Acid Phosphatases Do Not Contribute to the Pathogenesis of Type A Francisella tularensis▿ †

The intracellular pathogen Francisella tularensis is the causative agent of tularemia, a zoonosis that can affect humans with potentially lethal consequences. Essential to Francisella virulence is its ability to survive and proliferate within phagocytes through phagosomal escape and cytosolic replication. Francisella spp. encode a variety of acid phosphatases, whose roles in phagosomal escape and virulence have been documented yet remain controversial. Here we have examined in the highly virulent (type A) F. tularensis strain Schu S4 the pathogenic roles of three distinct acid phosphatases, AcpA, AcpB, and AcpC, that are most conserved between Francisella subspecies. Neither the deletion of acpA nor the combination of acpA, acpB, and acpC deletions affected the phagosomal escape or cytosolic growth of Schu S4 in murine and human macrophages, despite decreases in acid phosphatase activities by as much as 95%. Furthermore, none of these mutants were affected in their ability to cause lethality in mice upon intranasal inoculation. Hence, the acid phosphatases AcpA, AcpB, and AcpC do not contribute to intracellular pathogenesis and do not play a major role in the virulence of type A Francisella strains

Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella, indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host-pathogen interface to influence the intracellular fate of this pathogen

Construction of a Vero Cell Line Expressing Human ICAM1 for the Development of Rhinovirus Vaccines

Human rhinoviruses (HRVs) are small non-enveloped RNA viruses that belong to the Enterovirus genus within the Picornaviridae family and are known for causing the common cold. Though symptoms are generally mild in healthy individuals, the economic burden associated with HRV infection is significant. A vaccine could prevent disease. The Vero-cell-based viral vaccine platform technology was considered for such vaccine development. Unfortunately, most HRV strains are unable to propagate on Vero cells due to a lack of the major receptor of HRV group A and B, intercellular adhesion molecule (ICAM1, also known as CD54). Therefore, stable human ICAM1 expressing Vero cell clones were generated by transfecting the ICAM1 gene in Vero cells and selecting clones that overexpressed ICAM1 on the cell surface. Cell banks were made and expression of ICAM1 was stable for at least 30 passages. The Vero_ICAM1 cells and parental Vero cells were infected with four HRV prototypes, B14, A16, B37 and A57. Replication of all four viruses was detected in Vero_ICAM1, but not in the parental Vero cells. Altogether, Vero cells expressing ICAM1 could efficiently propagate the tested HRV strains. Therefore, ICAM1-expressing cells could be a useful tool for the development and future production of polyvalent HRV vaccines or other viruses that use ICAM1 as a receptor

Rickettsia australis Activates Inflammasome in Human and Murine Macrophages.

Rickettsiae actively escape from vacuoles and replicate free in the cytoplasm of host cells, where inflammasomes survey the invading pathogens. In the present study, we investigated the interactions of Rickettsia australis with the inflammasome in both mouse and human macrophages. R. australis induced a significant level of IL-1β secretion by human macrophages, which was significantly reduced upon treatment with an inhibitor of caspase-1 compared to untreated controls, suggesting caspase-1-dependent inflammasome activation. Rickettsia induced significant secretion of IL-1β and IL-18 in vitro by infected mouse bone marrow-derived macrophages (BMMs) as early as 8-12 h post infection (p.i.) in a dose-dependent manner. Secretion of these cytokines was accompanied by cleavage of caspase-1 and was completely abrogated in BMMs deficient in caspase-1/caspase-11 or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), suggesting that R. australis activate the ASC-dependent inflammasome. Interestingly, in response to the same quantity of rickettsiae, NLRP3-/- BMMs significantly reduced the secretion level of IL-1β compared to wild type (WT) controls, suggesting that NLRP3 inflammasome contributes to cytosolic recognition of R. australis in vitro. Rickettsial load in spleen, but not liver and lung, of R. australis-infected NLRP3-/- mice was significantly greater compared to WT mice. These data suggest that NLRP3 inflammasome plays a role in host control of bacteria in vivo in a tissue-specific manner. Taken together, our data, for the first time, illustrate the activation of ASC-dependent inflammasome by R. australis in macrophages in which NLRP3 is involved

DipA is exposed to the host cytosol during macrophage infection.

<p>(A) Representative confocal micrographs of BMMs infected for 10 h with SchuS4Δ<i>dipA</i>(p<i>dipA-HA</i>), SchuS4(p<i>iglI-HA</i>), or SchuS4(p<i>iglA-HA</i>). Using conditions that permeabilize host plasma membranes but not bacterial membranes (described in Materials and Methods), samples were processed for immunofluorescence labelling of HA-tagged proteins (green) and bacterial LPS (red), and counterstained with DAPI to label DNA (blue). Magnified insets show single channel images of the boxed area. Scale bars, 10 or 2 µm. (B) Quantification of CCF2/AM cleavage in J774A.1 cells that were either uninfected or infected with SchuS4, or SchuS4 expressing C-terminal TEM1 fusions with IglI, IglA or DipA. After 16 h, infected macrophages were loaded with CCF2/AM and analyzed by live cell microscopy for blue fluorescence emission. At least 100 cells were scored per experiment. Data are means ± SD from a representative experiment performed in triplicate out of three independent repeats. Asterisks indicate statistically significant differences compared to uninfected, SchuS4-infected, and SchuS4 expressing IglA-TEM1-infected controls (* <i>P < 0.05</i>, 1-way ANOVA, Tukey’s post-test). (C) Representative fluorescence micrographs of J774A.1 cells that were either uninfected or infected for 16 h with SchuS4, SchuS4 expressing IglI-TEM1, SchuS4 expressing IglA-TEM1, and SchuS4 expressing DipA-TEM1. Cells emitting blue fluorescence indicate delivery of TEM1 β-lactamase fusions to the cytosol and CCF2/AM cleavage. Intact CCF2/AM, indicating the absence of TEM1 β-lactamase activity in the cytosol, results in green fluorescence emission. Scale bar, 50 µm.</p

Identification of DipA interacting partners.

<p>(A) Code blue stained gel of proteins immunoprecipitated with anti-HA conjugated beads from lysates of SchuS4Δ<i>dipA</i> expressing DipA [SchuS4Δ<i>dipA</i>(p<i>dipA</i>)] or DipA-HA [SchuS4Δ<i>dipA</i>(p<i>dipA-HA</i>)]. Samples were resolved by SDS-PAGE and stained with GelCode® Blue. Protein bands migrating at ~40 kDa were subjected to mass spectrometry-based identification. FTT1407c, FTT1365c and FTT0583 were specifically co-immunoprecipitated with DipA-HA. (B and C) Verification of FopA interaction with DipA by immunoblot analysis. (B) Lysates from SchuS4Δ<i>dipA</i> expressing DipA derivatives (DipA, Dip-HA, DipAΔSel1ab-HA, DipAΔSel1cd-HA), or SchuS4Δ<i>flpA</i> expressing FlpA derivatives (FlpA, FlpA-HA) were subjected to immunoprecipitation (IP) using anti-HA conjugated beads followed by immunoblot (IB) analysis with anti-HA, anti-FopA and anti-FipB antibodies. I denotes sample input; B denotes bound fraction. (C) Lysate from SchuS4Δ<i>dipA</i> expressing DipA-HA was subjected to immunoprecipitation (IP) using anti-FopA antibodies followed by immunoblot (IB) analysis with anti-FopA antibodies to detect FopA and anti-HA antibodies to detect Dip-HA. I denotes sample input; B denotes bound fraction.</p

Viral RNA-dependent RNA polymerase mutants display an altered mutation spectrum resulting in attenuation in both mosquito and vertebrate hosts.

The presence of bottlenecks in the transmission cycle of many RNA viruses leads to a severe reduction of number of virus particles and this occurs multiple times throughout the viral transmission cycle. Viral replication is then necessary for regeneration of a diverse mutant swarm. It is now understood that any perturbation of the mutation frequency either by increasing or decreasing the accumulation of mutations in an RNA virus results in attenuation of the virus. To determine if altering the rate at which a virus accumulates mutations decreases the probability of a successful virus infection due to issues traversing host bottlenecks, a series of mutations in the RNA-dependent RNA polymerase of Venezuelan equine encephalitis virus (VEEV), strain 68U201, were tested for mutation rate changes. All RdRp mutants were attenuated in both the mosquito and vertebrate hosts, while showing no attenuation during in vitro infections. The rescued viruses containing these mutations showed some evidence of change in fidelity, but the phenotype was not sustained following passaging. However, these mutants did exhibit changes in the frequency of specific types of mutations. Using a model of mutation production, these changes were shown to decrease the number of stop codons generated during virus replication. This suggests that the observed mutant attenuation in vivo may be due to an increase in the number of unfit genomes, which may be normally selected against by the accumulation of stop codons. Lastly, the ability of these attenuated viruses to transition through a bottleneck in vivo was measured using marked virus clones. The attenuated viruses showed an overall reduction in the number of marked clones for both the mosquito and vertebrate hosts, as well as a reduced ability to overcome the known bottlenecks in the mosquito. This study demonstrates that any perturbation of the optimal mutation frequency whether through changes in fidelity or by alterations in the mutation frequency of specific nucleotides, has significant deleterious effects on the virus, especially in the presence of host bottlenecks

The SLR and CC domains of DipA are functionally distinct.

<p>(A) Ability of DipA variants to complement the intracellular growth defect of SchuS4Δ<i>dipA</i>. Viable intracellular bacteria were enumerated at 1 h and 16 h p.i. from BMMs infected with SchuS4, SchuS4Δ<i>dipA</i>, or SchuS4Δ<i>dipA</i> expressing HA-tagged DipA variants (DipA-HA, DipAΔSel1ab-HA, DipAΔSel1cd-HA, DipAΔCC-HA, DipACC(AIL ³D)-HA, or DipACC(LAL ³D)-HA). Fold change in replication was calculated by comparing CFUs at 16 h p.i. versus 1 h p.i. Data are means ± SD from three independent experiments. Asterisks indicate statistically significant differences compared to SchuS4-infected and SchuS4Δ<i>dipA</i> expressing DipA-HA-infected macrophages (* <i>P < 0.05</i>, 1-way ANOVA, Tukey’s post-test). (B) Subcellular localization of HA-tagged DipA variants as described in (A). Soluble (Sol), inner membrane (IM), and outer membrane (OM) enriched fractions were separated based on Sarkosyl solubility and subjected to immunoblot analysis with antibodies against HA. Each fraction was concentrated to the same volume and equal volumes were loaded. (C) Immunoblot analysis of purified surface biotinylated proteins from lysates of SchuS4Δ<i>dipA</i> strains expressing HA-tagged DipA variants as described in (A). Input and biotinylated (surface) samples were processed for CFU enumeration and immunoblotting as described in Materials and Methods. Samples were loaded based on CFU equivalents as follows: 1x10⁶ (Input) or 1x10⁸ (surface). (D) Quantification of J774A.1 cells emitting blue fluorescence that were either uninfected or infected with SchuS4, or SchuS4 expressing C-terminal TEM1 fusions with IglI, IglA, DipA, DipAΔSel1ab, DipAΔSel1cd, DipAΔCC, DipACC(AIL ³D), or DipACC(LAL ³D). Infected cells were analyzed for CCF2/AM cleavage at 16 h pi. At least 100 cells were scored per experiment. Data are means ± SD from a representative experiment performed in triplicate out of three independent repeats. Asterisks indicate statistically significant differences compared to uninfected, SchuS4-infected, and SchuS4 expressing IglA-TEM1-infected controls (* <i>P < 0.001</i>, 1-way ANOVA, Tukey’s post-test). (E) Representative fluorescence micrographs of J774A.1 macrophages infected for 16 h with SchuS4 expressing either DipA-TEM1, DipAΔSel1ab-TEM1, DipAΔSel1cd-TEM1, DipAΔCC-TEM1, DipACC(AIL ³D)-TEM1, or DipACC(LAL ³D)-TEM1. Cells emitting blue fluorescence indicate delivery of TEM1 β-lactamase fusions to the cytosol to cleave the CCF2/AM substrate. Intact CCF2/AM, indicating the absence of TEM1 β-lactamase activity in the cytosol, results in green fluorescence emission. Scale bar, 50 µm.</p

LD50 of SchuS4ΔFTT0369c and protective efficacy following increased challenge dose of wild type SchuS4.

<p>(A) Balb/c mice (n = 10/group) were infected intradermally with the indicated number of <i>F. tularensis</i> ΔFTT0369c. Mice were regularly monitored for up to 45 days after infection and euthanized at the first signs of irreversible illness. (B and C) Mice (n = 10/group) were challenged with approximately 5×10⁴ CFU intradermally. Forty-five days after infection mice were challenged intranasally with approximately 50 CFU (B) or 200 CFU (C) wild type SchuS4. Mice were regularly monitored for up to 30 days after infection and euthanized at the first signs of irreversible illness. * = p<0.05 compared to all other groups. ** = p<0.05 compared to mice receiving 7×10⁵, 1×10⁶ and 3×10⁶.</p