Biology subjects

Bliska, J. B.

Publications and source records attributed to Bliska, J. B..

5 recordsLinked to original sources

bioRxiv2023

A Type VI Secretion System in Burkholderia Species cenocepacia and orbicola Triggers Distinct Macrophage Death Pathways Independent of the Pyrin Inflammasome

The Burkholderia cepacia complex contains opportunistic pathogens that cause chronic infections and inflammation in lungs of people with cystic fibrosis. Two closely related species within this complex are Burkholderia cenocepacia and the recently classified Burkholderia orbicola. B. cenocepacia and B. orbicola encode a type VI secretion system and the effector TecA, which is detected by the pyrin/caspase-1 inflammasome, and triggers macrophage inflammatory death. In our earlier study the pyrin inflammasome was dispensable for lung inflammation in mice infected with B. orbicola AU1054, indicating this species activates an alternative pathway of macrophage inflammatory death. Notably, B. cenocepacia J2315 and K56-2 can damage macrophage phagosomes and K56-2 triggers activation of the caspase-11 inflammasome, which detects cytosolic LPS. Here we investigated inflammatory cell death in pyrin-deficient (Mefv-/-) mouse macrophages infected with B. cenocepacia J2315 or K56-2 or B. orbicola AU1054 or PC184. Macrophage inflammatory death was measured by cleavage of gasdermin D protein, release of cytokines IL-1 and IL-1{beta} and plasma membrane rupture. Findings suggest that J2315 and K56-2 are detected by the caspase-11 inflammasome in Mefv-/- macrophages, resulting in IL-1{beta} release. In contrast, inflammasome activation is not detected in Mefv-/- macrophages infected with AU1054 or PC184. Instead, AU1054 triggers an alternative macrophage inflammatory death pathway that requires TecA and results in plasma membrane rupture and IL-1 release. Amino acid variation between TecA isoforms in B. cenocepacia and B. orbicola may explain how the latter species triggers a non-inflammasome macrophage death pathway.

microbiology↗

bioRxiv2022

Phosphoprotein Phosphatase Activity Positively Regulates Oligomeric Pyrin to Trigger Inflammasome Assembly in Response to Bacterial Effectors and Toxins that Inactivate RhoA in Macrophages

Pyrin is a pattern-recognition receptor in phagocytes that triggers capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. Pyrin contains oligomerization domains and is negatively regulated by phosphorylation of two residues, S205 and S241 (murine) or S208 and S242 (human), via the kinases PKN1/2, which are activated by RhoA. Familial Mediterranean Fever (FMF) is caused by phagocyte production of pyrin gain of function variants, which have a lower threshold for inflammasome assembly upon RhoA-PKN axis inhibition. Inactivation of the RhoA-PKN axis removes negative regulation but a phosphoprotein phosphatase (PPP) is needed to positively regulate pyrin. No PPP that dephosphorylates pyrin has been identified, oligomerization of murine pyrin has not been studied, and the phosphorylation status of oligomeric pyrin is unknown. We used murine macrophages and FMF patients monocytes combined with the use of bacterial agonists and chemical inhibitors, native PAGE, phospho-specific antibodies and siRNA knockdowns to determine if a PPP positively regulates oligomeric pyrin. Results with broadly-specific inhibitors indicate that PPP activity is required to dephosphorylate murine and human pyrin in wild type or FMF patients phagocytes. Findings from native PAGE show that murine pyrin forms oligomers that are phosphorylated on S205 prior to RhoA inactivation. Inhibitors cause reduced mobility of murine pyrin on native PAGE and hyperphosphorylation of S242 in human pyrin, suggesting a PPP constitutively counterbalances PKN to keep the second site hypophosphorylated. Data from siRNA knockdown experiments implicate PP2A in dephosphorylation of S205 and positive regulation of pyrin in response to RhoA inactivation. Key pointsMurine pyrin is oligomeric and phosphorylated on S205 prior to inflammasome assembly PPP activity positively regulates pyrin inflammasome assembly in mice and humans The alpha and beta subunits of PP2A dephosphorylate murine pyrin S205 in macrophages

immunology↗

bioRxiv2022

Intestinal Bacteroides Modulates Systemic Inflammation and the Microbial Ecology in a Mouse Model of CF: Evidence for Propionate and other Short Chain Fatty Acids Reducing Systemic Inflammatory Cytokines

Persons with cystic fibrosis, starting in early life, have intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid (SCFA) propionate. We demonstrate here that CFTR-/- Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1{beta}-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. Bacteroides isolates also produce low levels of butyrate; this SCFA is positively correlated with inhibition of the inflammatory response. Finally, the introduction of Bacteroides-supplemented stool from infants with CF into the gut of CftrF508del mice results in an increase in propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of E. coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. Together, our data indicate the important role of Bacteroides and Bacteroides-derived propionate in the context of the developing microbiome in infants and children with CF, which could help explain the observed gut-lung axis in CF.

microbiology↗

bioRxiv2021

The Burkholderia cenocepacia type VI secretion system effector TecA is a virulence factor during lung infection

Burkholderia cenocepacia (Bc) is a member of the Burkholderia cepacia complex (Bcc), a group of bacteria with members responsible for causing lung infections in cystic fibrosis (CF) patients. The most severe outcome of Bcc infection in CF patients is cepacia syndrome, a disease characterized by necrotizing pneumonia with bacteremia and sepsis. Bc is strongly associated with cepacia syndrome making it one of the most virulent members of the Bcc. Mechanisms underlying the pathogenesis of Bc in lung infections and cepacia syndrome remain to be uncovered. Bc is primarily an intracellular pathogen, and encodes the type VI secretion system (T6SS) anti-host effector TecA, which is translocated into host cells. TecA is a deamidase that inactivates multiple Rho GTPases, including RhoA. Inactivation of RhoA by TecA triggers assembly of the pyrin inflammasome, leading to secretion of proinflammatory cytokines such as IL-1{beta} from macrophages. Previous work with the Bc clinical isolate J2315 showed that TecA increases immunopathology during acute lung infection in C57BL/6 mice and suggested that this effector acts as a virulence factor by triggering assembly of the pyrin inflammasome. Here, we extend these results using a second Bc clinical isolate, AU1054, to demonstrate that TecA exacerbates weight loss and lethality during lung infection in C57BL/6 mice and CF mice. Unexpectedly, pyrin was dispensable for TecA virulence activity in both mouse infection models. Our findings establish that TecA is a Bc virulence factor that exacerbates lung inflammation, weight loss, and lethality in a mouse lung infection model. ImportanceBc is often considered the most virulent species in the Bcc because of its close association with cepacia syndrome in addition to its capacity to cause chronic lung infections in CF patients (Loutet and Valvano 2010). Prior to this study virulence factors of Bc important for causing lethal disease had not been identified in a CF animal model of lung infection. Results of this study describe a CF mouse model and its use in demonstrating that the T6SS effector TecA of Bc exacerbates inflammatory cell recruitment and weight loss and is required for lethality and thus acts as a key virulence factor during lung infection. This model will be important in further studies to better understand TecAs role as a virulence factor and in investigating ways to prevent or treat Bc infections in CF patients. Additionally, TecA may be the founding member of a family of virulence factors in opportunistic pathogens.

microbiology↗

bioRxiv2021

Precursor abundance influences divergent antigen specific CD8+ T cell responses after Yersinia pseudotuberculosis foodborne infection

Primary infection of C57BL/6 mice with the bacterial pathogen Yersinia pseudotuberculosis elicits an unusually large H-2Kb-restricted CD8+ T cell response to the endogenous and protective bacterial epitope YopE69-77. To better understand the basis for this large response, the model OVA257-264 epitope was inserted into YopE in Y. pseudotuberculosis and antigen specific CD8+ T cells in mice were characterized after foodborne infection with the resulting strain. The epitope YopE69-77 elicited significantly larger CD8+ T cell populations in the small intestine, mesenteric lymph nodes (MLNs), spleen and liver between 7- and 30-days post infection, despite residing in the same protein and having a similar affinity for H-2Kb as OVA257-264. YopE-specific CD8+ T cell precursors were ~4.6 times as abundant as OVA-specific precursors in the MLNs, spleen and other lymph nodes of naive mice, explaining the dominance of YopE69-77 over OVA257-264 at early infection times. However, other factors contributed to this dominance as the ratio of YopE-specific to OVA-specific CD8+ T cells increased between 7- and 30-days post infection. We also compared the YopE-specific and OVA-specific CD8+ T cells generated during infection for effector and memory phenotypes. Significantly higher percentages of YopE-specific cells were characterized as short short-lived effectors while higher percentages of OVA-specific cells were memory precursor effectors at day 30 post infection in spleen and liver. Our results suggest that a large precursor number contributes to the dominance and effector and memory functions of CD8+ T cells generated to the protective YopE69-77 epitope during Y. pseudotuberculosis infection of C57BL/6 mice.

immunology↗