Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling.

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.

Effect of Iron Limitation, Elevated Temperature, and Florfenicol on the Proteome and Vesiculation of the Fish Pathogen Aeromonas salmonicida.

We analyzed the proteomic response of the Gram-negative fish pathogen A. salmonicida to iron limitation, an elevated incubation temperature, and the antibiotic florfenicol. Proteins from different subcellular fractions (cytosol, inner membrane, outer membrane, extracellular and outer membrane vesicles) were enriched and analyzed. We identified several iron-regulated proteins that were not reported in the literature for A. salmonicida before. We could also show that hemolysin, an oxidative-stress-resistance chaperone, a putative hemin receptor, an M36 peptidase, and an uncharacterized protein were significantly higher in abundance not only under iron limitation but also with an elevated incubation temperature. This may indicate that these proteins involved in the infection process of A. salmonicida are induced by both factors. The analysis of the outer membrane vesicles (OMVs) with and without applied stresses revealed significant differences in the proteomes. OMVs were smaller and contained more cytoplasmic proteins after antibiotic treatment. After cultivation with low iron availability, several iron-regulated proteins were found in the OMVs, indicating that A. salmonicida OMVs potentially have a function in iron acquisition, as reported for other bacteria. The presence of iron-regulated transporters further indicates that OMVs obtained from 'stressed' bacteria might be suitable vaccine candidates that induce a protective anti-virulence immune response.

Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence.

Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.

Yields and Immunomodulatory Effects of Pneumococcal Membrane Vesicles Differ with the Bacterial Growth Phase.

Streptococcus pneumoniae infections are a leading cause of death worldwide. Bacterial membrane vesicles (MVs) are promising vaccine candidates because of the antigenic components of their parent microorganisms. Pneumococcal MVs exhibit low toxicity towards several cell lines, but their clinical translation requires a high yield and strong immunogenic effects without compromising immune cell viability. MVs are isolated during either the stationary phase (24 h) or death phase (48 h), and their yields, immunogenicity and cytotoxicity in human primary macrophages and dendritic cells have been investigated. Death-phase vesicles showed higher yields than stationary-phase vesicles. Both vesicle types displayed acceptable compatibility with primary immune cells and several cell lines. Both vesicle types showed comparable uptake and enhanced release of the inflammatory cytokines, tumor necrosis factor and interleukin-6, from human primary immune cells. Proteomic analysis revealed similarities in vesicular immunogenic proteins such as pneumolysin, pneumococcal surface protein A, and IgA1 protease in both vesicle types, but stationary-phase MVs showed significantly lower autolysin levels than death-phase MVs. Although death-phase vesicles produced higher yields, they lacked superiority to stationary-phase vesicles as vaccine candidates owing to their similar antigenic protein cargo and comparable uptake into primary human immune cells.

Peptide-based quorum sensing systems in Paenibacillus polymyxa.

Paenibacillus polymyxa is an agriculturally important plant growth-promoting rhizobacterium. Many Paenibacillus species are known to be engaged in complex bacteria-bacteria and bacteria-host interactions, which in other species were shown to necessitate quorum sensing communication. However, to date, no quorum sensing systems have been described in Paenibacillus Here, we show that the type strain P. polymyxa ATCC 842 encodes at least 16 peptide-based communication systems. Each of these systems is comprised of a pro-peptide that is secreted to the growth medium and processed to generate a mature short peptide. Each peptide has a cognate intracellular receptor of the RRNPP family, and we show that external addition of P. polymyxa communication peptides leads to reprogramming of the transcriptional response. We found that these quorum sensing systems are conserved across hundreds of species belonging to the Paenibacillaceae family, with some species encoding more than 25 different peptide-receptor pairs, representing a record number of quorum sensing systems encoded in a single genome.

Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1 immunopathogenesis through modulation of the cytokine release and active inhibition of immune responses.

The immunosuppressive domain of the transmembrane envelope protein gp41 of HIV-1 binds to human monocytes and B cells.

The induction of the acquired immunodeficiency syndrome by the human immunodeficiency virus-1 (HIV-1) is a complex process which is not yet understood in full detail. Still open is the question whether the highly conserved so-called immunosuppressive (Isu) domain in the transmembrane envelope (TM) protein gp41 of HIV-1 is actively participating in immunopathogenesis. Inactivated virus particles, recombinant gp41 and peptides corresponding to the Isu domain have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. Here we demonstrate, using fluorescence-activated cell sorting and competition experiments, that homopolymers of the Isu peptide of HIV-1 are binding specifically to human peripheral blood mononuclear cells, mainly to monocytes and B cells. These data suggest that a putative receptor might be involved in the immunomodulatory effects observed previously.