Biology subjects

Bliska, J. B.

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

10 recordsLinked to original sources

bioRxiv2026

A bistable type III secretion system is primed and loaded in Pseudomonas aeruginosa hypervirulent isolates with positively selected codon changes in exsA or exsD

Pseudomonas aeruginosa has multiple virulence factors that promote airway infections in people with immune deficiencies. Most clinical isolates from human infections have functional type III secretion systems (T3SSs) that are subject to bistable regulation and secrete the cytotoxic effector ExoS into neutrophils. During airway infection in people with cystic fibrosis (CF), P. aeruginosa maintains bistable regulation or genetically adapts by inactivating or hyperactivating T3SS expression. CF clinical isolates with hyperactive T3SSs have codon changes in the transcriptional regulators, exsA or exsD. It is not known how these mutations: 1) arise, 2) promote hypervirulence, or 3) impact bistable T3SS regulation. To understand how these mutants arise, ~24,000 genomes in the NCBI Pathogens database were searched for positively selected mutations in exs genes based on nonsynonomous to synonymous codon change ratios. Missense and nonsense mutations were detected in exsA (47) or exsD (45) the majority of which are predicted to be positively selected. To determine how one of these codon changes promotes hypervirulence, an exsAT48I isolate was used to establish lung infections in mice engineered with a CF genotype. The exsAT48I mutant and an exsA+ control had similar lung burdens, but the former caused exacerbated weight loss and lung inflammation, suggesting immunopathology from phagocyte death as the cause of hypervirulence. Two additional hyperactive mutants were identified by screening a small collection of isolates from children with CF. Immunoblotting and fluorescence microscopy showed that hyperactive exsA or exsD mutants increased production of T3SS injectisomes and effectors, indicating they have locked on (primed and loaded) phenotypes. ExsA dimer modeling revealed that codon changes like T48I in the {beta}-barrel domain cause a conformational change that hyperactivates the transcription factor. These results define that hyperactive mutants are positively selected, hypervirulent due to lung inflammation, and have primed and loaded T3SSs.

microbiology↗

bioRxiv2026

Inflammasome Activation and IL-1β Release in Alveolar Macrophages Infected with Pseudomonas aeruginosa is Reduced in Hypoxia

Pseudomonas aeruginosa is an important agent of acute or chronic airway infections. A complex set of genetic and environmental factors determine the outcome of a P. aeruginosa airway infection. P. aeruginosa can undergo genetic adaptation to fine tune the expression of a type III secretion system (T3SS) and other virulence factors in response to selective pressures encountered during infection. Genetic and environmental factors predispose many patient groups for P. aeruginosa infections, including people with cystic fibrosis (pwCF). CF is a genetic disorder resulting in areas of hypoxia and thick mucus that fosters P. aeruginosa airway infection. Resident alveolar macrophages (AMs) help coordinate immune responses to P. aeruginosa in airways by producing proinflammatory cytokines such as IL-1{beta}, which can be important for infection resistance. AMs infected with laboratory strains of P. aeruginosa detect the T3SS and activate the NLRC4 inflammasome, resulting in IL-1{beta} release. Studies of inflammasome responses in AMs to a clinical CF isolate of P. aeruginosa have not been reported. Here, we characterized the CF clinical isolate DH1137 and found that it has a downregulated but functional T3SS, is adapted to grow in hypoxia, and induces significant production of the inflammasome cytokines IL-1{beta} and IL-1 during lung infection in a CF mouse model. Inflammasome gene expression was primed in AMs by LPS stimulation, and upon DH1137 infection these cells released IL-1[beta]. Intriguingly, we found that infection of AMs with DH1137 in hypoxia resulted in significantly dampened inflammasome activation compared to normoxia. These data describe a new mechanism that P. aeruginosa may exploit to evade immune detection by tissue-resident lung macrophages in the context of CF.

immunology↗

bioRxiv2026

The Yersinia pestis virulence effector YopM binds to a key regulatory site on the human pyrin death domain to inhibit inflammasome activation and effector-triggered immunity

Bacteria in the genus Yersinia use a type III secretion system to inject several effectors into host cells to disrupt multiple signaling processes. Two effectors disrupt host signaling by inactivating RhoA GTPases. Inactivation of RhoA triggers pyrin inflammasome assembly in infected phagocytes leading to a protective immune response. A third protein, YopM, is an essential virulence factor that counteracts effector-triggered immunity by inactivating pyrin. YopM has a leucine-rich repeat (LRR) domain and hijacks host kinases to inactivate pyrin by phosphorylation. Previously, the LRR domain of Yersinia pestis YopM was implicated in binding to an N-terminal region of human pyrin, which includes the eponymous pyrin domain (PYD), a member of the death domain family. However, the interaction mechanism was undefined. Using a bacterial two-hybrid assay, protein biochemistry and X-ray crystallography, we determined that the concave surface of the YopM LRR domain binds to the PYD. Identification of critical residues in the interaction revealed that YopM binds PYD R42, an amino acid important for positive and negative regulation of pyrin. YopM binding to the PYD was reduced by R42W, a codon change associated with the autoinflammatory disease Familial Mediterranean Fever (FMF). Furthermore, we show that YopM codon substitution variants defective for PYD binding fail to inhibit the pyrin inflammasome in human monocytes infected with Yersinia. In addition, we found that PYD binding is dispensable for YopM to inhibit the inflammasome in murine macrophages, suggesting this effector uses a distinct mechanism to target mouse pyrin. These results define how Y. pestis YopM binds the human PYD and provide insights into how this interaction likely selected for pyrin gain of function variants resulting in FMF. SIGNIFICANCEPyrin is an inflammasome sensor encoded by the MEFV gene that is notable for its role in the autoinflammatory disease Familial Mediterranean Fever (FMF) and immunity to the plague agent Yersinia pestis. Y. pestis normally prevents inflammasome activation using the virulence factor YopM which binds pyrin and inhibits effector-triggered immunity. Gain of function mutations in MEFV that cause FMF were likely selected during historic plague pandemics to counteract YopM. Here, we defined the molecular mechanism of the YopM-pyrin interaction, which revealed that the effector binds to a key site of positive and negative regulation on the N-terminal death domain. These findings have important implications for understanding how YopM promotes pathogenesis and likely selected for gain of function mutations in MEFV.

microbiology↗

bioRxiv2025

Sex and regional effects of Bacteroides in the gut

Bacteroides spp. is a key immune-programming microbe in healthy individuals - these bacteria have been shown to be reduced in abundance across a variety of disease states. Our study investigated the systemic and region-specific responses to Bacteroides colonization in the gut, including sex-related differences, in mice. Utilizing C57BL/6 mice, we administered Bacteroides to conventional, antibiotic-treated mice, then assessed this microbes influence on the gut microbiota composition and inflammatory responses following an airway lipopolysaccharide challenge to assess effects on the gut-lung axis. We found that Bacteroides successfully colonizes the intestinal tract of antibiotic-treated mice, particularly the colon lumen of the large intestine as evidenced by 16S rRNA amplicon gene sequencing and culturing. Differential gene expression analysis using NanoString technology revealed significant immune response variations across the gut regions, with notable differences in adaptive immune response genes. A striking sex-dependent outcome was noted in the regulation of atg12 in the cecum, potentially enhancing autophagic function, particularly in female mice. Additionally, Bacteroides intestinal colonization was associated with altered expression of macrophage markers such as cd163, cd84, and ms4a4a, which may reflect shifts in the macrophage profile within the cecum. These findings pave the way for novel therapeutic approaches that leverage microbial impacts on gut and systemic health, offering a deeper understanding of Bacteroides role in human health and disease. Our study highlights the necessity for further research to elucidate the intricate relationships between gut microbiota, host immunity, biological sex and their interplay. ImportanceThis research marks an investigation into how specific microbiota, like Bacteroides, regulate host responses across different gut regions to influence systemic health. By dissecting the impact of Bacteroides across multiple regions of the intestinal tract, this study offers new insights into the localized and whole-body effects of this important immune-programming microbe. Such an understanding is crucial as it helps in unraveling the complex interplay between gut microbes and the hosts immune system. This research helps bridge the gap between local intestinal ecology and overall systemic health, addresses important questions relevant to the gut-lung axis, and helps pave the way for innovative therapies.

microbiology↗

bioRxiv2024

ExoS Effector in Pseudomonas aeruginosa Hyperactive Type III Secretion System Mutant Promotes Enhanced Plasma Membrane Rupture in Neutrophils

Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a large percentage of airway infections that cause morbidity and mortality in immunocompromised patients, especially those with cystic fibrosis (CF). One important P. aeruginosa virulence factor is a type III secretion system (T3SS) that translocates effectors into host cells. ExoS is a T3SS effector with ADP ribosyltransferase (ADPRT) activity. The ADPRT activity of ExoS promotes P. aeruginosa virulence by inhibiting phagocytosis and limiting the oxidative burst in neutrophils. The P. aeruginosa T3SS also translocates flagellin, which can activate the NLRC4 inflammasome, resulting in: 1) gasdermin-D (GSDMD) pores, release of IL-1{beta} and pyroptosis; and 2) histone 3 citrullination (CitH3) and decondensation and expansion of nuclear DNA into the cytosol. However, recent studies with the P. aeruginosa laboratory strain PAO1 indicate that ExoS ADPRT activity inhibits activation of the NLRC4 inflammasome in neutrophils. Here, an ExoS+ CF clinical isolate of P. aeruginosa with a hyperactive T3SS was identified. Variants of the hyperactive T3SS mutant or PAO1 were used to infect neutrophils from C57BL/6 mice or mice engineered to have a CF genotype or a defect in inflammasome assembly. Responses to NLRC4 inflammasome assembly or ExoS ADPRT activity were assayed, results of which were found to be similar for C57BL/6 or CF neutrophils. The hyperactive T3SS mutant had enhanced resistance to neutrophil killing, like previously identified hypervirulent P. aeruginosa isolates. ExoS ADPRT activity in the hyperactive T3SS mutant regulated inflammasome and nuclear DNA decondensation responses like PAO1 but promoted enhanced CitH3 and plasma membrane rupture (PMR). Glycine supplementation inhibited PMR caused by the hyperactive T3SS mutant, suggesting ninjurin-1 is required for this process. These results identify enhanced neutrophil PMR as a pathogenic activity of ExoS ADPRT in a hypervirulent P. aeruginosa isolate.

microbiology↗

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↗