Production of a Functional Factor, p40, by Lactobacillus rhamnosus GG Is Promoted by Intestinal Epithelial Cell-Secreted Extracellular Vesicles.

The symbiotic relationship between the gut microbiome and the host provides a nutrient-rich environment for gut microbes and has beneficial effects on host health. Although the composition of the gut microbiome is known to be influenced by both host genetics and environmental factors, host effects on the activities and functions of the gut microbial communities remain poorly understood. Intestinal epithelial cells exert front-line responses to gut microbes and contribute to maintaining a healthy intestinal homeostasis. Here, seeking to elucidate whether intestinal epithelial cells modulate Lactobacillus rhamnosus GG (LGG) functions, we examined the production of p40, an LGG-derived secretory protein that protects intestinal epithelial cells against inflammation. We found that growth medium conditioned with colonic epithelial cell-derived components promotes p40 protein synthesis and secretion by LGG and enhances LGG-stimulated protective responses in intestinal epithelial cells. Furthermore, when LGG was cultured with the colonic luminal contents from healthy mice, p40 production was upregulated but was attenuated with luminal contents from mice with intestinal inflammation. Importantly, the colonic epithelial cell-derived components potentiated LGG-produced p40 levels in a mouse model of colitis and enhanced LGG-mediated amelioration of intestinal inflammation in this model. Notably, we found that colonic epithelial cell-secreted extracellular vesicles participate in communicating with LGG and that heat shock protein 90 (HSP90) in these vesicles might mediate the promotion of p40 production. These results reveal a previously unrecognized mechanism by which the anti-inflammatory effect of LGG is reinforced by intestinal epithelial cells and thereby maintains intestinal health.

Tobramycin reduces key virulence determinants in the proteome of Pseudomonas aeruginosa outer membrane vesicles.

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.

Group A Streptococcus Prevents Mast Cell Degranulation to Promote Extracellular Trap Formation.

The resurgence of Group A Streptococcus (GAS) infections in the past two decades has been a rising major public health concern. Due to a large number of GAS infections occurring in the skin, mast cells (MCs), innate immune cells known to localize to the dermis, could play an important role in controlling infection. MCs can exert their antimicrobial activities either early during infection, by degranulation and release of antimicrobial proteases and the cathelicidin-derived antimicrobial peptide LL-37, or by forming antibacterial MC extracellular traps (MCETs) in later stages of infection. We demonstrate that MCs do not directly degranulate in response to GAS, reducing their ability to control bacterial growth in early stages of infection. However, MC granule components are highly cytotoxic to GAS due to the pore-forming activity of LL-37, while MC granule proteases do not significantly affect GAS viability. We therefore confirmed the importance of MCETs by demonstrating their capacity to reduce GAS survival. The data therefore suggests that LL-37 from MC granules become embedded in MCETs, and are the primary effector molecule by which MCs control GAS infection. Our work underscores the importance of a non-traditional immune effector cell, utilizing a non-conventional mechanism, in the defense against an important human pathogen.

Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation.

Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhibition of effector CD4+ T cell responses by M. tuberculosis may contribute to immune evasion. TCR signaling is inhibited by M. tuberculosis cell envelope lipoglycans, such as lipoarabinomannan and lipomannan, but a mechanism for lipoglycans to traffic from M. tuberculosis within infected macrophages to reach T cells is unknown. In these studies, we found that membrane vesicles produced by M. tuberculosis and released from infected macrophages inhibited the activation of CD4+ T cells, as indicated by reduced production of IL-2 and reduced T cell proliferation. Flow cytometry and Western blot demonstrated that lipoglycans from M. tuberculosis-derived bacterial vesicles (BVs) are transferred to T cells, where they inhibit T cell responses. Stimulation of CD4+ T cells in the presence of BVs induced expression of GRAIL, a marker of T cell anergy; upon restimulation, these T cells showed reduced ability to proliferate, confirming a state of T cell anergy. Furthermore, lipoarabinomannan was associated with T cells after their incubation with infected macrophages in vitro and when T cells were isolated from lungs of M. tuberculosis-infected mice, confirming the occurrence of lipoarabinomannan trafficking to T cells in vivo. These studies demonstrate a novel mechanism for the direct regulation of CD4+ T cells by M. tuberculosis lipoglycans conveyed by BVs that are produced by M. tuberculosis and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.

Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type.

Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins.

Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.

Entry of Porphyromonas gingivalis outer membrane vesicles into epithelial cells causes cellular functional impairment.

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

Effect of lipopolysaccharide (LPS) and peptidoglycan (PGN) on human mast cell numbers, cytokine production, and protease composition.

BACKGROUND: Human mast cell (HuMC) maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. It is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor (TLR) ligands, such as lipopolysaccharide (LPS) or peptidoglycan (PGN), influences HuMC biology. RESULTS: Over 6 wks of culture, LPS had minimal effect on HuMC numbers but increased CD117, tryptase and chymase expression. PGN inhibited HuMC development. For mature mast cells, LPS in the presence of rhSCF (10 ng/ml) increased CD117, tryptase, chymase and carboxypeptidase expression, primarily in CD117low HuMC. LPS decreased FcepsilonRI expression and beta-hexosaminidase release; but had no effect on LTC4 and PGD2 production. PGN reduced HuMC numbers; and CD117 and tryptase expression. IL-1beta and IL-6 (in addition to IL-8 and IL-12) were detected in short-term culture supernatants of LPS treated cells, and reproduced the increases in CD117, tryptase, chymase, and carboxypeptidase expression observed in the presence of LPS. Comparative studies with mouse bone marrow-derived mast cells from wild type, but not TLR4 knockout mice, showed increases in mRNA of mouse mast cell chymases MMCP-1, MMCP-2 and MMCP-4. CONCLUSION: PGN inhibits HuMC growth, while LPS exerts its primary effects on mature HuMC by altering cytokine production and protease composition, particularly at low concentrations of SCF. These data demonstrate the ability of bacterial products to alter HuMC mediator production, granular content, and number which may be particularly relevant at mucosal sites where HuMC are exposed to these products.

Exosomes secreted by bacterially infected macrophages are proinflammatory.

Exosomes are small vesicles that are secreted from cells. They are derived from multivesicular endosomes that fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular environment. Exosomes from antigen-presenting cells contain a range of immunostimulatory molecules that activate T cells, which suggests that they may have an important role in the propagation of immune responses. Of considerable interest is the finding that exosomes derived from bacterially infected macrophages carry bacterial coat components and use these to stimulate bystander macrophages and neutrophils to secrete proinflammatory mediators, including tumor necrosis factor-alpha, the chemokine regulated upon activation, normal T cell-expressed and -secreted (RANTES, also known as CCL5), and inducible nitric oxide synthase. Here, we address these studies in relation to other findings on dendritic cell-derived exosomes that are also powerful immunoregulators.

RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics.

Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER-Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner.

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.

Involvement of VAMP-2 in exocytosis of IL-1 beta in turbot (Scophthalmus maximus) leukocytes after Vibrio anguillarum infection.

Vibrio anguillarum is a major pathogen threatening the fish aquaculture in China. Infection of cultivated turbot (Scophthalmus maximus) with V. anguillarum induced rapid synthesis and secretion of IL-1beta, which initiates the innate immune response. SNARE proteins are known to regulate vesicular trafficking and fusion in all eukaryotes. Here, we determined whether SNARE proteins, specifically vesicle-associated membrane protein-2 (VAMP-2), are involved in regulated exocytosis of IL-1beta of leukocytes in marine fish. We show that VAMP-2 is present in turbot blood leukocytes, with nucleotide sequence identity of 88.2% and 93.0% to those of zebra fish and sea bass, respectively. After Vibrio infection, turbot leukocyte VAMP-2 was increased at the levels of transcription and translation in a temporal pattern coinciding with leukocyte IL-1beta secretion. Confocal microscopy localized VAMP-2 to vesicle structures in leukocytes. Taken together, our results suggest that VAMP-2 is involved in regulated exocytosis of cytokines in immunocytes in fish.

C57BL/6 mice lacking Muc1 show no ocular surface phenotype.

PURPOSE: To test the hypothesis that a membrane-spanning mucin, Muc1, facilitates the spread of tear film and protects against bacterial adherence. METHODS: Age-matched, Muc1 null mice and wild-type mice of C57BL/6 genetic background were used for comparison. Eyes were examined by slit lamp biomicroscopy with fluorescein solution to assess epithelial damage and tear film stability. Structure of the ocular surface epithelia was examined by light microscopy, scanning and transmission electron microscopy, and wholemount confocal microscopy. Bacterial adherence assay was performed on in vivo corneas with Pseudomonas aeruginosa containing a plasmid encoding green fluorescent protein, followed by wholemount confocal microscopy. Real-time reverse transcription-polymerase chain reaction was performed using Muc4-specific primers to quantitate Muc4 mRNA expression in ocular surface tissues. RESULTS: No differences were found between Muc1 null and control mice in any parameter tested. Ocular surface epithelia of Muc1 null mice of the C57BL/6 strain had a normal appearance of surface microplicae, a well-developed glycocalyx on the apical cell membrane, and a normal appearance of goblet cell mucin packets. There was no convincing evidence that bacterial adherence on the cornea was increased in Muc1 null mice. Muc4 mRNA expression was not upregulated in Muc1 null mice compared with control. No ocular surface infections were observed in Muc1 null mice of the C57BL/6 strain (n = 204), which were housed in the animal facility over a period of 26 months. CONCLUSIONS: Muc1 null mice of C57BL/6 background appeared normal in all respects tested. These data differ from the reported phenotype in the mice of the C57BL/6 x SVJ129 background, which show development of blepharitis and conjunctivitis.