Direct Culture-Independent Strain Typing of Burkholderia cepacia Complex in Sputum Samples from Patients with Cystic Fibrosis ▿

We examined if multilocus sequence typing (MLST), a method for genotyping and species identification of Burkholderia cepacia complex bacteria, could be applied directly to cystic fibrosis sputum. The redesigned nested-PCR MLST format was successfully used to accurately identify strains in 23 sputum samples, of which 8 were culture negative

Widespread clone of Burkholderia cenocepacia in cystic fibrosis patients in the Czech Republic

The morbidity and mortality rates in patients with cystic fibrosis (CF) are significantly affected by infections with Burkholderia cepacia complex. In a Czech CF Centre, the prevalence of the infection reached up to 30 %, with the majority of patients found to be infected with Burkholderia cenocepacia (formerly genomovar III of the Burkholderia cepacia complex). Since B. cenocepacia is associated with patient-to-patient transmission and epidemic outbreaks among CF patients, this study sought to examine the epidemiological relatedness between the Czech isolates belonging to the genomovar-homogeneous group. Eighty-three clinical isolates recovered from 67 CF patients were analysed using a random amplified polymorphic DNA (RAPD) assay and macrorestriction typing (SpeI and XbaI) followed by PFGE. A single predominant banding pattern shared by multiple isolates was detected, although SpeI-generated PFGE results yielded a higher rate of inter-pattern variability in comparison to the more uniform RAPD and XbaI-generated PFGE results for this clone. Both typing systems also showed that only three out of 67 patients harboured strains distinct from the major strain type. The dominant clone was characterized by PCR positivity for the B. cepacia epidemic strain marker, PCR negativity for the cable pilin subunit gene and close genetic relatedness to the epidemic strain of RAPD 01 type previously identified in Canada

Oxidative stress of Burkholderia cenocepacia induces insertion sequence-mediated genomic rearrangements that interfere with macrorestriction-based genotyping

Burkholderia cenocepacia can cause serious infections and epidemics in patients with cystic fibrosis (CF). A CF population in the Czech Republic experienced an epidemic outbreak caused by a B. cenocepacia ST-32 strain. The clonality of the isolates was evident by multilocus sequence typing; however, fingerprinting profiles obtained by pulsed-field gel electrophoresis (PFGE) showed substantial band variability. We investigated whether the PFGE pattern diversity resulted from genomic rearrangements mediated by insertion sequences (IS); in addition, we determined whether stressful growth conditions altered the transposition activity of these IS. DNA probes for IS commonly found in B. cenocepacia were designed using the B. cenocepacia J2315 genome. Southern hybridization analysis of ST-32 isolates demonstrated diversity in both the copy number and the insertion site for a homologue of ISBcen20. Movement of the ISBcen20 homologue was detected when the ST-32 isolate CZ1238 was exposed to oxidative stress (growth in the presence of H2O2). PFGE analysis of CZ1238 derivatives exposed to oxidative stress demonstrated genomic rearrangements. Interestingly, when the closely related B. cenocepacia strain J2315 was exposed to oxidative stress, no movement of ISBcen20 was detected. Since frameshift mutations are present within the transposases of all copies of this IS in J2315, our data suggest that the transposase is inactive. In summary, we have demonstrated for the first time that IS movement can be mediated by oxidative stress and can lead to genomic rearrangements in the CF pathogen B. cenocepacia. These IS movements may alter the PFGE fingerprints of isolates that are clonal by other typing methods

Caspase-2 and oxidative stress underlie the immunogenic potential of high hydrostatic pressure-induced cancer cell death

High hydrostatic pressure (HHP) promotes key characteristics of immunogenic cell death (ICD), in thus far resembling immunogenic chemotherapy and ionizing irradiation. Here, we demonstrate that cancer cells succumbing to HHP induce CD4(+) and CD8(+) T cell-dependent protective immunity in vivo. Moreover, we show that cell death induction by HHP relies on the overproduction of reactive oxygen species (ROS), causing rapid establishment of the integrated stress response, eIF2α phosphorylation by PERK, and sequential caspase-2, -8 and -3 activation. Non-phosphorylatable eIF2α, depletion of PERK, caspase-2 or -8 compromised calreticulin exposure by cancer cells succumbing to HHP but could not inhibit death. Interestingly, the phagocytosis of HHP-treated malignant cells by dendritic cells was suppressed by the knockdown of caspase-2 in the former. Thus, caspase-2 mediates a key function in the interaction between dying cancer cells and antigen presenting cells. Our results indicate that the ROS→PERK→eIF2α→caspase-2 signaling pathway is central for the perception of HHP-driven cell death as immunogenic.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=koni20status: publishe

Caspase-2 and oxidative stress underlie the immunogenic potential of high hydrostatic pressure-induced cancer cell death

International audienceHigh hydrostatic pressure (HHP) promotes key characteristics of immunogenic cell death (ICD), in thus far resembling immunogenic chemotherapy and ionizing irradiation. Here, we demonstrate that cancer cells succumbing to HHP induce CD4+ and CD8+ T cell-dependent protective immunity in vivo. Moreover, we show that cell death induction by HHP relies on the overproduction of reactive oxygen species (ROS), causing rapid establishment of the integrated stress response, eIF2α phosphorylation by PERK, and sequential caspase-2, -8 and -3 activation. Non-phosphorylatable eIF2α, depletion of PERK, caspase-2 or -8 compromised calreticulin exposure by cancer cells succumbing to HHP but could not inhibit death. Interestingly, the phagocytosis of HHP-treated malignant cells by dendritic cells was suppressed by the knockdown of caspase-2 in the former. Thus, caspase-2 mediates a key function in the interaction between dying cancer cells and antigen presenting cells. Our results indicate that the ROS→PERK→eIF2α→caspase-2 signaling pathway is central for the perception of HHP-driven cell death as immunogenic

M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer

International audienceBackground: The immunological microenvironment of primary high-grade serous carcinomas (HGSCs) has a major impact on disease outcome. Conversely, little is known on the microenvironment of metastatic HGSCs and its potential influence on patient survival. Here, we explore the clinical relevance of the immunological configuration of HGSC metastases.Methods: RNA sequencing was employed on 24 paired primary tumor microenvironment (P-TME) and metastatic tumor microenvironment (M-TME) chemotherapy-naive HGSC samples. Immunohistochemistry was used to evaluate infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ (lysosomal-associated membrane protein 3) dendritic cells (DCs), NKp46+ (natural killer) cells and CD68+CD163+ M2-like tumor-associated macrophages (TAMs), abundance of PD-1+ (programmed cell death 1), LAG-3+ (lymphocyte-activating gene 3) cells, and PD-L1 (programmed death ligand 1) expression in 80 samples. Flow cytometry was used for functional assessments on freshly resected HGSC samples.Results: 1468 genes were differentially expressed in the P-TME versus M-TME of HGSCs, the latter displaying signatures of extracellular matrix remodeling and immune infiltration. M-TME infiltration by immune effector cells had little impact on patient survival. Accordingly, M-TME-infiltrating T cells were functionally impaired, but not upon checkpoint activation. Conversely, cytokine signaling in favor of M2-like TAMs activity appeared to underlie inhibited immunity in the M-TME and poor disease outcome.Conclusions: Immunosuppressive M2-like TAM infiltrating metastatic sites limit clinically relevant immune responses against HGSCs