The YcnI protein from Bacillus subtilis contains a copper-binding domain.

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram-positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through the ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized Domain of Unknown Function 1775 (DUF1775) containing YcnI. DUF1775 domains are found across bacterial phylogeny, and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance and inductively coupled plasma-MS, found that this protein can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper-binding site featuring a unique monohistidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

An in-depth exploration of the multifaceted roles of EVs in the context of pathogenic single-cell microorganisms.

SUMMARYExtracellular vesicles (EVs) have been recognized throughout scientific communities as potential vehicles of intercellular communication in both eukaryotes and prokaryotes, thereby influencing various physiological and pathological functions of both parent and recipient cells. This review provides an in-depth exploration of the multifaceted roles of EVs in the context of bacteria and protozoan parasite EVs, shedding light on their contributions to physiological processes and disease pathogenesis. These studies highlight EVs as a conserved mechanism of cellular communication, which may lead us to important breakthroughs in our understanding of infection, mechanisms of pathogenesis, and as indicators of disease. Furthermore, EVs are involved in host-microbe interactions, offering insights into the strategies employed by bacteria and protozoan parasites to modulate host responses, evade the immune system, and establish infections.

Multivariate Analysis of Individual Bacterial Outer Membrane Vesicles Using Fluorescence Microscopy.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. OMVs have emerged as promising therapeutic agents for various biological applications such as vaccines and targeted drug delivery. However, the full potential of OMVs is currently constrained by inherent heterogeneities, such as size and cargo differences, and traditional ensemble assays are limited in their ability to reveal OMV heterogeneity. To overcome this issue, we devised an innovative approach enabling the identification of various characteristics of individual OMVs. This method, employing fluorescence microscopy, facilitates the detection of variations in size and surface markers. To demonstrate our method, we utilize the oral bacterium Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) which produces OMVs with a bimodal size distribution. As part of its virulence, A. actinomycetemcomitans secretes leukotoxin (LtxA) in two forms: soluble and surface associated with the OMVs. We observed a correlation between the size and toxin presence where larger OMVs were much more likely to possess LtxA compared to the smaller OMVs. In addition, we noted that, among the smallest OMVs (<100 nm diameter), the fractions that are toxin positive range from 0 to 30%, while the largest OMVs (>200 nm diameter) are between 70 and 100% toxin positive.

Identifying size-dependent toxin sorting in bacterial outer membrane vesicles.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. Despite being isolated from a single population of bacteria, OMVs can exhibit heterogeneous size and toxin content, which can be obscured by assays that measure ensemble properties. To address this issue, we utilize fluorescence imaging of individual OMVs to reveal size-dependent toxin sorting. Our results showed that the oral bacterium Aggregatibacter actinomycetemcomitans (A.a.) produces OMVs with a bimodal size distribution, where larger OMVs were much more likely to possess leukotoxin (LtxA). Among the smallest OMVs (< 100 nm diameter), the fraction that are toxin positive ranges from 0-30%, while the largest OMVs (> 200 nm diameter) are between 70-100% toxin positive. Our single OMV imaging method provides a non-invasive way to observe OMV surface heterogeneity at the nanoscale level and determine size-based heterogeneities without the need for OMV fraction separation.

Imaging Giant Vesicle Membrane Domains with a Luminescent Europium Tetracycline Complex.

Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in fluorescence contrast. Here, we show that membrane microdomains in giant unilamellar vesicles (GUVs) can be visualized with europium luminescence using a complex of europium III (Eu3+) and tetracycline (EuTc). EuTc is unlike typical organic lipid probes in that it is a coordination complex with a unique excitation/emission wavelength combination (396/617 nm), a very large Stokes shift (221 nm), and a very narrow emission bandwidth (8 nm). The probe preferentially interacts with liquid disordered domains in GUVs, which results in intensity contrast across the surface of phase-separated GUVs. Interestingly, EuTc also alters GM1 ganglioside partitioning. GM1 typically partitions into liquid ordered domains, but after labeling phase-separated GUVs with EuTc, cholera toxin B-subunit (CTxB), which binds GM1, labels liquid disordered domains. We also demonstrate that EuTc, but not free Eu3+ or Tc, significantly reduces lipid diffusion coefficients. Finally, we show that EuTc can be used to label cellular membranes similar to a traditional membrane probe. EuTc may find utility as a membrane imaging probe where its large Stokes shift and sharp emission band would enable multicolor imaging.