Heterogeneity and Compositional Diversities of <i>Campylobacter jejuni</i> Outer Membrane Vesicles (OMVs) Drive Multiple Cellular Uptake Processes

Naturally secreted outer membrane vesicles (OMVs) from gut microbes carry diverse cargo, including proteins, nucleic acids, toxins, and many unidentified secretory factors. Bacterial OMVs can shuttle molecules across different cell types as a generalized secretion system, facilitating bacterial pathogenicity and self-survival. Numerous mucosal pathogens, including Campylobacter jejuni (C. jejuni), share a mechanism of harmonized secretion of major virulence factors. Intriguingly, as a common gut pathogen, C. jejuni lacks some classical virulence-associated secretion systems; alternatively, it often employs nanosized lipid-bound OMVs as an intensive strategy to deliver toxins, including secretory proteins, into the target cells. To better understand how the biophysical and compositional attributes of natural OMVs of C. jejuni regulate their cellular interactions to induce a biologically relevant host response, we conducted an in-depth morphological and compositional analysis of naturally secreted OMVs of C. jejuni. Next, we focused on understanding the mechanism of host cell-specific OMVs uptake from the extracellular milieu. We showed that intracellular perfusion of OMVs is mediated by cytosolic as well as multiple endocytic uptake processes due to the heterogenic nature, abundance of surface proteins, and membrane phospholipids acquired from the source bacteria. Furthermore, we used human and avian cells as two different host targets to provide evidence of target cell-specific preferential uptake of OMVs. Together, the present study provides insight into the unique functionality of natural OMVs of C. jejuni at the cellular interface, upholding their potential for multimodal use as prophylactic and therapeutic carriers

Organization and Dynamics of Hippocampal Membranes in a Depth-Dependent Manner: An Electron Spin Resonance Study

Organization and dynamics of neuronal membranes represent crucial determinants for the function of neuronal receptors and signal transduction. Previous work from our laboratory has established hippocampal membranes as a convenient natural source for studying neuronal receptors. In this work, we have monitored the organization and dynamics of hippocampal membranes and their modulation by cholesterol and protein content utilizing location (depth)-specific spin-labeled phospholipids by ESR spectroscopy. The choice of ESR spectroscopy is appropriate due to slow diffusion encountered in crowded environments of neuronal membranes. Analysis of ESR spectra shows that cholesterol increases hippocampal membrane order while membrane proteins increase lipid dynamics resulting in disordered membranes. These results are relevant in understanding the complex organization and dynamics of hippocampal membranes. Our results are significant in the overall context of membrane organization under low cholesterol conditions and could have implications in neuronal diseases characterized by low cholesterol conditions due to defective cholesterol metabolism

Lipidated Lysine and Fatty Acids Assemble into Protocellular Membranes to Assist Regioselective Peptide Formation: Correlation to the Natural Selection of Lysine over Nonproteinogenic Lower Analogues

The self-assembly of prebiotically plausible amphiphiles (fatty acids) to form a bilayer membrane for compartmentalization is an important factor during protocellular evolution. Such fatty acid-based membranes assemble at relatively high concentrations, and they lack robust stability. We have demonstrated that a mixture of lipidated lysine (cationic) and prebiotic fatty acids (decanoic acid, anionic) can form protocellular membranes (amino acid-based membranes) at low concentrations via electrostatic, hydrogen bonding, and hydrophobic interactions. The formation of vesicular membranes was characterized by dynamic light scattering (DLS), pyrene and Nile Red partitioning, cryo-transmission electron microscopy (TEM) images, and glucose encapsulation studies. The lipidated nonproteinogenic analogues of lysine (Lys), such as ornithine (Orn) and 2,4-diaminobutyric acid (Dab), also form membranes with decanoate (DA). Time-dependent turbidimetric and 1H NMR studies suggested that the Lys-based membrane is more stable than the membranes prepared from nonproteinogenic lower analogues. The Lys-based membrane embeds a model acylating agent (aminoacyl-tRNA mimic) and facilitates the colocalization of substrates to support regioselective peptide formation via the α-amine of Lys. These membranes thereby assist peptide formation and control the positioning of the reactants (model acylating agent and −NH2 of amino acids) to initiate biologically relevant reactions during early evolution

Differential scanning calorimetry.

<p>DSC thermograms of C-214-HrpZ_Pss (A) and full length HrpZ_Pss (B). The scan rate was 60^o.h⁻¹. The change in heat capacity (ΔC_p) of the native protein and the denatured state are shown. Deconvolution of DSC thermogram of C-214-HrpZ_Pss (C) and full length HrpZ_Pss (D). The experimentally obtained thermograms are shown as solid lines, individual transitions deduced from deconvolution analysis are shown as dashed lines and the sum of the transitions obtained from deconvolution analysis are shown as dotted lines.</p

Tryptophan exposure and thermal unfolding.

<p>(A) Stern-Volmer plots for the quenching of the intrinsic fluorescence of C-214-HrpZ_Pss with acrylamide (□), iodide ion (○) and cesium ion (▴). Temperature dependence of (B) tryptophan emission maximum, and (C) fluorescence intensity at emission maximum, of C-214-HrpZ_Pss.</p

Circular dichroism spectroscopy.

<p>A) Far-UV CD spectra of C-214-HrpZ_Pss (solid line) and full length HrpZ_Pss (dashed line) at 25°C. The dotted line corresponds to the calculated fit obtained by using the CDSSTR program for C-214-HrpZ_Pss. B) Temperature dependence of α-helical content in C-214-HrpZ_Pss.</p

Conformational stability curves of C-214HrpZ_Pss and HrpZ_Pss.

<p>Solid lines correspond to <i>dimer - unfolded state</i> transition (ΔG_Di-U). Values of ΔG₂ and ΔG₃ for HrpZ_Pss corresponding to transition 2 and 3 were calculated using equations 1 and 2 (see text for more details).</p

Results of CD spectral analysis of C-214-HrpZ_Pss.

<p>The secondary structural components of the full-length HrpZ_Pss<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109871#pone.0109871-Tarafdar1" target="_blank">[15]</a> are given in parentheses.</p><p>Results of CD spectral analysis of C-214-HrpZ_Pss.</p

Thermodynamic parameters for the thermal unfolding of C-214-HrpZ_Pss.

<p>Values given are averages obtained from 3 independent measurements. Values in parentheses correspond to the thermal unfolding of full length HrpZ_Pss<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109871#pone.0109871-Tarafdar1" target="_blank">[15]</a>. *C-214-HrpZ_Pss does not have transition 3.</p><p>Thermodynamic parameters for the thermal unfolding of C-214-HrpZ_Pss.</p

Differential oligomerization of C-214-HrpZ_Pss and full length HrpZ_Pss studied by AFM.

<p>C-214-HrpZ_Pss forms non specific aggregates (<b>A</b>), whereas full length HrpZ_Pss is characterized by the formation of both non-specific and fibrillar aggregates (<b>B</b>). Scale bar: 0.5 µm.</p