Formation of Amyloid-Like Conformational States of ß-Structured Membrane Proteins on the Example of OMPF Porin from the Yersinia pseudotuberculosis Outer Membrane.

The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with ß-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total ß-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to ß-barrel.

Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore.

Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis porin A (MspA) nanopore with the N91H substitution, which enables direct identification of all 20 proteinogenic amino acids when combined with a machine-learning algorithm. The validation accuracy reaches 99.1%, with 30.9% signal recovery. The feasibility of ultrasensitive quantification of amino acids was also demonstrated at the nanomolar range. Furthermore, the capability of this system for real-time analyses of two representative post-translational modifications (PTMs), one unnatural amino acid and ten synthetic peptides using exopeptidases, including clinically relevant peptides associated with Alzheimer's disease and cancer neoantigens, was demonstrated. Notably, our strategy successfully distinguishes peptides with only one amino acid difference from the hydrolysate and provides the possibility to infer the peptide sequence.

Solute Transport through Mitochondrial Porins In Vitro and In Vivo.

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article.

Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein.

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity. In this study, we report visible light control over the cytolytic activity of a protein. A water-soluble visible-light-operated tetra-ortho-fluoro-azobenzene photoswitch was synthesized by utilizing the nucleophilic aromatic substitution reaction for installing a solubilizing sulfonate group onto the electron-poor photoswitch structure. The azobenzene was attached to two cysteine mutants of the pore-forming protein fragaceatoxin C (FraC), and their respective activities were evaluated on red blood cells. For both mutants, the green-light-irradiated sample, containing predominantly the cis-azobenzene isomer, was more active compared to the blue-light-irradiated sample. Ultimately, the same modulation of the cytolytic activity pattern was observed toward a hypopharyngeal squamous cell carcinoma. These results constitute the first case of using low energy visible light to control the biological activity of a toxic protein.

Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore.

Natural proteins are composed of 20 proteinogenic amino acids and their post-translational modifications (PTMs). However, due to the lack of a suitable nanopore sensor that can simultaneously discriminate between all 20 amino acids and their PTMs, direct sequencing of protein with nanopores has not yet been realized. Here, we present an engineered hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole Ni2+ modification. It enables full discrimination of all 20 proteinogenic amino acids and 4 representative modified amino acids, Nω,N'ω-dimethyl-arginine (Me-R), O-acetyl-threonine (Ac-T), N4-(ß-N-acetyl-D-glucosaminyl)-asparagine (GlcNAc-N) and O-phosphoserine (P-S). Assisted by machine learning, an accuracy of 98.6% was achieved. Amino acid supplement tablets and peptidase-digested amino acids from peptides were also analyzed using this strategy. This capacity for simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs suggests the potential to achieve protein sequencing using this nanopore-based strategy.

Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex ßγ-CAT evolved to drive cell vesicular delivery and transport.

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. ßγ-CAT, a complex of pore-forming protein BmALP1 (two ßγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the ßγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for ßγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the ßγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of ßγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

Pore-Forming Proteins: From Pore Assembly to Structure by Quantitative Single-Molecule Imaging.

Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is their ability to form pores that disrupt the membrane permeability barrier and ion homeostasis and generally induce cell death. Some PFPs are part of the genetically encoded machinery of eukaryotic cells that are activated against infection by pathogens or in physiological programs to carry out regulated cell death. PFPs organize into supramolecular transmembrane complexes that perforate membranes through a multistep process involving membrane insertion, protein oligomerization, and finally pore formation. However, the exact mechanism of pore formation varies from PFP to PFP, resulting in different pore structures with different functionalities. Here, we review recent insights into the molecular mechanisms by which PFPs permeabilize membranes and recent methodological advances in their characterization in artificial and cellular membranes. In particular, we focus on single-molecule imaging techniques as powerful tools to unravel the molecular mechanistic details of pore assembly that are often obscured by ensemble measurements, and to determine pore structure and functionality. Uncovering the mechanistic elements of pore formation is critical for understanding the physiological role of PFPs and developing therapeutic approaches.

The Small RNA NcS25 Regulates Biological Amine-Transporting Outer Membrane Porin BCAL3473 in Burkholderia cenocepacia.

Regulation of porin expression in bacteria is complex and often involves small-RNA regulators. Several small-RNA regulators have been described for Burkholderia cenocepacia, and this study aimed to characterize the biological role of the conserved small RNA NcS25 and its cognate target, outer membrane protein BCAL3473. The B. cenocepacia genome carries a large number of genes encoding porins with yet-uncharacterized functions. Expression of the porin BCAL3473 is strongly repressed by NcS25 and activated by other factors, such as a LysR-type regulator and nitrogen-depleted growth conditions. The porin is involved in transport of arginine, tyrosine, tyramine, and putrescine across the outer membrane. Porin BCAL3473, with NcS25 as a major regulator, plays an important role in the nitrogen metabolism of B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is a Gram-negative bacterium which causes infections in immunocompromised individuals and in people with cystic fibrosis. A low outer membrane permeability is one of the factors giving it a high level of innate resistance to antibiotics. Porins provide selective permeability for nutrients, and antibiotics can also traverse the outer membrane by this means. Knowing the properties and specificities of porin channels is therefore important for understanding resistance mechanisms and for developing new antibiotics and could help in overcoming permeability issues in antibiotic treatment.

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection.

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.

Conformational Dynamics of Loop L3 in OmpF: Implications toward Antibiotic Translocation and Voltage Gating.

In the present work, we delineate the molecular mechanism of a bulky antibiotic permeating through a bacterial channel and uncover the role of conformational dynamics of the constriction loop in this process. Using the temperature accelerated sliced sampling approach, we shed light onto the dynamics of the L3 loop, in particular the F118 to S125 segment, at the constriction regions of the OmpF porin. We complement the findings with single channel electrophysiology experiments and applied-field simulations, and we demonstrate the role of hydrogen-bond stabilization in the conformational dynamics of the L3 loop. A molecular mechanism of permeation is put forward wherein charged antibiotics perturb the network of stabilizing hydrogen-bond interactions and induce conformational changes in the L3 segment, thereby aiding the accommodation and permeation of bulky antibiotic molecules across the constriction region. We complement the findings with single channel electrophysiology experiments and demonstrate the importance of the hydrogen-bond stabilization in the conformational dynamics of the L3 loop. The generality of the present observations and experimental results regarding the L3 dynamics enables us to identify this L3 segment as the source of gating. We propose a mechanism of OmpF gating that is in agreement with previous experimental data that showed the noninfluence of cysteine double mutants that tethered the L3 tip to the barrel wall on the OmpF gating behavior. The presence of similar loop stabilization networks in porins of other clinically relevant pathogens suggests that the conformational dynamics of the constriction loop is possibly of general importance in the context of antibiotic permeation through porins.

Single-molecule approaches reveal outer membrane protein biogenesis dynamics.

Outer membrane proteins (OMPs) maintain the viability of Gram-negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi-protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single-molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis.

Salmonella Typhimurium outer membrane protein A (OmpA) renders protection from nitrosative stress of macrophages by maintaining the stability of bacterial outer membrane.

Bacterial porins are highly conserved outer membrane proteins used in the selective transport of charged molecules across the membrane. In addition to their significant contributions to the pathogenesis of Gram-negative bacteria, their role(s) in salmonellosis remains elusive. In this study, we investigated the role of outer membrane protein A (OmpA), one of the major outer membrane porins of Salmonella, in the pathogenesis of Salmonella Typhimurium (STM). Our study revealed that OmpA plays an important role in the intracellular virulence of Salmonella. An ompA deficient strain of Salmonella (STM ΔompA) showed compromised proliferation in macrophages. We found that the SPI-2 encoded virulence factors such as sifA and ssaV are downregulated in STM ΔompA. The poor colocalization of STM ΔompA with LAMP-1 showed that disruption of SCV facilitated its release into the cytosol of macrophages, where it was assaulted by reactive nitrogen intermediates (RNI). The enhanced recruitment of nitrotyrosine on the cytosolic population of STM ΔompAΔsifA and ΔompAΔssaV compared to STM ΔsifA and ΔssaV showed an additional role of OmpA in protecting the bacteria from host nitrosative stress. Further, we showed that the generation of greater redox burst could be responsible for enhanced sensitivity of STM ΔompA to the nitrosative stress. The expression of several other outer membrane porins such as ompC, ompD, and ompF was upregulated in STM ΔompA. We found that in the absence of ompA, the enhanced expression of ompF increased the outer membrane porosity of Salmonella and made it susceptible to in vitro and in vivo nitrosative stress. Our study illustrates a novel mechanism for the strategic utilization of OmpA by Salmonella to protect itself from the nitrosative stress of macrophages.

Characterization of Treponema denticola Major Surface Protein (Msp) by Deletion Analysis and Advanced Molecular Modeling.

Treponema denticola, a keystone pathogen in periodontitis, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp forms an oligomeric outer membrane complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular processes in host cells. T. denticola msp is an ortholog of the Treponema pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. We recently identified the primary Msp surface-exposed epitope and proposed a model of the Msp protein as a ß-barrel protein similar to Gram-negative bacterial porins. Here, we report fine-scale Msp mutagenesis demonstrating that both the N and C termini as well as the centrally located Msp surface epitope are required for native Msp oligomer expression. Removal of as few as three C-terminal amino acids abrogated Msp detection on the T. denticola cell surface, and deletion of four residues resulted in complete loss of detectable Msp. Substitution of a FLAG tag for either residues 6 to 13 of mature Msp or an 8-residue portion of the central Msp surface epitope resulted in expression of full-length Msp but absence of the oligomer, suggesting roles for both domains in oligomer formation. Consistent with previously reported Msp N-glycosylation, proteinase K treatment of intact cells released a 25 kDa polypeptide containing the Msp surface epitope into culture supernatants. Molecular modeling of Msp using novel metagenome-derived multiple sequence alignment (MSA) algorithms supports the hypothesis that Msp is a large-diameter, trimeric outer membrane porin-like protein whose potential transport substrate remains to be identified. IMPORTANCE The Treponema denticola gene encoding its major surface protein (Msp) is an ortholog of the T. pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. Using a combined strategy of fine-scale mutagenesis and advanced predictive molecular modeling, we characterized the Msp protein and present a high-confidence model of its structure as an oligomer embedded in the outer membrane. This work adds to knowledge of Msp-like proteins in oral treponemes and may contribute to understanding the evolutionary and potential functional relationships between T. denticola Msp and the orthologous T. pallidum Tpr proteins.

Gasdermins: Pore-forming Proteins as a Potential Therapeutic Target.

Gasdermins are novel pore forming proteins that comprise Gasdermin A, Gasdermin B, Gasdermin C, Gasdermin D, Gasdermin E and Pejvakin (DFNB59). Recently, pyroptosis has been redefined as "Gasdermin mediated necrosis", as gasdermins are key regulators of apoptosis, necrosis, and pyroptosis. The discovery of the gasdermin family has broadened the field of pyroptosis studies. Studies have correlated gasdermins with several diseases. This review summarizes the physiological roles and signal transduction of gasdermins. It further highlights the role of gasdermins in pathological conditions like autoimmune disease, kidney diseases, and central nervous system diseases.

Non-binary Encoded Nucleic Acid Barcodes Directly Readable by a Nanopore.

A large collection of unique molecular barcodes is useful in the simultaneous sensing or screening of molecular analytes. Though the sequence of DNA has been widely applied to encode for molecular barcodes, decoding of these barcodes is normally assisted by sequencing. We here demonstrate a barcode system based solely on self-assembly of synthetic nucleic acids and direct nanopore decoding. Each molecular barcode is composed of "n" distinct information nodes in a non-binary manner and can be sequentially scanned and decoded by a Mycobacterium smegmatis porin A (MspA) nanopore. Nanopore events containing step-shaped features were consistently reported. 14 unique information nodes were developed which in principle could encode for 14n unique molecular barcodes in a barcode containing "n" information nodes. These barcode probes were adapted to detect different antibody proteins or cancer-related microRNAs, suggesting their immediate application in a wide variety of sensing applications.

Pharmacological characterization of a structural hybrid P2X7R antagonist using ATP and LL-37.

Antagonists of the P2X7 receptor (P2X7R) have the potential to treat diseases where neuroinflammation is present such as depression, chronic pain and Alzheimer's disease. We recently developed a structural hybrid (C1; 1-((adamantan-1-yl)methyl)-2-cyano-3-(quinolin-5-yl)guanidine) of a purported competitive P2X7R antagonist (C2; 2-cyano-1-((1S)-1-phenylethyl)-3-(quinolin-5-yl)guanidine) and a likely negative allosteric modulator (NAM) of the P2X7R (C3; N-((adamantan-1-yl)methyl)-2-chloro-5-methoxybenzamide). Here we aimed to pharmacologically characterize C1, to gain insights into how select structural components impact antagonist interaction with the P2X7R. A second aim was to examine the role of the peptide LL-37, an apparent activator of the P2X7R, and compare the ability of multiple P2X7R antagonists to block its effects. Compounds 1, 2 and 3 were characterised using washout, Schild and receptor protection studies, all using dye uptake assays in HEK293 cells expressing the P2X7R. LL-37 was examined in the same HEK293 cells and THP-1 monocytes. Compounds 2 and 3 acted as a BzATP-competitive antagonist and NAM of the P2X7R respectively. Compound 1 was a slowly reversible NAM of the P2X7R suggesting the incorporation of an appropriately positioned adamantane promotes binding to the allosteric site of the P2X7R. LL-37 was shown to potentiate the ability of ATP to induce dye uptake at low concentrations (1-3 µg mL-1) or induce dye uptake alone at higher concentrations (10-20 µg mL-1). None of the P2X7R antagonists studied were able to block LL-37-induced dye uptake bringing in to question the ability of current P2X7R antagonists to inhibit the inflammatory action of LL-37 in vivo.

Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function.

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

Major Outer Membrane Protein from Legionella pneumophila Inhibits Phagocytosis but Enhances Chemotaxis of RAW 264.7 Macrophages by Regulating the FOXO1/Coronin-1 Axis.

Legionella pneumophila is an intracellular pathogen that can cause Legionnaire's disease by invading alveolar epithelial cells and macrophages. The major outer membrane protein (MOMP) plays an important role in the interaction between bacteria and host cells. However, the role of MOMP in the process of L. pneumophila invasion of macrophages and its working mechanism remain unknown. We aimed to explore the effects of MOMP on phagocytosis and chemotaxis of RAW 264.7 macrophages. The chemotactic activity, toxicity, and phagocytosis of RAW 264.7 cocultured with different concentrations of MOMP were determined by Transwell, CCK-8, and neutral red uptake assays, respectively. Target genes were detected by double-luciferase and pull down assays. qRT-PCR and Western blot were performed to analyze the expression of several important proteins involved in the immune response pathway, including coronin-1, interleukins (IL-10), forkhead transcription factor 1 (FOXO1), nucleotide-binding oligomerization domain protein (NOD) 1, NOD2, and receptor-interacting protein (RIP) 2. After coculturing with MOMP, cytological observation indicated a decrease of phagocytosis and a marked increase of chemotaxis in RAW 264.7 macrophages. The phagocytosis degree of RAW 264.7 macrophage varied with the concentration gradient of MOMP in a time-dependent manner. MOMP could increase the expression levels of MCP-1, IL-10, NOD2, and RIP2 and decrease the expression levels of FOXO1 and coronin-1 in cell culture supernatants. In addition, we found that FOXO1 could promote its transcription by binding to the promoter of coronin-1. The results of the present study suggested that MOMP could inhibit phagocytosis and facilitate chemotaxis of RAW 264.7 macrophage, which might be associated with the FOXO1/coronin-1 axis.