In Vitro Reconstruction of Bacterial ß-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of ß-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli (E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented ß-barrel membrane proteins via intact ß-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled ß-barrel membrane proteins by demonstrating four representative ß-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

Age-Associated Reduction of Sinus Macrophages in Human Mesenteric Lymph Nodes.

There are numerous macrophages and dendritic cells in lymph nodes (LNs). Recent studies have highlighted that sinus macrophages (SMs) in LNs possess antigen-presenting capabilities and are related to anti-cancer immune responses. In this study, we assessed the distribution of SMs in mesenteric LNs removed during surgery for colorectal cancer. A marked reduction of SMs was noted in elderly patients, particularly those over 80 years old. We observed a disappearance of CD169-positive cells in LNs where SMs were reduced. In silico analysis of publicly available single-cell RNA sequencing data from LNs revealed that CD169-positive macrophages express numerous genes associated with antigen presentation and lymphocyte proliferation, similar to dendritic cells' functions. In conclusion, our study demonstrates that SMs, potentially crucial for immune activation, diminish in the LNs of elderly patients. This reduction of SMs may contribute to the immune dysfunction observed in the elderly.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'ß-signal' imprinted in the final ß-strand of the OMP engages the ß-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the ß-signal are repeated in other, internal ß-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the ß-signal, arranging several ß-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Identification of genes involved in enhanced membrane vesicle formation in Pseudomonas aeruginosa biofilms: surface sensing facilitates vesiculation.

Membrane vesicles (MVs) are small spherical structures (20-400 nm) produced by most bacteria and have important biological functions including toxin delivery, signal transfer, biofilm formation, and immunomodulation of the host. Although MV formation is enhanced in biofilms of a wide range of bacterial species, the underlying mechanisms are not fully understood. An opportunistic pathogen, Pseudomonas aeruginosa, causes chronic infections that can be difficult to treat due to biofilm formation. Since MVs are abundant in biofilms, can transport virulence factors to the host, and have inflammation-inducing functions, the mechanisms of enhanced MV formation in biofilms needs to be elucidated to effectively treat infections. In this study, we evaluated the characteristics of MVs in P. aeruginosa PAO1 biofilms, and identified factors that contribute to enhanced MV formation. Vesiculation was significantly enhanced in the static culture; MVs were connected to filamentous substances in the biofilm, and separation between the outer and inner membranes and curvature of the membrane were observed in biofilm cells. By screening a transposon mutant library (8,023 mutants) for alterations in MV formation in biofilms, 66 mutants were identified as low-vesiculation strains (2/3 decrease relative to wild type), whereas no mutant was obtained that produced more MVs (twofold increase). Some transposons were inserted into genes related to biofilm formation, including flagellar motility (flg, fli, and mot) and extracellular polysaccharide synthesis (psl). ΔpelAΔpslA, which does not synthesize the extracellular polysaccharides Pel and Psl, showed reduced MV production in biofilms but not in planktonic conditions, suggesting that enhanced vesiculation is closely related to the synthesis of biofilm matrices in P. aeruginosa. Additionally, we found that blebbing occurred during bacterial attachment. Our findings indicate that biofilm-related factors are closely involved in enhanced MV formation in biofilms and that surface sensing facilitates vesiculation. Furthermore, this work expands the understanding of the infection strategy in P. aeruginosa biofilms.

Expression of macrophage/dendritic cell-related molecules in lymph node sinus macrophages.

The role of sinus macrophages (SMs) in anticancer immune responses has received considerable interest in recent years, but the types of molecules that are expressed in human SMs have not yet been clarified in detail. We therefore sought to identify dendritic cell (DC)- or macrophage-related molecules in SMs in human lymph nodes (LNs). SMs are strongly positive for Iba-1, CD163, CD169, and CD209. CD169 (clone SP216) reacted with almost all SMs, mainly in the cell surface membrane, while CD169 (clone HSn 7D2) reacted with a subpopulation of SMs, mainly in the cytoplasm, with a significant increase observed after IFN-α stimulation. The immunoreactivity of clone HSn 7D2 was markedly reduced after transfection with small interfering RNA against CD169, while that of clone SP216 was slightly reduced. The induction of CCL8 and CXCL10 messenger RNA (mRNA) expression by IFN-α was confirmed using cultured macrophages and RT-qPCR, but fluorescence in situ hybridization did not detect CCL8 and CXCL10 mRNA expression in SMs. Single-cell RNA sequence data of LNs indicated that the highest level of CXCL10 gene expression occurred in monocytes. In conclusion, we found that CD209, also known as DC-related molecule, was expressed in human SMs. The heterogeneity observed in CD169 reacted with cone HSn 7D2 and SP216 was potentially due to the modification of CD169 protein by IFN stimulation. Further, no expression of CXCL10 mRNA in SMs suggested that SMs might be resident macrophages.

Molecular characterization of SrSTP14, a sugar transporter from thermogenic skunk cabbage, and its possible role in developing pollen.

In floral thermogenesis, sugars play an important role not only as energy providers but also as growth and development facilitators. Yet, the mechanisms underlying sugar translocation and transport in thermogenic plants remain to be studied. Asian skunk cabbage (Symplocarpus renifolius) is a species that can produce durable and intense heat in its reproductive organ, the spadix. Significant morphological and developmental changes in the stamen are well-characterized in this plant. In this study, we focused on the sugar transporters (STPs), SrSTP1 and SrSTP14, whose genes were identified by RNA-seq as the upregulated STPs during thermogenesis. Real-time PCR confirmed that mRNA expression of both STP genes was increased from the pre-thermogenic to the thermogenic stage in the spadix, where it is predominantly expressed in the stamen. SrSTP1 and SrSTP14 complemented the growth defects of a hexose transporter-deficient yeast strain, EBY4000, on media containing 0.02, 0.2, and 2% (w/v) glucose and galactose. Using a recently developed transient expression system in skunk cabbage leaf protoplasts, we revealed that SrSTP1 and SrSTP14-GFP fusion proteins were mainly localized to the plasma membrane. To dig further into the functional analysis of SrSTPs, tissue-specific localization of SrSTPs was investigated by in situ hybridization. Using probes for SrSTP14, mRNA expression was observed in the microspores within the developing anther at the thermogenic female stage. These results indicate that SrSTP1 and SrSTP14 transport hexoses (e.g., glucose and galactose) at the plasma membrane and suggest that SrSTP14 may play a role in pollen development through the uptake of hexoses into pollen precursor cells.

Categorization of Escherichia coli outer membrane proteins by dependence on accessory proteins of the ß-barrel assembly machinery complex.

The outer membrane (OM) of gram-negative bacteria is populated by various outer membrane proteins (OMPs) that fold into a unique ß-barrel transmembrane domain. Most OMPs are assembled into the OM by the ß-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is composed of two essential proteins (BamA and BamD) and three nonessential accessory proteins (BamB, BamC, and BamE). The currently proposed molecular mechanisms of the BAM complex involve only essential subunits, with the function of the accessory proteins remaining largely unknown. Here, we compared the accessory protein requirements for the assembly of seven different OMPs, 8- to 22-stranded, by our in vitro reconstitution assay using an E. coli mid-density membrane. BamE was responsible for the full efficiency of the assembly of all tested OMPs, as it enhanced the stability of essential subunit binding. BamB increased the assembly efficiency of more than 16-stranded OMPs, whereas BamC was not required for the assembly of any tested OMPs. Our categorization of the requirements of BAM complex accessory proteins in the assembly of substrate OMPs enables us to identify potential targets for the development of new antibiotics.

Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila.

Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3-only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. Here, we identify the first Drosophila BH3-only protein and name it sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonara-mediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. To our knowledge, this is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.

GM-CSF derived from the inflammatory microenvironment potentially enhanced PD-L1 expression on tumor-associated macrophages in human breast cancer.

Ever since immune checkpoint inhibitors have been approved for anti-cancer therapy in several cancers, including triple-negative breast cancer, the significance of programmed death-1 ligand 1 (PD-L1) expression in the tumor immune microenvironment has been a topic of interest. In the present study, we investigated the detailed mechanisms of PD-L1 overexpression on tumor-associated macrophages (TAMs) in breast cancer. In in vitro culture studies using human monocyte-derived macrophages, lymphocytes, and breast cancer cell lines, PD-L1 overexpression on macrophages was induced by the conditioned medium (CM) of activated lymphocytes, but not that of cancer cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from activated lymphocytes was found to be involved in PD-L1 overexpression, in addition to interferon (IFN)-γ, via STAT3 pathway activation. Macrophages suppressed lymphocyte activation, and this inhibition was impaired by PD-1 blocking. The CM of activated lymphocytes also induced the overexpression of PD-L2, but GM-CSF did not affect PD-L2 expression. In the murine E0771 breast cancer model, anti-GM-CSF therapy did not affect PD-L1 expression on TAMs, and the mechanisms of PD-L1 expression on TAMs might differ between humans and mice. However, not only PD-L1, but also PD-L2 was overexpressed on TAMs in the E0771 tumor model, and their expression levels were significantly lower in the tumors in nude mice than in wild-type mice. Anti-PD-L1 antibody and anti-PD-L2 antibody synergistically inhibited E0771 tumor development. In conclusion, PD-L1 and PD-L2 were overexpressed on TAMs, and they potentially contributed to immunosuppression. The GM-CSF-STAT3 pathway is thought to represent a new mechanism of PD-L1 overexpression on TAMs in human breast cancer microenvironment.

Soluble Factors Involved in Cancer Cell-Macrophage Interaction Promote Breast Cancer Growth.

BACKGROUND/AIM: Recent studies have indicated the clinical significance of tumor-associated macrophages (TAMs) in breast cancer; however, the detailed mechanisms of cell-cell interactions between TAMs and cancer cells remain unclear. MATERIALS AND METHODS: In vitro cell culture studies using human monocyte-derived macrophages and breast cancer cell lines were performed to test which cytokines would be involved in cell-cell interactions between cancer cells and macrophages. In addition, studies using human resected samples and animal breast cancer models were performed to examine the significance of TAMs in cancer development. RESULTS: Osteopontin, HB-EGF, and IL-6 were suggested to be macrophage-derived growth factors for breast cancer cells. FROUNT inhibitor significantly blocked TAM infiltration and subcutaneous tumor growth in an E0771 mouse breast cancer model. CONCLUSION: TAMs express growth factors, such as osteopontin, for cancer cells, and targeting of TAM infiltration might be a promising approach for anti-breast cancer therapy.

Substrate-dependent arrangements of the subunits of the BAM complex determined by neutron reflectometry.

In Gram-negative bacteria, the ß-barrel assembly machinery (BAM) complex catalyses the assembly of ß-barrel proteins into the outer membrane, and is composed of five subunits: BamA, BamB, BamC, BamD and BamE. Once assembled, - ß-barrel proteins can be involved in various functions including uptake of nutrients, export of toxins and mediating host-pathogen interactions, but the precise mechanism by which these ubiquitous and often essential ß-barrel proteins are assembled is yet to be established. In order to determine the relative positions of BAM subunits in the membrane environment we reconstituted each subunit into a biomimetic membrane, characterizing their interaction and structural changes by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and neutron reflectometry. Our results suggested that the binding of BamE, or a BamDE dimer, to BamA induced conformational changes in the polypeptide transported-associated (POTRA) domains of BamA, but that BamB or BamD alone did not promote any such changes. As monitored by neutron reflectometry, addition of an unfolded substrate protein extended the length of POTRA domains further away from the membrane interface as part of the mechanism whereby the substrate protein was folded into the membrane.

Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane.

In Gram-negative bacteria, the multi-protein ß-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling ß-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl ß-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-l-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.

Multiple heterochronic gastrointestinal stromal tumors in the stomach detected 6 years after resection: a case report.

BACKGROUND: To date, only a few cases of multiple GISTs with different clones in different organs have been published. However, a case of multiple GISTs with different clones occurring in a single organ has never been reported. CASE PRESENTATION: A 41-year-old patient underwent laparoscopic partial gastrectomy for gastric gastrointestinal stromal tumor (GIST) in 2012. The pathological findings showed high-risk characteristics for recurrence, so he received adjuvant therapy with imatinib for 3 years. In 2018, 3 years after completing the adjuvant therapy, tumor lesions at residual gastric cardia were incidentally identified by follow-up computed tomography (CT). The pathological findings of the tumor biopsy revealed gastric GIST. He underwent secondary laparoscopic partial gastrectomy and was diagnosed with high-risk GIST. Adjuvant therapy with imatinib was restarted immediately. The two gastric GISTs had the same exon 11 mutations in the c-kit gene, but they had different missense mutations. This molecular heterogeneity suggested that they were derived from different origins. CONCLUSION: We reported a multiple heterochronic GIST in the stomach detected 6 years after resection. There may be a possibility that another heterochronic GIST will occur in the remnant stomach in the future, so close follow-up will be needed.

Maf expression in human macrophages and lymph node sinus macrophages in patients with esophageal cancer.

The large Maf transcription factors are expressed in immune cells including macrophages and lymphocytes. To investigate the distribution of Maf expression in human organs, immunostaining for Maf was performed using sections of several human organs. High Maf expression was seen in the nucleus of macrophages in the gastrointestinal tract and lymph node sinus macrophages (LySMs). Then, we assessed whether Maf expression in LySMs was correlated with CD169 expression and the clinical prognosis in patients with esophageal cancer. Maf expression was associated with CD169 expression, but Maf expression in LySMs was not associated with the clinical course in patients with esophageal cancer. We determined which cytokines stimulate Maf expression using cultured macrophages. Immunocytochemistry showed that Maf expression was significantly elevated by interferon-γ. These results are the first report of Maf expression in human samples. Maf expression may be a marker for the macrophage population in humans.

Structure of the mitochondrial import gate reveals distinct preprotein paths.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 ß-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1-3. Each Tom40 ß-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

Porin Associates with Tom22 to Regulate the Mitochondrial Protein Gate Assembly.

Mitochondria import nearly all of their resident proteins from the cytosol, and the TOM complex functions as their entry gate. The TOM complex undergoes a dynamic conversion between the majority population of a three-channel gateway ("trimer") and the minor population that lacks Tom22 and has only two Tom40 channels ("dimer"). Here, we found that the porin Por1 acts as a sink to bind newly imported Tom22. This Por1 association thereby modulates Tom22 integration into the TOM complex, guaranteeing formation of the functional trimeric TOM complex. Por1 sequestration of Tom22 dissociated from the trimeric TOM complex also enhances the dimeric TOM complex, which is preferable for the import of TIM40/MIA-dependent proteins into mitochondria. Furthermore, Por1 appears to contribute to cell-cycle-dependent variation of the functional trimeric TOM complex by chaperoning monomeric Tom22, which arises from the cell-cycle-controlled variation of phosphorylated Tom6.

High CD169 expression in lymph node macrophages predicts a favorable clinical course in patients with esophageal cancer.

Recent findings indicate CD169-positive lymph node sinus macrophages (LySMs) in the regional lymph nodes (RLNs) play an important role in anti-cancer immunity. In the present study, we investigated the correlation between CD169 expression in RLNs and clinicopathologic factors. Higher CD169 expression in LySMs was significantly associated with longer cancer-specific survival (CSS). The density of tumor-infiltrating lymphocytes (TILs) in the cancer nest and CD169 expression on LySMs were positively associated in patients who underwent pretreatment. As CD169 expression is thought to reflect a high interferon signature in RLNs, we tried to identify immunity-related genes that are up-regulated by interferon in macrophages as well as CD169. Indoleamine 2,3-dioxygenase (IDO1) was found to be elevated by interferon, and expression of IDO1 was tested using immunohistochemistry. IDO1 expression on LySMs was positively correlated with CD169 expression; however, there was no significant correlation between IDO1 and clinicopathologic factors. These results suggest that high expression of CD169 in LySMs reflects a high potential for anti-cancer immune responses in esophageal cancer patients and that monitoring CD169 expression would be useful for evaluating the potential of anti-cancer immune reactions.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

The ß-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.

Natural compounds that regulate lymph node sinus macrophages: Inducing an anti-tumor effect by regulating macrophage activation.

Recent progress in anti-tumor therapy has revealed the significance of anti-tumor immune responses in tumor progression and clinical course in several kinds of malignant tumors. The draining lymph node is an important immune system component that contains a number of antigen-presenting cells, which induce rapid immune responses to foreign antigens. Current studies have shown that higher expression of CD169 on lymph node sinus macrophages is associated with the induction of anti-tumor immunity. In the present study, we searched for natural compounds that regulate the CD169-positive phenotype in macrophages to identify potential new anti-cancer agents targeting macrophage activation. Among 50 natural compounds, aculeatiside A, naringin, and onionin A significantly induced the CD169-positive phenotype in human monocyte-derived macrophages. These compounds also induced CD169 overexpression and secretion of inflammatory cytokines, including interleukin (IL)-1ß and IL-12, in murine macrophages. Subcutaneous injection of aculeatiside A and naringin enhanced mRNA expression of IL-1ß, IL12, and CD169 in regional lymph nodes in mice. These findings suggest aculeatiside A and naringin may enhance anti-tumor immune responses by inducing CD169-positive macrophages in lymph nodes.

CD169-positive sinus macrophages in the lymph nodes determine bladder cancer prognosis.

CD169+ macrophages are suggested to play a pivotal role in establishing anti-tumor immunity. They capture dead tumor cell-associated antigens and transfer their information to lymphocsytes, including CD8+ T cells, which is important for successful tumor suppression. This study aimed to determine the prognostic significance of CD169+ macrophages residing in the tumor-draining lymph nodes from cases of bladder cancer. In this retrospective study, 44 bladder cancer patients who received radical cystectomy were examined. The abundance of CD169+ macrophages in the regional lymph nodes and the number of CD8+ T cells in the primary tumor were investigated by immunohistochemistry. A CD169 score was calculated based on the intensity of CD169 staining and the proportion of CD169+ macrophages, and the scores were compared to the patients' clinicopathological parameters. A high CD169 score was significantly associated with low T stage and with a high number of CD8+ T cells infiltrating into the tumor. The group with high CD169 expression had significantly longer cancer-specific survival than the group with low CD169 expression (5-year cancer-specific survival rate: 83.3% vs 31.3%). In a multivariate analysis, the CD169 score was identified as a strong and independent favorable prognostic factor for cancer-specific survival. Our findings suggest that CD169+ macrophages in the lymph nodes enhance anti-tumor immunity by expanding CD8+ T cells in bladder cancer. The CD169 score may serve as a novel marker for the evaluation of bladder cancer prognosis.