Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice.

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global health threat. The only available vaccine Bacille Calmette-Guérin (BCG) does not prevent adult pulmonary TB. New effective TB vaccines should aim to stimulate robust T cell responses in the lung mucosa to achieve high protective efficacy. We have previously developed a novel viral vaccine vector based on recombinant Pichinde virus (PICV), a non-pathogenic arenavirus with low seroprevalence in humans, and have demonstrated its efficacy to induce strong vaccine immunity with undetectable anti-vector neutralization activity. Methods: Using this tri-segmented PICV vector (rP18tri), we have generated viral vectored TB vaccines (TBvac-1, TBvac-2, and TBvac-10) encoding several known TB immunogens (Ag85B, EsxH, and ESAT-6/EsxA). A P2A linker sequence was used to allow for the expression of two proteins from one open-reading-frame (ORF) on the viral RNA segments. The immunogenicity of TBvac-2 and TBvac-10 and the protective efficacy of TBvac-1 and TBvac-2 were evaluated in mice. Results: Both viral vectored vaccines elicited strong antigen-specific CD4 and CD8 T cells through intramuscular (IM) and intranasal (IN) routes as evaluated by MHC-I and MHC-II tetramer analyses, respectively. The IN inoculation route helped to elicit strong lung T cell responses. The vaccine-induced antigen-specific CD4 T cells are functional, expressing multiple cytokines as detected by intracellular cytokine staining. Finally, immunization with TBvac-1 or TBvac-2, both expressing the same trivalent antigens (Ag85B, EsxH, ESAT6/EsxA), reduced Mtb lung tissue burden and dissemination in an aerosol challenge mouse model. Conclusions: The novel PICV vector-based TB vaccine candidates can express more than two antigens via the use of P2A linker sequence and elicit strong systemic and lung T cell immunity with protective efficacy. Our study suggests the PICV vector as an attractive vaccine platform for the development of new and effective TB vaccine candidates.

Microbial Communities Under Distinct Thermal and Geochemical Regimes in Axial and Off-Axis Sediments of Guaymas Basin.

Cold seeps and hydrothermal vents are seafloor habitats fueled by subsurface energy sources. Both habitat types coexist in Guaymas Basin in the Gulf of California, providing an opportunity to compare microbial communities with distinct physiologies adapted to different thermal regimes. Hydrothermally active sites in the southern Guaymas Basin axial valley, and cold seep sites at Octopus Mound, a carbonate mound with abundant methanotrophic cold seep fauna at the Central Seep location on the northern off-axis flanking regions, show consistent geochemical and microbial differences between hot, temperate, cold seep, and background sites. The changing microbial actors include autotrophic and heterotrophic bacterial and archaeal lineages that catalyze sulfur, nitrogen, and methane cycling, organic matter degradation, and hydrocarbon oxidation. Thermal, biogeochemical, and microbiological characteristics of the sampling locations indicate that sediment thermal regime and seep-derived or hydrothermal energy sources structure the microbial communities at the sediment surface.

Characteristics and Evolution of sill-driven off-axis hydrothermalism in Guaymas Basin - the Ringvent site.

The Guaymas Basin spreading center, at 2000 m depth in the Gulf of California, is overlain by a thick sedimentary cover. Across the basin, localized temperature anomalies, with active methane venting and seep fauna exist in response to magma emplacement into sediments. These sites evolve over thousands of years as magma freezes into doleritic sills and the system cools. Although several cool sites resembling cold seeps have been characterized, the hydrothermally active stage of an off-axis site was lacking good examples. Here, we present a multidisciplinary characterization of Ringvent, an ~1 km wide circular mound where hydrothermal activity persists ~28 km northwest of the spreading center. Ringvent provides a new type of intermediate-stage hydrothermal system where off-axis hydrothermal activity has attenuated since its formation, but remains evident in thermal anomalies, hydrothermal biota coexisting with seep fauna, and porewater biogeochemical signatures indicative of hydrothermal circulation. Due to their broad potential distribution, small size and limited life span, such sites are hard to find and characterize, but they provide critical missing links to understand the complex evolution of hydrothermal systems.

Mycobacterium tuberculosis Requires Regulation of ESX-5 Secretion for Virulence in Irgm1-Deficient Mice.

The Mycobacterium tuberculosis type VII secretion system ESX-5, which has been implicated in virulence, is activated at the transcriptional level by the phosphate starvation-responsive Pst/SenX3-RegX3 signal transduction system. Deletion of pstA1, which encodes a Pst phosphate transporter component, causes constitutive activation of the response regulator RegX3, hypersecretion of ESX-5 substrates and attenuation in the mouse infection model. We hypothesized that constitutive activation of ESX-5 secretion causes attenuation of the ΔpstA1 mutant. To test this, we uncoupled ESX-5 from regulation by RegX3. Using electrophoretic mobility shift assays, we defined a RegX3 binding site in the esx-5 locus. Deletion or mutation of the RegX3 binding site reversed hypersecretion of the ESX-5 substrate EsxN by the ΔpstA1 mutant and abrogated induction of EsxN secretion in response to phosphate limitation by wild-type M. tuberculosis The esx-5 RegX3 binding site deletion (ΔBS) also suppressed attenuation of the ΔpstA1 mutant in Irgm1-/- mice. These data suggest that constitutive ESX-5 secretion sensitizes M. tuberculosis to an immune response that still occurs in Irgm1-/- mice. However, the ΔpstA1 ΔBS mutant remained attenuated in both NOS2-/- and C57BL/6 mice, suggesting that factors other than ESX-5 secretion also contribute to attenuation of the ΔpstA1 mutant. In addition, a ΔpstA1 ΔesxN mutant lacking the hypersecreted ESX-5 substrate EsxN remained attenuated in Irgm1-/- mice, suggesting that ESX-5 substrates other than EsxN cause increased susceptibility to host immunity. Our data indicate that while M. tuberculosis requires ESX-5 for virulence, it tightly controls secretion of ESX-5 substrates to avoid elimination by host immune responses.

Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity.

Mycobacterium tuberculosis releases membrane vesicles (MV) that modulate host immune responses and aid in iron acquisition, although they may have additional unappreciated functions. MV production appears to be a regulated process, but virR remains the only characterized genetic regulator of vesiculogenesis. Here, we present data supporting a role for the M. tuberculosis Pst/SenX3-RegX3 signal transduction system in regulating MV production. Deletion of pstA1, which encodes a transmembrane component of the phosphate-specific transport (Pst) system, causes constitutive activation of the SenX3-RegX3 two-component system, leading to increased protein secretion via the specialized ESX-5 type VII secretion system. Using proteomic mass spectrometry, we identified several additional proteins hyper-secreted by the ΔpstA1 mutant, including LpqH, an MV-associated lipoprotein. Nanoparticle tracking analysis revealed a 15-fold increase in MV production by the ΔpstA1 mutant. Both hyper-secretion of LpqH and increased MV release required RegX3 but were independent of VirR, suggesting that Pst/SenX3-RegX3 controls MV release by a novel mechanism. Prior proteomic analysis identified ESX-5 substrates associated with MV. We therefore hypothesized that MV release requires ESX-5 activity. We constructed strains that conditionally express eccD5 , which encodes the predicted ESX-5 transmembrane channel. Upon EccD5 depletion, we observed reduced secretion of the ESX-5 substrates EsxN and PPE41, but MV release was unaffected. Our data suggest that ESX-5 does not affect vesicle production and imply that further characterization of the Pst/SenX3-RegX3 regulon might reveal novel mechanisms of M. tuberculosis vesicle biogenesis.IMPORTANCE In Gram-negative bacteria, MV derived from the outer membrane have diverse functions in bacterial physiology and pathogenesis, and several factors regulating their production have been identified. Though Gram-positive bacteria and mycobacteria that lack an outer membrane also produce vesicles with described roles in pathogenesis, the mechanisms of MV biogenesis in these organisms remain poorly characterized. Defining mechanisms of MV biogenesis might yield significant insights into the importance of MV production during infection. In M. tuberculosis, only a single genetic element, virR, is known to regulate MV production. Our work reveals that the Pst/SenX3-RegX3 signal transduction system is a novel regulator of MV biogenesis that controls MV production by a mechanism that is independent of both VirR and activation of the specialized ESX-5 protein secretion system. Understanding which genes in the RegX3 regulon cause increased MV production might reveal novel molecular mechanisms of MV release.

Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence.

A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the capacity to survive within host macrophages. Although several factors required for this survival have been identified, a comprehensive knowledge of such factors and how they work together to manipulate the host environment to benefit bacterial survival are not well understood. To systematically identify Mtb factors required for intracellular growth, we screened an arrayed, non-redundant Mtb transposon mutant library by high-content imaging to characterize the mutant-macrophage interaction. Based on a combination of imaging features, we identified mutants impaired for intracellular survival. We then characterized the phenotype of infection with each mutant by profiling the induced macrophage cytokine response. Taking a systems-level approach to understanding the biology of identified mutants, we performed a multiparametric analysis combining pathogen and host phenotypes to predict functional relationships between mutants based on clustering. Strikingly, mutants defective in two well-known virulence factors, the ESX-1 protein secretion system and the virulence lipid phthiocerol dimycocerosate (PDIM), clustered together. Building upon the shared phenotype of loss of the macrophage type I interferon (IFN) response to infection, we found that PDIM production and export are required for coordinated secretion of ESX-1-substrates, for phagosomal permeabilization, and for downstream induction of the type I IFN response. Multiparametric clustering also identified two novel genes that are required for PDIM production and induction of the type I IFN response. Thus, multiparametric analysis combining host and pathogen infection phenotypes can be used to identify novel functional relationships between genes that play a role in infection.

Small molecule inhibitors of the Candida albicans budded-to-hyphal transition act through multiple signaling pathways.

The ability of the pathogenic yeast Candida albicans to interconvert between budded and hyphal growth states, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans development and virulence. The BHT is under the control of multiple cell signaling pathways that respond to external stimuli, including nutrient availability, high temperature, and pH. Previous studies identified 21 small molecules that could inhibit the C. albicans BHT in response to carbon limitation in Spider media. However, the studies herein show that the BHT inhibitors had varying efficacies in other hyphal-inducing media, reflecting their varying abilities to block signaling pathways associated with the different media. Chemical epistasis analyses suggest that most, but not all, of the BHT inhibitors were acting through either the Efg1 or Cph1 signaling pathways. Notably, the BHT inhibitor clozapine, a FDA-approved drug used to treat atypical schizophrenia by inhibiting G-protein-coupled dopamine receptors in the brain, and several of its functional analogs were shown to act at the level of the Gpr1 G-protein-coupled receptor. These studies are the first step in determining the target and mechanism of action of these BHT inhibitors, which may have therapeutic anti-fungal utility in the future.