Deletion of ripA Alleviates Suppression of the Inflammasome and MAPK by Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis

Adherence of <i>Y. pestis</i> biofilm.

<p>The <i>Y. pestis</i> A) wild-type CO92 and B) Δ<i>tatA</i> mutant cultures were grown in HI broth at 26°C. Arrows indicate biofilm formation. C) To measure biofilm adherence, the biofilm of wild-type CO92, Δ<i>tatA</i> mutant, and complemented Δ<i>tatA</i> mutant was stained with crystal violet and assayed by absorbance as described in the Materials and Methods. The standard deviation was derived from quadruplicate samples. These data represent three separate experiments.</p

Pathology of mice challenged intranasally with <i>Y. pestis</i>.

<p>A and B) Skull sections (4×) of a mouse challenged with the Δ<i>tatA</i> mutant (2,800 CFU). The mouse was moribund and euthanized on day 8 postchallenge. Note inflammation (indicated by *) that extends into the cranial vault and meninges. Panel A shows HE staining, and Panel B shows IHC staining with antibody to the F1 capsule. C) HE stained section from a mouse (2×) challenged with wild-type <i>Y. pestis</i> (5,500 CFU) that succumbed to infection on day 3 post challenge. In contrast to panel A, the inflammation for mice challenged with the wild-type strain, is contained in the inner ear area and did not extend into the brain (arrow). D and E) Lung sections of mice challenged intranasally with <i>Y. pestis</i>. Overall, lung lesion development and character are the same between strains with necrosuppurative inflammation surrounding large colonies of bacteria. Panel D is of a lung section stained with HE (20×) from a mouse challenged with the Δ<i>tatA</i> mutant. Panel E is of a lung section stained with HE (20×) challenged with wild-type <i>Y. pestis</i>. IE = inner ear; CRB = cerebrum.</p

Localizing the F1 capsule by microscopy.

<p><i>Y. pestis</i> strains were grown at 37°C in HI broth containing CaCl₂ and exposed to an antibody against the F1 capsule. IFM samples: A) wild-type, B) Δ<i>tatA</i> mutant, and C) complemented Δ<i>tatA</i> mutant. The images (100×) were captured for all samples at identical camera settings to maintain relative fluorescence. IEM samples: D) wild-type (micron bar = 0.1 µm), E) Δ<i>tatA</i> mutant (micron bar = 0.1 µm), and F) C12, F1 negative (micron bar = 0.5 µm).</p

Primers.

<p>Primers.</p

Pathology of mice challenged with <i>Y. pestis</i> by small particle aerosol.

<p>A and B) HE stained skull sections of aerosol challenged mice. A) Mouse (2×) challenged with wild-type CO92 (6.9×10⁵ CFU) that was moribund and euthanized on day 3 postchallenge. B) Mouse (4×) challenged with Δ<i>tatA</i> mutant (3.3×10⁶ CFU) that was moribund and euthanized on day 7 postchallenge. No inner ear (IE) or meningeal involvement was detected for mice aerosol challenged with either strain of <i>Y. pestis</i>. HE stained lung sections (10×) of mice aerosol challenged with CO92 (C) or Δ<i>tatA</i> mutant (D). There was no difference in lesion character or severity in mice challenged with either strain.</p

Strains and Plasmids.

<p>Strains and Plasmids.</p

Growth assays.

<p><i>Y. pestis</i> wild type and Δ<i>tatA</i> mutant strains were grown in HI broth at 28°C (A and B), 37°C with CaCl₂ (C and D), or 37°C with MOX (E and F). Growth was monitored by both optical density (A, C, and E) and CFU counts (B, D, and F). OD measurements are based upon quadruplicate samples and bars represent standard deviation. CFU measurements are based upon triplicate samples and bars represent standard deviation. These data represent at least two separate experiments.</p

Dissemination studies of mice challenged intranasally with <i>Y. pestis</i>.

<p>Mice were challenged with CO92 (113,000 CFU) or Δ<i>tatA</i> mutant (76,000 CFU). At set time points, mice were euthanized, and the lungs and spleens were harvested. The A) lungs and B) spleens were homogenized and plated to determine bacterial recovery. For each time point, five mice were assayed, except for day 3 for wild-type challenged mice. Only four mice were tested as the remaining challenged mice had succumbed to infection. The lines (solid = CO92 and hashed = Δ<i>tatA</i>) are connecting at the geometrical means at the data points of CFU recovery from the respective organs are represent the overall trend during the course of infection.</p

Calculated LD₅₀ for the CO92 wild-type and Δ<i>tatA</i> mutant <i>Y. pestis</i> strains.

a<p>Calculated inhaled dose.</p>b<p>A significant difference was observed between the LD₅₀ determinations between the Δ<i>tatA</i> mutant and complemented strain (<i>p</i><0.0001).</p>c<p>Not determined.</p