Assembly of a unique membrane complex in type VI secretion systems of Bacteroidota.

The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis (tssNQOPR) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.

Rocket-miR, a translational launchpad for miRNA-based antimicrobial drug development.

IMPORTANCE: Antimicrobial-resistant infections contribute to millions of deaths worldwide every year. In particular, the group of bacteria collectively known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) pathogens are of considerable medical concern due to their virulence and exceptional ability to develop antibiotic resistance. New kinds of antimicrobial therapies are urgently needed to treat patients for whom existing antibiotics are ineffective. The Rocket-miR application predicts targets of human miRNAs in bacterial and fungal pathogens, rapidly identifying candidate miRNA-based antimicrobials. The application's target audience are microbiologists that have the laboratory resources to test the application's predictions. The Rocket-miR application currently supports 24 recognized human pathogens that are relevant to numerous diseases including cystic fibrosis, chronic obstructive pulmonary disease (COPD), urinary tract infections, and pneumonia. Furthermore, the application code was designed to be easily extendible to other human pathogens that commonly cause hospital-acquired infections.

Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis.

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.

Lysine acetylation of cytoskeletal proteins: Emergence of an actin code.

Reversible lysine acetylation of nuclear proteins such as histones is a long-established important regulatory mechanism for chromatin remodeling and transcription. In the cytoplasm, acetylation of a number of cytoskeletal proteins, including tubulin, cortactin, and the formin mDia2, regulates both cytoskeletal assembly and stability. More recently, acetylation of actin itself was revealed to regulate cytoplasmic actin polymerization through the formin INF2, with downstream effects on ER-to-mitochondrial calcium transfer, mitochondrial fission, and vesicle transport. This finding raises the possibility that actin acetylation, along with other post-translational modifications to actin, might constitute an "actin code," similar to the "histone code" or "tubulin code," controlling functional shifts to these central cellular proteins. Given the multiple roles of actin in nuclear functions, its modifications might also have important roles in gene expression.

Tumor microtubes connect pancreatic cancer cells in an Arp2/3 complex-dependent manner.

Actin-based tubular connections between cells have been observed in many cell types. Termed "tunneling nanotubes (TNTs)," "membrane nanotubes," "tumor microtubes (TMTs)," or "cytonemes," these protrusions interconnect cells in dynamic networks. Structural features in these protrusions vary between cellular systems, including tubule diameter and the presence of microtubules. We find tubular protrusions, which we classify as TMTs, in a pancreatic cancer cell line, Dartmouth-Hitchcock Pancreatic Cancer (DHPC)-018. TMTs are present in DHPC-018-derived tumors in mice, as well as in a mouse model of pancreatic cancer and a subset of primary human tumors. DHPC-018 TMTs have heterogeneous diameter (0.39-5.85 µm, median 1.92 µm) and contain actin filaments, microtubules, and cytokeratin 19-based intermediate filaments. TMTs do not allow intercellular transfer of cytoplasmic GFP. Actin filaments are cortical within the protrusion, as opposed to TNTs, in which filaments run down the center. TMTs are dynamic in length, but are long lived (median >60 min). Inhibition of actin polymerization, but not microtubules, results in TMT loss. Extracellular calcium is necessary for TMT maintenance. A second class of tubular protrusion, which we term cell-substrate protrusion, has similar width range and cytoskeletal features but makes contact with the substratum as opposed to another cell. Similar to previous work on TNTs, we find two assembly mechanisms for TMTs, which we term "pull-away" and "search-and-capture." Inhibition of Arp2/3 complex inhibits TMT assembly by both mechanisms. This work demonstrates that the actin architecture of TMTs in pancreatic cancer cells is fundamentally different from that of TNTs and demonstrates the role of Arp2/3 complex in TMT assembly.

Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly.

Filopodia are actin-dependent finger-like structures that protrude from the plasma membrane. Actin filament barbed-end-binding proteins localized to filopodial tips are key to filopodial assembly. Two classes of barbed-end-binding proteins are formins and Ena/VASP proteins, and both classes have been localized to filopodial tips in specific cellular contexts. Here, we examine the filopodial roles of the FMNL formins and Ena/VASP proteins in U2OS cells. FMNL3 suppression reduces filopodial assembly by 90%, and FMNL3 is enriched at >95% of filopodial tips. Suppression of VASP or Mena (also known as ENAH) reduces filopodial assembly by >75%. However, VASP and Mena do not display consistent filopodial tip localization, but are enriched in focal adhesions (FAs). Interestingly, >85% of FMNL3-containing filopodia are associated with FAs. Two situations increase Ena/VASP filopodial localization: (1) expression of myosin-X, and (2) actively spreading cells. In spreading cells, filopodia often mark sites of nascent adhesions. Interestingly, VASP suppression in spreading cells causes a significant increase in adhesion assembly at filopodial tips. This work demonstrates that, in U2OS cells, Ena/VASP proteins play roles in filopodia beyond those at filopodial tips.This article has an associated First Person interview with the first author of the paper.

Heme sensing in Bacillus thuringiensis: a supplementary HssRS-regulated heme resistance system.

Several Gram-positive pathogens scavenge host-derived heme to satisfy their nutritional iron requirement. However, heme is a toxic molecule capable of damaging the bacterial cell. Gram-positive pathogens within the phylum Firmicutes overcome heme toxicity by sensing heme through HssRS, a two-component system that regulates the heme detoxification transporter HrtAB. Here we show that heme sensing by HssRS and heme detoxification by HrtAB occur in the insect pathogen Bacillus thuringiensis We find that in B. thuringiensis, HssRS directly regulates an operon, hrmXY, encoding hypothetical membrane proteins that are not found in other Firmicutes with characterized HssRS and HrtAB systems. This novel HssRS-regulated operon or its orthologs BMB171_c3178 and BMB171_c3330 are required for maximal heme resistance. Furthermore, the activity of HrmXY is not dependent on expression of HrtAB. These results suggest that B. thuringiensis senses heme through HssRS and induces expression of separate membrane-localized systems capable of overcoming different aspects of heme toxicity.