Legionella eukaryotic-like type IV substrates interfere with organelle trafficking.

Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes (leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.

The two-component regulatory system senX3-regX3 regulates phosphate-dependent gene expression in Mycobacterium smegmatis.

Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (P(i)) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in P(i) acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a P(i)-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon P(i) availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3 approximately P) as a transcriptional activator of phoA. Furthermore, purified RegX3 approximately P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3 approximately P. Based upon our findings, we propose a model for P(i)-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.

Local delivery of recombinant vaccinia virus expressing secondary lymphoid chemokine (SLC) results in a CD4 T-cell dependent antitumor response.

Secondary lymphoid chemokine (SLC) attracts mature dendritic cells (DCs) and naïve T cells. Co-localization of these cells within local tumor environments may enhance the induction of tumor-specific T cells. However, the presence of danger signals or other DC maturation signals are required to optimize T-cell priming. We hypothesized that expression of SLC in vaccinia virus would provide local chemokine delivery and adjuvant factors. A recombinant vaccinia virus expressing murine SLC (rVmSLC) was constructed and characterized. SLC expression was confirmed by Western blot analysis and functional activity was determined by in vitro chemotaxis assay. Supernatants from rVmSLC-infected cells attracted CD4 T cells, and also induced the migration of CD8 T cells and DCs. Although poxviruses are known to express several chemokine-binding proteins, systemic injection of rVmSLC was well tolerated in mice up to a dose of 1 x 10(7) pfu and did not significantly alter vaccinia-specific T-cell immunity. Local injection of rVmSLC into established tumors derived from the murine colon cancer line, CT26, resulted in enhanced infiltration of CD4 T cells, which correlated with inhibition of tumor growth. The central role of CD4 T cells was further demonstrated by loss of anti-tumor activity in CD4 T-cell depleted mice. Intratumoral delivery of SLC using a poxviral vaccine extends the use of SLC in anti-tumor therapies and may present an effective alternative for improving the immunotherapy of cancer alone or in combination with other anti-tumor agents for clinical therapy.