Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens

The innate immune system is the first line of host defense against invading pathogens. In multicellular organisms, specialized innate immune cells recognize conserved pathogen-associated molecular patters (PAMPs) with germ-line encoded pattern recognition receptors (PRR). Thereby, the organism discriminates between self and non-self and engages mechanisms to eliminate the invader. Beside PAMPs, PRRs recognize mislocalized self-molecules, so called danger-associated molecular patterns (DAMPs), which are indicators of tissue or cellular damage. Upon PAMP or DAMP recognition, PRRs induce innate immune signaling pathways leading to the activation of pro-inflammatory genes and interferon production, which are important mediators of inflammation. Therefore the recognition of invading pathogen and thereby activation of innate immune signaling pathways determines the success of the immune system to eliminate the potential threat. Innate immune signaling pathways largely depend on phosphorylation cascades. Today, global phosphorylation changes are analyzed by mass spectrometry, however the number of detected phosphopeptides remains unchanged despite technical improvements. Therefore, we investigated the issue of phosphopeptide detection in mass spectrometry. The analyses of phosphopeptide-enriched samples have revealed lower signal intensities in MS1 spectra compared to total cell lysate samples, which results in poor phosphopeptide detection with mass spectrometry. Based on these observations, we hypothesized that the phosphate groups of phosphopeptides account for this poor detection. Indeed, we significantly increase the signal intensities in MS1 spectra after enzymatic removal of phosphate groups from phosphopeptides, and consequently we detect three-times more peptides in phosphatase-treated samples. Validation experiments elucidate that most of the newly detected peptides have been initially phosphorylated. Moreover, the newly detected peptides enlarge the activated signaling network upon Salmonella infection. Importantly, we identify known innate immune signaling pathways, which were missing in the analyses of phospho-enriched samples. Taken together, the phosphate groups of phosphopeptides globally suppress peptide ionization efficacy and therefore account for the low phosphopeptide detection rate by mass spectrometry. By removing the phosphate groups, we identify three times more peptides after phosphatase treatment. The newly detected peptides enlarge the network of activated innate immune signaling pathways upon Salmonella infection and include signaling pathways that are important but have not been detected in phospho-enriched samples. Therefore our findings improve the analyses of innate immune signaling pathways by mass spectrometry and consequently the understanding of innate immunity. One of the main mechanisms to eliminate invading microbes is by phagocytosis and degradation within phago-lysosomes. However, professional pathogens have developed various defense mechanisms to resist intracellular killing and can even use innate immune cells as replicative niches. For example, the bacterial pathogen Francisella tularensis causes a severe and life-threatening disease called tularemia in humans, because Francisella can survive and replicate in macrophages and dendritic cells. Critical for Francisella pathogenicity is the ability of the phagocytosed bacteria to escape from the phagosome to the host cytosol. Even though we know that genes encoded on the Francisella pathogenicity island (FPI) are essential for escaping from the phagosome, the mechanism is unknown. Homology analyses have suggested that the FPI encodes a type 6 secretion system (T6SS). However experimental evidence is missing, which show that the FPI encode a functional T6SS. Therefore, we investigated whether the FPI encodes a functional T6SS and what impact a functional T6SS has on Francisella virulence in vitro and in vivo. We show that the FPI of Francisella novicida (F. novicida) encodes a functional T6SS that assembles exclusively at bacterial poles. T6SS function depends on the unfoldase ClpB, which specifically recognizes contracted T6SS sheaths leading to their disassembly. Furthermore we have characterized FPI genes that show no homology with known T6SSs. We have identified IglF, IglG, IglI and IglJ as structural components of the T6SS and PdpC, PdpD, PdpE and AnmK as potential T6SS effector proteins. Whereas PdpE and AnmK are dispensable for phagosomal escape, AIM2 inflammasome activation and virulence in mice, pdpC- and pdpD-deficient bacteria are impaired in all aforementioned analyses. This suggests that PdpC and PdpD are bacterial effector proteins involved in phagosomal escape and thereby in the establishment of a F. novicida infection. Taken together, F. novicida uses its T6SS to deliver the effector proteins PdpC and PdpD into host cells. PdpC and PdpD are involved in phagosomal rupture and consequently in bacterial escape to the cytosol. These findings are a major breakthrough in the understanding of Francisella pathogenicity and could lead to new vaccination strategies to eradicate the life-threatening human disease Tularemia

Type-3 Secretion System-induced pyroptosis protects Pseudomonas against cell-autonomous immunity

Inflammasome-induced pyroptosis comprises a key cell-autonomous immune process against intracellular bacteria, namely the generation of dying cell structures. These so-called pore-induced intracellular traps (PITs) entrap and weaken intracellular microbes. However, the immune importance of pyroptosis against extracellular pathogens remains unclear. Here, we report that Type-3 secretion system (T3SS)-expressing Pseudomonas aeruginosa ( P. aeruginosa ) escaped PIT immunity by inducing a NLRC4 inflammasome-dependent macrophage pyroptosis response in the extracellular environment. To the contrary, phagocytosis of Salmonella Typhimurium promoted NLRC4-dependent PIT formation and the subsequent bacterial caging. Remarkably, T3SS-deficient Pseudomonas were efficiently sequestered within PIT-dependent caging, which favored exposure to neutrophils. Conversely, both NLRC4 and caspase-11 deficient mice presented increased susceptibility to T3SS-deficient P. aeruginosa challenge, but not to T3SS-expressing P. aeruginosa. Overall, our results uncovered that P. aeruginosa uses its T3SS to overcome inflammasome-triggered pyroptosis, which is primarily effective against intracellular invaders. Importance Although innate immune components confer host protection against infections, the opportunistic bacterial pathogen Pseudomonas aeruginosa ( P. aeruginosa ) exploits the inflammatory reaction to thrive. Specifically the NLRC4 inflammasome, a crucial immune complex, triggers an Interleukin (IL)-1β and -18 deleterious host response to P. aeruginosa . Here, we provide evidence that, in addition to IL-1 cytokines, P. aeruginosa also exploits the NLRC4 inflammasome-induced pro-inflammatory cell death, namely pyroptosis, to avoid efficient uptake and killing by macrophages. Therefore, our study reveals that pyroptosis-driven immune effectiveness mainly depends on P. aeruginosa localization. This paves the way toward our comprehension of the mechanistic requirements for pyroptosis effectiveness upon microbial infections and may initiate targeted approaches in order to ameliorate the innate immune functions to infections. Graphical abstract Macrophages infected with T3SS-expressing P. aeruginosa die in a NLRC4-dependent manner, which allows bacterial escape from PIT-mediated cell-autonomous immunity and neutrophil efferocytosis. However, T3SS-deficient P. aeruginosa is detected by both NLRC4 and caspase-11 inflammasomes, which promotes bacterial trapping and subsequent efferocytosis of P. aeruginosa -containing-PITs by neutrophils

Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture

Dual endothelin antagonist aprocitentan for resistant hypertension (PRECISION): a multicentre, blinded, randomised, parallel-group, phase 3 trial

BACKGROUND: Resistant hypertension is associated with increased cardiovascular risk. The endothelin pathway has been implicated in the pathogenesis of hypertension, but it is currently not targeted therapeutically, thereby leaving this relevant pathophysiological pathway unopposed with currently available drugs. The aim of the study was to assess the blood pressure lowering efficacy of the dual endothelin antagonist aprocitentan in patients with resistant hypertension. METHODS: PRECISION was a multicentre, blinded, randomised, parallel-group, phase 3 study, which was done in hospitals or research centres in Europe, North America, Asia, and Australia. Patients were eligible for randomisation if their sitting systolic blood pressure was 140 mm Hg or higher despite taking standardised background therapy consisting of three antihypertensive drugs, including a diuretic. The study consisted of three sequential parts: part 1 was the 4-week double-blind, randomised, and placebo-controlled part, in which patients received aprocitentan 12·5 mg, aprocitentan 25 mg, or placebo in a 1:1:1 ratio; part 2 was a 32-week single (patient)-blind part, in which all patients received aprocitentan 25 mg; and part 3 was a 12-week double-blind, randomised, and placebo-controlled withdrawal part, in which patients were re-randomised to aprocitentan 25 mg or placebo in a 1:1 ratio. The primary and key secondary endpoints were changes in unattended office systolic blood pressure from baseline to week 4 and from withdrawal baseline to week 40, respectively. Secondary endpoints included 24-h ambulatory blood pressure changes. The study is registered on ClinicalTrials.gov, NCT03541174. FINDINGS: The PRECISION study was done from June 18, 2018, to April 25, 2022. 1965 individuals were screened and 730 were randomly assigned. Of these 730 patients, 704 (96%) completed part 1 of the study; of these, 613 (87%) completed part 2 and, of these, 577 (94%) completed part 3 of the study. The least square mean (SE) change in office systolic blood pressure at 4 weeks was -15·3 (SE 0·9) mm Hg for aprocitentan 12·5 mg, -15·2 (0·9) mm Hg for aprocitentan 25 mg, and -11·5 (0·9) mm Hg for placebo, for a difference versus placebo of -3·8 (1·3) mm Hg (97·5% CI -6·8 to -0·8, p=0·0042) and -3·7 (1·3) mm Hg (-6·7 to -0·8; p=0·0046), respectively. The respective difference for 24 h ambulatory systolic blood pressure was -4·2 mm Hg (95% CI -6·2 to -2·1) and -5·9 mm Hg (-7·9 to -3·8). After 4 weeks of withdrawal, office systolic blood pressure significantly increased with placebo versus aprocitentan (5·8 mm Hg, 95% CI 3·7 to 7·9, p<0·0001). The most frequent adverse event was mild-to-moderate oedema or fluid retention, occurring in 9%, 18%, and 2% for patients receiving aprocitentan 12·5 mg, 25 mg, and placebo, during the 4-week double-blind part, respectively. This event led to discontinuation in seven patients treated with aprocitentan. During the trial, a total of 11 treatment-emergent deaths occurred, none of which were regarded by the investigators to be related to study treatment. INTERPRETATION: In patients with resistant hypertension, aprocitentan was well tolerated and superior to placebo in lowering blood pressure at week 4 with a sustained effect at week 40. FUNDING: Idorsia Pharmaceuticals and Janssen Biotech

Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol