. agalactiae was also investigated and different patterns were identified downstream the T7SS, which were related to the diversity of T7SS putative effectors. The T7SS demonstrated possible sequence type (ST)-dependent diversity in both datasets. This work elucidated detailed genetic characteristics of T7SS and its genetic environment in S. agalactiae and further identified its possible ST-dependent diversity, which gave a clue of its mode of transmission. Further investigations are required to elucidate the underlying mechanisms and its functions.

Streptococcus suis can cause severe infections in pigs and humans. The tonsils of pigs are major niches for S. suis, and different serotypes of S. suis can be found in the same tonsil. Pig tonsil colonization by S. suis is believed to be an important source of infection for humans and pigs. However, how S. suis competes for a stable tonsil niche is unknown. Here, we found that S. suis strain WUSS351, isolated from a healthy pig tonsil, is virulent and multidrug-resistant. The ABC transporter system SstFEG, conferring resistance to bacitracin, was reported to confer a competitive survival advantage in vivo. In addition, strain WUSS351 has several antimicrobial systems, including a novel type VII secretion system (T7SS), lantibiotic bacteriocin, and lactococcin972-like bacteriocin Lcn351. Bacterial competition experiments demonstrated T7SS-mediated cell contact-dependent antagonism of S. suis. Antibacterial activity analysis and 16S rRNA gene sequencing of the culture-independent and culture-dependent pig tonsillar microbiome revealed that Lcn351 mainly targets S. suis, one of the core microbiomes in pig tonsils. Taken together, our results revealed the mechanism of the stable persistence of S. suis in the tonsil niche, which might have important implications for S. suis epidemiology, potentially influencing strain prevalence and disease progression.

Type VII secretion systems (T7SSs) are found in many disease related bacteria including Mycobacterium tuberculosis (Mtb). ESX-1 [early secreted antigen 6 kilodaltons (ESAT-6) system 1] is one of the five subtypes (ESX-1~5) of T7SSs in Mtb, where it delivers virulence factors into host macrophages during infection. However, little is known about the molecular details as to how this occurs. Here, we provide high-resolution crystal structures of the C-terminal ATPase3 domains of EccC subunits from four different Mtb T7SS subtypes. These structures adopt a classic RecA-like ɑ/β fold with a conserved Mg-ATP binding site. The structure of EccCb1 in complex with the C-terminal peptide of EsxB identifies the location of substrate recognition site and shows how the specific signaling module \"LxxxMxF\" for Mtb ESX-1 binds to this site resulting in a translation of the bulge loop. A comparison of all the ATPase3 structures shows there are significant differences in the shape and composition of the signal recognition pockets across the family, suggesting that distinct signaling sequences of substrates are required to be specifically recognized by different T7SSs. A hexameric model of the EccC-ATPase3 is proposed and shows the recognition pocket is located near the central substrate translocation channel. The diameter of the channel is ~25-Å, with a size that would allow helix-bundle shaped substrate proteins to bind and pass through. Thus, our work provides new molecular insights into substrate recognition for Mtb T7SS subtypes and also a possible transportation mechanism for substrate and/or virulence factor secretion.

Streptococcus suis (SS), an important pathogen for pigs, is not only considered as a zoonotic agent for humans, but is also recognized as a major reservoir of antimicrobial resistance contributing to the spread of resistance genes to other pathogenic Streptococcus species. In addition to serotype 2 (SS2), serotype 9 (SS9) is another prevalent serotype isolated from diseased pigs. Although many SS strains have been sequenced, the complete genome of a non-SS2 virulent strain has been unavailable to date. Here, we report the complete genome of GZ0565, a virulent strain of SS9, isolated from a pig with meningitis. Comparative genomic analysis revealed five new putative virulence or antimicrobial resistance-associated genes in strain GZ0565 but not in SS2 virulent strains. These five genes encode a putative triacylglycerol lipase, a TipAS antibiotic-recognition domain protein, a putative TetR family transcriptional repressor, a protein containing a LPXTG domain and a G5 domain, and a type VII secretion system (T7SS) putative substrate (EsxA), respectively. Western blot analysis showed that strain GZ0565 can secrete EsxA. We generated an esxA deletion mutant and showed that EsxA contributes to SS virulence in a mouse infection model. Additionally, the antibiotic resistance gene vanZSS was identified and expression of vanZSS conferred resistance to teicoplanin and dalbavancin in Streptococcus agalactiae. We believe this is the first experimental demonstration of the existence of the T7SS putative substrate EsxA and its contribution to bacterial virulence in SS. Together, our results contribute to further understanding of the virulence and antimicrobial resistance characteristics of SS.

The protein EccB1, a core component of the type VII secretion system (T7SS) of Mycobacterium tuberculosis, has been identified as an ATPase and is essential for the secretion of virulence factors by the ESX-1 system. In a previous study, EccB1 structures were determined in two different conformations. Here, two new conformations are identified and described. These four conformations present snapshots of the swinging movement of the membrane-distal domain A2. The movement of this domain involves conformational changes in two flexible loops (loop A, residues 243-264, and loop B, residues 324-341) which are rich in proline and glycine residues and connect domain A2 to domains C1 and B2. It is proposed that the movement of this domain is related to the ATPase activity of EccB1 and its homologues, as well as to the substrate transport of ESX secretion systems.

Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.