Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.

Listeria monocytogenes is a ubiquitous bacterial pathogen that threatens the food chain and human health. In this study, whole-genome sequencing (WGS) was used for the genomic characterization of L. monocytogenes (n = 24) from beef and beef-based products. Multilocus Sequence Type (MLST) analysis revealed that ST204 of CC204 was the most common sequence type (ST). Other sequence types detected included ST1 and ST876 of CC1, ST5 of CC5, ST9 of CC9, ST88 of CC88, ST2 and ST1430 of CC2, and ST321 of CC321. Genes encoding for virulence factors included complete LIPI-1 (pfrA-hly-plcA-plcB-mpl-actA) from 54% (13/24) of the isolates of ST204, ST321, ST1430, and ST9 and internalin genes inlABC that were present in all the STs. All the L. monocytogenes STs carried four intrinsic/natural resistance genes, fosX, lin, norB, and mprF, conferring resistance to fosfomycin, lincosamide, quinolones, and cationic peptides, respectively. Plasmids pLGUG1 and J1776 were the most detected (54% each), followed by pLI100 (13%) and pLM5578 (7%). The prophage profile, vB_LmoS_188, was overrepresented amongst the isolates, followed by LP_101, LmoS_293_028989, LP_030_2_021539, A006, and LP_HM00113468. Listeria genomic island 2 (LGI-2) was found to be present in all the isolates, while Listeria genomic island 3 (LGI-3) was present in a subset of isolates (25%). The type VII secretion system was found in 42% of the isolates, and sortase A was present in all L. monocytogenes genomes. Mobile genetic elements and genomic islands did not harbor any virulence, resistance, or environmental adaptation genes that may benefit L. monocytogenes. All the STs did not carry genes that confer resistance to first-line antibiotics used for the treatment of listeriosis. The characterization of L. monocytogenes in our study highlighted the environmental resistance and virulence potential of L. monocytogenes and the risk posed to the public, as this bacterium is frequently found in food and food processing environments.

Group B Streptococcus (GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Firmicutes and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiont Enterococcus faecalis both in vitro using predator-prey assays and in vivo in the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named group B streptococcal LXG Toxin A) that contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.

Successful occupancy of a given niche requires the colonising bacteria to interact extensively with the biotic and abiotic environment, including other resident microbes. Bacteria have evolved a range of protein secretion machines for this purpose with eleven such systems identified to date. The type VIIb secretion system (T7SSb) is utilised by Bacillota to secrete a range of protein substrates, including antibacterial toxins targeting closely related strains, and the system as a whole has been implicated in a range of activities such as iron acquisition, intercellular signalling, host colonisation and virulence. This review covers the components and secretion mechanism of the T7SSb, the substrates of these systems and their roles in Gram-positive bacteria, with a focus on interbacterial competition.

OBJECTIVE: Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.

The type VIIb protein secretion system (T7SSb) plays a role in interbacterial competition in Gram-positive Firmicute bacteria and secretes various toxic effector proteins. The mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by the Staphylococcus aureus T7SSb, which forms a complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins, EsxB, EsxC and EsxD. Here we show that EsxBCD bind to the transport domain of EsaD and function as EsaD export factors. We report the first structural information for a complete T7SSb substrate pre-secretion complex. Cryo-EM of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers a conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export.

Type VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group B Streptococcus (GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intraspecies diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low interspecies and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxA in vitro and that in subtype I strain CJB111, esxA1 appears to be differentially transcribed from the T7SS operon. Furthermore, we observe subtype-specific effects of GBS T7SS on host colonization, as CJB111 subtype I but not CNCTC 10/84 subtype III T7SS promotes GBS vaginal colonization. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

Mycobacterium kansasii (Mk) is an opportunistic pathogen that is frequently isolated from urban water systems, posing a health risk to susceptible individuals. Despite its ability to cause tuberculosis-like pulmonary disease, very few studies have probed the genetics of this opportunistic pathogen. Here, we report a comprehensive essentiality analysis of the Mk genome. Deep sequencing of a high-density library of Mk Himar1 transposon mutants revealed that 86.8% of the chromosomal thymine-adenine (TA) dinucleotide target sites were permissive to insertion, leaving 13.2% TA sites unoccupied. Our analysis identified 394 of the 5,350 annotated open reading frames (ORFs) as essential. The majority of these essential ORFs (84.8%) share essential mutual orthologs with Mycobacterium tuberculosis (Mtb). A comparative genomics analysis identified 139 Mk essential ORFs that share essential orthologs in four other species of mycobacteria. Thirteen Mk essential ORFs share orthologs in all four species that were identified as being not essential, while only two Mk essential ORFs are absent in all species compared. We used the essentiality data and a comparative genomics analysis reported here to highlight differences in essentiality between candidate Mtb drug targets and the corresponding Mk orthologs. Our findings suggest that the Mk genome encodes redundant or additional pathways that may confound validation of potential Mtb drugs and drug target candidates against the opportunistic pathogen. Additionally, we identified 57 intergenic regions containing four or more consecutive unoccupied TA sites. A disproportionally large number of these regions were located upstream of pe/ppe genes. Finally, we present an essentiality and orthology analysis of the Mk pRAW-like plasmid, pMK1248. IMPORTANCE Mk is one of the most common nontuberculous mycobacterial pathogens associated with tuberculosis-like pulmonary disease. Drug resistance emergence is a threat to the control of Mk infections, which already requires long-term, multidrug courses. A comprehensive understanding of Mk biology is critical to facilitate the development of new and more efficacious therapeutics against Mk. We combined transposon-based mutagenesis with analysis of insertion site identification data to uncover genes and other genomic regions required for Mk growth. We also compared the gene essentiality data set of Mk to those available for several other mycobacteria. This analysis highlighted key similarities and differences in the biology of Mk compared to these other species. Altogether, the genome-wide essentiality information generated and the results of the cross-species comparative genomics analysis represent valuable resources to assist the process of identifying and prioritizing potential Mk drug target candidates and to guide future studies on Mk biology.

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.

The 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 coproduction 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 homologs at the tspA locus, suggestive of additional roles in intraspecies 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 homologs.