Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

UpFrontPSMA: a randomized phase 2 study of sequential 177 Lu-PSMA-617 and docetaxel vs docetaxel in metastatic hormone-naïve prostate cancer (clinical trial protocol).

OBJECTIVE: To assess the activity and safety of sequential lutetium-177 (177 Lu)-PSMA-617 and docetaxel vs docetaxel on a background of androgen deprivation therapy (ADT) in men with de novo metastatic hormone-naïve prostate cancer (mHNPC). PATIENTS AND METHODS: UpFrontPSMA (NCT04343885) is an open-label, randomized, multicentre, phase 2 trial, recruiting 140 patients at 12 Australian centres. Key eligibility criteria include: prostate cancer with a histological diagnosis within 12 weeks of screening commencement; prostate-specific antigen (PSA) >10 ng/mL at diagnosis; ≤4 weeks on ADT; evidence of metastatic disease on computed tomography (CT) and/or bone scan; high-volume prostate-specific membrane antigen (PSMA)-avid disease with a maximum standardized uptake value >15; and absence of extensive discordant fluorodeoxyglcuose (FDG)-positive, PSMA-negative disease. 68 Ga-PSMA-11 and 18 F-FDG positron-emission tomography (PET)/CT undergo central review to determine eligibility. Patients are randomized 1:1 to experimental treatment, Arm A (177 Lu-PSMA-617 7.5GBq q6w × 2 cycles followed by docetaxel 75 mg/m2 q3w × 6 cycles), or standard-of-care treatment, Arm B (docetaxel 75 mg/m2 q3w × 6 cycles). All patients receive continuous ADT. Patients are stratified based on disease volume on conventional imaging and duration of ADT at time of registration. The primary endpoint is the proportion of patients with undetectable PSA (≤0.2 ng/L) at 12 months after study treatment commencement. Secondary endpoints include safety, time to castration resistance, overall survival, PSA and radiographic progression-free survival, objective tumour response rate, early PSMA PET response, health-related quality of life, and frequency and severity of adverse events. Enrolment commenced in April 2020. RESULTS AND CONCLUSIONS: The results of this trial will generate data on the activity and safety of 177 Lu-PSMA-617 in men with de novo mHNPC in a randomized phase 2 design.

A phase I/II study of stereotactic radiotherapy and pembrolizumab for oligometastatic renal tumours (RAPPORT): Clinical trial protocol.

BACKGROUND: The management of oligometastatic clear cell renal cell carcinoma (ccRCC) varies widely, ranging from observation to resection or systemic therapies. Prolonged survival has been observed following resection or stereotactic ablative body radiotherapy (SABR). Immunotherapy combinations have shown survival benefits, however, toxicity is higher than that for monotherapy and complete response rates remain less than 10%. The combination of effective local therapies in conjunction with immunotherapy may provide more durable control and pre-clinical models have suggested a synergistic immune-priming effect of SABR. OBJECTIVES: and Methods: RAPPORT is a prospective, single arm, phase I/II study assessing the safety, efficacy and biological effects of single fraction SABR followed by pembrolizumab for oligometastatic ccRCC. The study will include 30 patients with histological confirmed ccRCC and 1-5 oligometastases, one or more of which must be suitable for SABR. Patients can have received prior systemic therapy but not prior immunotherapy. A single 20Gy of SABR is followed 5 days later by 8 cycles of 200 mg pembrolizumab, every 3 weeks. Adverse events are recorded using CTCAE V4.03 and tumour response evaluated by Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1). Tumour tissue and peripheral blood samples will be collected pre-, during and post-treatment to assess longitudinal changes in immune subsets. OUTCOMES AND SIGNIFICANCE: The RAPPORT study will provide important safety and early efficacy data on the combination of SABR and pembrolizumab in oligometastatic ccRCC and will provide an insight into the underlying biological effects of combination therapy. TRIAL REGISTRATION: clinicaltrials.gov ID NCT02855203.

EirA Is a Novel Protein Essential for Intracellular Replication of Coxiella burnetii.

The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication. This protein, subsequently named EirA (essential for intracellular replication A), is indispensable for intracellular replication and virulence, as demonstrated by infection of human cell lines and in vivo infection of Galleria mellonella The putative N-terminal signal peptide is essential for protein function but is not required for localization of EirA to the bacterial inner membrane compartment and axenic culture supernatant. In the absence of EirA, C. burnetii remains viable but nonreplicative within the host phagolysosome, as coinfection with C. burnetii expressing native EirA rescues the replicative defect in the mutant strain. In addition, while the bacterial ultrastructure appears to be intact, there is an altered metabolic profile shift in the absence of EirA, suggesting that EirA may impact overall metabolism. Most strikingly, in the absence of EirA, Dot/Icm effector translocation was inhibited even when EirA-deficient C. burnetii replicated in the wild type (WT)-supported Coxiella containing vacuoles. EirA may therefore have a novel role in the control of Dot/Icm activity and represent an important new therapeutic target.

Coxiella burnetii utilizes both glutamate and glucose during infection with glucose uptake mediated by multiple transporters.

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome. In order to understand how C. burnetii survives within this intracellular niche, we have investigated the carbon metabolism of both intracellular and axenically cultivated bacteria. Both bacterial populations were shown to assimilate exogenous [13C]glucose or [13C]glutamate, with concomitant labeling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilization but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters which may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labeling approach, we identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C-label incorporation but did not impact intracellular replication or virulence. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected.

De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever.

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative l-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant WT GST-NadB has l-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.