Structural Insights into the Regulation of <i>Staphylococcus aureus</i> Phosphofructokinase by Tetramer–Dimer Conversion

Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states. We report that the Pfk from <i>Staphylococcus aureus</i> NCTC 8325 (<i>Sa</i>Pfk) exists as both an active tetramer and an inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes <i>Sa</i>Pfk tetrameric assembly. Crystal structures of <i>Sa</i>Pfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer–dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between the tetramer and dimer in addition to the well-characterized R-state/T-state mechanism

Table_1_Human adenovirus infection induces pulmonary inflammatory damage by triggering noncanonical inflammasomes activation and macrophage pyroptosis.docx

IntroductionHuman adenovirus (HAdV) is a common respiratory virus, which can lead to severe pneumonia in children and immunocompromised persons, and canonical inflammasomes are reported to be involved in anti-HAdV defense. However, whether HAdV induced noncanonical inflammasome activation has not been explored. This study aims to explore the broad roles of noncanonical inflammasomes during HAdV infection to investigate the regulatory mechanism of HAdV-induced pulmonary inflammatory damage. MethodsWe mined available data on GEO database and collected clinical samples from adenovirus pneumonia pediatric patients to investigate the expression of noncanonical inflammasome and its clinical relevance. An in vitro cell model was employed to investigate the roles of noncanonical inflammasomes in macrophages in response to HAdV infection.ResultsBioinformatics analysis showed that inflammasome-related genes, including caspase-4 and caspase-5, were enriched in adenovirus pneumonia. Moreover, caspase-4 and caspase-5 expression levels were significantly increased in the cells isolated from peripheral blood and broncho-alveolar lavage fluid (BALF) of pediatric patients with adenovirus pneumonia, and positively correlated with clinical parameters of inflammatory damage. In vitro experiments revealed that HAdV infection promoted caspase-4/5 expression, activation and pyroptosis in differentiated THP-1 (dTHP-1) human macrophages via NF-κB, rather than STING signaling pathway. Interestingly, silencing of caspase-4 and caspase-5 in dTHP-1 cells suppressed HAdV-induced noncanonical inflammasome activation and macrophage pyroptosis, and dramatically decreased the HAdV titer in cell supernatants, by influencing virus release rather than other stages of virus life cycle.DiscussionIn conclusion, our study demonstrated that HAdV infection induced macrophage pyroptosis by triggering noncanonical inflammasome activation via a NF-kB-dependent manner, which may explore new perspectives on the pathogenesis of HAdV-induced inflammatory damage. And high expression levels of caspase-4 and caspase-5 may be a biomarker for predicting the severity of adenovirus pneumonia.</p

Expression of IFNs in mock-treated or poly(I∶C) pretreated HepG2 cells.

<p>After pretreatment with poly(I∶C) for 9 h, HepG2 cells were infected with DENV2 at an MOI 1. The cells were harvested at 0, 6, 12, and 24 h p.i. for RNA extraction and real-time RT-PCR for IFN-β (A) or IL-28A/B (B). Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, **, p<0.01; ***, p<0.001.</p

Image_1_Human adenovirus infection induces pulmonary inflammatory damage by triggering noncanonical inflammasomes activation and macrophage pyroptosis.pdf

IntroductionHuman adenovirus (HAdV) is a common respiratory virus, which can lead to severe pneumonia in children and immunocompromised persons, and canonical inflammasomes are reported to be involved in anti-HAdV defense. However, whether HAdV induced noncanonical inflammasome activation has not been explored. This study aims to explore the broad roles of noncanonical inflammasomes during HAdV infection to investigate the regulatory mechanism of HAdV-induced pulmonary inflammatory damage. MethodsWe mined available data on GEO database and collected clinical samples from adenovirus pneumonia pediatric patients to investigate the expression of noncanonical inflammasome and its clinical relevance. An in vitro cell model was employed to investigate the roles of noncanonical inflammasomes in macrophages in response to HAdV infection.ResultsBioinformatics analysis showed that inflammasome-related genes, including caspase-4 and caspase-5, were enriched in adenovirus pneumonia. Moreover, caspase-4 and caspase-5 expression levels were significantly increased in the cells isolated from peripheral blood and broncho-alveolar lavage fluid (BALF) of pediatric patients with adenovirus pneumonia, and positively correlated with clinical parameters of inflammatory damage. In vitro experiments revealed that HAdV infection promoted caspase-4/5 expression, activation and pyroptosis in differentiated THP-1 (dTHP-1) human macrophages via NF-κB, rather than STING signaling pathway. Interestingly, silencing of caspase-4 and caspase-5 in dTHP-1 cells suppressed HAdV-induced noncanonical inflammasome activation and macrophage pyroptosis, and dramatically decreased the HAdV titer in cell supernatants, by influencing virus release rather than other stages of virus life cycle.DiscussionIn conclusion, our study demonstrated that HAdV infection induced macrophage pyroptosis by triggering noncanonical inflammasome activation via a NF-kB-dependent manner, which may explore new perspectives on the pathogenesis of HAdV-induced inflammatory damage. And high expression levels of caspase-4 and caspase-5 may be a biomarker for predicting the severity of adenovirus pneumonia.</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p

Pretreatment of poly(I∶C) suppresses the DENV2 replication in HepG2 cells.

<p>HepG2 cells were mock- or pretreated with 5 µg poly (I∶C) for 9 h, then infected with DENV2. (A) Real-time PCR to measure viral mRNA levels. Total RNAs were harvested at 2, 6, 12, and 24 h p.i., and used for real-time RT-PCR. (B) Immunofluorescence microscopy. Infected cells were fixed at 24 h p.i. and incubated with DENV2 prM antibody. (C) Percentages of positive-stained cells determined by PicCnt 100×. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, *, p<0.05; ***, p<0.001.</p

DENV2 replication levels in IFN-β neutralizing antibody treated HepG2 cells.

<p>500 unit/ml IFN-β neutralizing antibody was added at 1 h before poly(I∶C) treatment and kept through poly(I∶C) treatment and virus infection. (A) Real-time PCR detecting the expression of viral mRNA; (B) Extracellular viral production determined by TCID50. Supernatants were harvested at 24 h p.i. and titered on C6/36 cells. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, **, p<0.01; ***, p<0.001.</p

IKK inhibitor reversed the IFN-β induction and virus replication suppression mediated by poly(I∶C) pretreatment.

<p>HepG2 cells were incubated in control or IKK inhibitor for 30 min before poly(I∶C) pretreatment and virus infection. (A) Cells were harvested for measurement of IFN-β expression level; (B) Immunofluorescence microscopy. Cells were fixed at 24 h p.i. and incubated with DENV2 prM antibody. Panel a, mock infected; b, DENV2; c, DMSO+poly(I∶C)+DENV2; d, BMS345541+poly(I∶C)+DENV2; (C) Percentages of positive-stained cells determined by PicCnt 100×; (D) Extracellular viral production determined by TCID50. Supernatants were harvested at 24 h p.i. and titered on C6/36 cells. Error bars represent the standard error of mean from the average of three experiments. Student's <i>t</i> test, *, p<0.05; **, p<0.01; ***, p<0.001.</p

image_1_Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis.tif

<p>Triggering receptors expressed on myeloid cells 2 (TREM2) is a novel cell surface receptor and functions as an immunomodulatory receptor in infectious diseases. In this study, we investigated the function and regulatory mechanism of TREM2 in Pseudomonas aeruginosa (P. aeruginosa) keratitis. We found that P. aeruginosa keratitis was more severe in Trem2^−/− versus wild type C57BL/6 mice as indicated by the increased clinical scores, bacterial load, and cornea pathology. The exacerbated disease progression caused by TREM2 deficiency was associated with boosted activation of caspase-1 and subsequent pyroptosis as well as increased expression of IL-1β. In addition, blockage of pyroptosis by caspase-1 inhibitor not only recovered the severe cornea pathology developed in Trem2^−/− mice but also restored the P. aeruginosa clearance suppressed by TREM2 deficiency. Our study demonstrated that TREM2 promotes host resistance against P. aeruginosa keratitis by inhibiting caspase-1-dependent pyroptosis, which provides new insights of TREM2-mediated anti-bacterial immunity.</p

table_1_Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis.docx

<p>Triggering receptors expressed on myeloid cells 2 (TREM2) is a novel cell surface receptor and functions as an immunomodulatory receptor in infectious diseases. In this study, we investigated the function and regulatory mechanism of TREM2 in Pseudomonas aeruginosa (P. aeruginosa) keratitis. We found that P. aeruginosa keratitis was more severe in Trem2^−/− versus wild type C57BL/6 mice as indicated by the increased clinical scores, bacterial load, and cornea pathology. The exacerbated disease progression caused by TREM2 deficiency was associated with boosted activation of caspase-1 and subsequent pyroptosis as well as increased expression of IL-1β. In addition, blockage of pyroptosis by caspase-1 inhibitor not only recovered the severe cornea pathology developed in Trem2^−/− mice but also restored the P. aeruginosa clearance suppressed by TREM2 deficiency. Our study demonstrated that TREM2 promotes host resistance against P. aeruginosa keratitis by inhibiting caspase-1-dependent pyroptosis, which provides new insights of TREM2-mediated anti-bacterial immunity.</p