Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Dual Membrane-spanning Anti-Sigma Factors Regulate Vesiculation in Gut Bacteroidota.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by O uter M embrane V esicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron ( Bt ). Our screening led us to the identification of a novel family of D ual M embrane-spanning A nti-sigma factors (Dma), which regulate OMV biogenesis in Bt . We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal ß-barrel embedded in the OM; its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Human gut bacteria tailor extracellular vesicle cargo for the breakdown of diet- and host-derived glycans.

Extracellular vesicles are produced in all three domains of life, and their biogenesis has common ancient origins in eukaryotes and archaea. Although bacterial vesicles were discovered several decades ago and multiple roles have been attributed to them, no mechanism has been established for vesicles biogenesis in bacteria. For this reason, there is a significant level of skepticism about the biological relevance of bacterial vesicles. Bacteroides thetaiotaomicron (Bt), a prominent member of the human intestinal microbiota, produces significant amounts of outer membrane vesicles (OMVs) which have been proposed to play key physiological roles. Here, we employed a dual marker system, consisting of outer membrane- and OMV-specific markers fused to fluorescent proteins to visualize OMV biogenesis by time-lapse microscopy. Furthermore, we performed comparative proteomic analyses to show that, in Bt, the OMV cargo is adapted for the optimal utilization of different polysaccharides. We also show that a negatively charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. Our results demonstrate that OMV production is the result of a highly regulated process in Bt.

Human gut bacteria tailor extracellular vesicle cargo for the breakdown of diet- and host-derived glycans.

Extracellular vesicles (EV) are produced in all three domains of life, and their biogenesis have common ancient origins in eukaryotes and archaea. Although bacterial vesicles were discovered several decades ago and multiple roles have been attributed to them, no mechanism has been established for vesicles biogenesis in bacteria. For this reason, there is a significant level of skepticism about the biological relevance of bacterial vesicles. In Bacteroides thetaiotaomicron ( Bt ), a prominent member of the human intestinal microbiota, outer membrane vesicles (OMVs) have been proposed to play key physiological roles. By employing outer membrane- and OMV-specific markers fused to fluorescent proteins we visualized OMV biogenesis in live-cells. We performed comparative proteomic analyses to demonstrate that Bt actively tailors its vesicle cargo to optimize the breakdown of diet- and host-derived complex glycans. Surprisingly, our data suggests that OMV are not employed for mucin degradation. We also show that, in Bt , a negatively-charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. We conclude that OMVs are the result of an exquisitely orchestrated mechanism. This work lays the foundation for further investigations into the physiological relevance of OMVs and their roles in gut homeostasis. Furthermore, our work constitutes a roadmap to guide EV biogenesis research in other bacteria.

InvL, an Invasin-Like Adhesin, Is a Type II Secretion System Substrate Required for Acinetobacter baumannii Uropathogenesis.

Acinetobacter baumannii is an opportunistic pathogen of growing concern, as isolates are commonly multidrug resistant. While A. baumannii is most frequently associated with pulmonary infections, a significant proportion of clinical isolates come from urinary sources, highlighting its uropathogenic potential. The type II secretion system (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in urinary tract infection (UTI) remains unknown. Here, we used a catheter-associated UTI (CAUTI) model to demonstrate that a modern urinary isolate, UPAB1, requires the T2SS for full virulence. A proteomic screen to identify putative UPAB1 T2SS effectors revealed an uncharacterized lipoprotein with structural similarity to the intimin-invasin family, which serve as type V secretion system (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the ß-barrel domain associated with T5SSs but was confirmed to require the T2SS for both surface localization and secretion. This makes InvL the first identified T2SS effector belonging to the intimin-invasin family. InvL was confirmed to be an adhesin, as the protein bound to extracellular matrix components and mediated adhesion to urinary tract cell lines in vitro. Additionally, the invL mutant was attenuated in the CAUTI model, indicating a role in Acinetobacter uropathogenesis. Finally, bioinformatic analyses revealed that InvL is present in nearly all clinical isolates belonging to international clone 2, a lineage of significant clinical importance. In all, we conclude that the T2SS substrate InvL is an adhesin required for A. baumannii uropathogenesis. IMPORTANCE While pathogenic Acinetobacter can cause various infections, we recently found that 20% of clinical isolates come from urinary sources. Despite the clinical relevance of Acinetobacter as a uropathogen, few virulence factors involved in urinary tract colonization have been defined. Here, we identify a novel type II secretion system effector, InvL, which is required for full uropathogenesis by a modern urinary isolate. Although InvL has predicted structural similarity to the intimin-invasin family of autotransporter adhesins, InvL is predicted to be anchored to the membrane as a lipoprotein. Similar to other invasin homologs, however, we demonstrate that InvL is a bona fide adhesin capable of binding extracellular matrix components and mediating adhesion to urinary tract cell lines. In all, this work establishes InvL as an adhesin important for Acinetobacter's urinary tract virulence and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.

Bacterial Outer Membrane Vesicles: From Discovery to Applications.

Secretion of cellular components across the plasma membrane is an essential process that enables organisms to interact with their environments. Production of extracellular vesicles in bacteria is a well-documented but poorly understood process. Outer membrane vesicles (OMVs) are produced in gram-negative bacteria by blebbing of the outer membrane. In addition to their roles in pathogenesis, cell-to-cell communication, and stress responses, OMVs play important roles in immunomodulation and the establishment and balance of the gut microbiota. In this review, we discuss the multiple roles of OMVs and the current knowledge of OMV biogenesis. We also discuss the growing and promising biotechnological applications of OMV.