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Sequencing of plasma microbial cell-free DNA (mcfDNA) has gained increased acceptance as a valuable adjunct to standard-of-care testing for diagnosis of infections throughout the body. Here, we report the analytical and clinical validation of a novel application of mcfDNA sequencing, the non-invasive detection of seven common antimicrobial resistance (AMR) genetic markers in 18 important pathogens. The AMR markers include SCC mec , mecA, mecC , vanA, vanB , bla CTX-M , and bla KPC . The AMR markers were computationally linked to the pathogens detected. Analytical validation showed high reproducibility (100%), inclusivity (54 to 100%), and exclusivity (100%). Clinical accuracy was assessed with 114 unique plasma samples from patients at seven study sites with concordant culture results for target bacteria from a variety of specimen types and correlated with available phenotypic antimicrobial susceptibility test results and genotypic results. The positive percent agreement (PPA), negative percent agreement (NPA), and diagnostic yield (DY) were estimated for each AMR marker. DY was defined as the percentage of tests that yielded an actionable result of either detected or not detected. The results for the combination of SCC mec and mecA for staphylococci were PPA 19/20 (95.0%), NPA 21/22 (95.4%), DY 42/60 (70.0%); vanA for enterococci were PPA 3/3 (100%), NPA 2/2 (100%), DY 5/6 (83.3%); bla CTX-M for gram-negative bacilli were PPA 5/6 (83.3%), NPA 29/29 (100%), DY 35/49 (71.4%); and bla KPC for gram-negative bacilli were PPA 0/2 (0%), NPA: 23/23 (100%), DY 25/44 (56.8%). The addition of AMR capability to plasma mcfDNA sequencing should provide clinicians with an effective new culture-independent tool for optimization of therapy., Importance: This manuscript is ideally suited for the Innovative Diagnostic Methods sections as it reports the analytical and clinical validation of a novel application of plasma microbial cell-free DNA sequencing for direct detection of seven selected antimicrobial resistance markers in 18 target pathogens. Clearly, it has potential clinical utility in optimizing therapy and was incorporated into the Karius test workflow in September 2023. In addition, the workflow could readily be adapted to expand the number of target bacteria and antimicrobial resistance markers as needed., Competing Interests: All employees of Karius, which markets the commercial test evaluated in this study, shared responsibilities for study design, analysis of the data, and writing and editing of the manuscript. John Joseph Farrell (OSF Healthcare), Sanjeet Dadwal (City of Hope), Amy L. Carr (AdventHealth Orlando), Arryn Craney (Orlando Health), Matthew Pike (Carle Foundation Hospital), and James B. Wood (Indiana University School of Medicine) received grant support from Karius to support the collection and curation of orthogonal data that were fundamental to the clinical validation and reviewed the manuscript.

Background: Plasma microbial cell-free DNA (mcfDNA) sequencing can establish the etiology of multiple infectious syndromes by identifying microbial DNA in plasma. However, data are needed to define the clinical scenarios where this tool offers the highest clinical benefit., Methods: We conducted a prospective multicenter observational study that evaluated the impact of plasma mcfDNA sequencing compared with usual care testing among adults with hematologic malignancies. This is a secondary analysis of an expanded cohort that evaluated the clinical utility of plasma mcfDNA sequencing across prespecified and adjudicated outcomes. We examined the percentage of participants for whom plasma mcfDNA sequencing identified a probable cause of pneumonia or clinically relevant nonpneumonia infection. We then assessed potential changes in antimicrobial therapy based on plasma mcfDNA sequencing results and the potential for early mcfDNA testing to avoid bronchoscopy and its associated adverse events., Results: Of 223 participants, at least 1 microbial detection by plasma mcfDNA sequencing was adjudicated as a probable cause of pneumonia in 57 (25.6%) and a clinically relevant nonpneumonia infection in 88 (39.5%). A probable cause of pneumonia was exclusively identified by plasma mcfDNA sequencing in 23 (10.3%) participants. Antimicrobial therapy would have changed for 41 (18.4%) participants had plasma mcfDNA results been available in real time. Among the 57 participants with a probable cause of pneumonia identified by plasma mcfDNA sequencing, bronchoscopy identified no additional probable cause of pneumonia in 52 (91.2%)., Conclusions: Plasma mcfDNA sequencing could improve management of both pneumonia and other concurrent infections in immunocompromised patients with suspected pneumonia., Competing Interests: Potential conflicts of interest. Deng B. Madut reports receiving consulting fees from Karius. Roy F. Chemaly reports being a scientific advisor or consultant for ADMA Biologics, Pulmotec, Janssen, Merck/MSD, Partner Therapeutics, Takeda, Shinoigi, Genentech, Astellas, Adagio Therapeutics, Oxford Immunotec, Qiagen, Karius, Moderna, and Ansun Pharmaceuticals and received research grants paid to his institution from NIH/NCI, Merck/MSD, Karius, AiCuris, Ansun Pharmaceuticals, Takeda, Genentech, Oxford Immunotec, Freestyle, and Eurofins-Viracor. Sanjeet Dadwal reports research grants paid to his institution from Karius, Merck, Gilead, Ansun Biopharma, Allovir. He has served on advisory boards for Merck, Takeda, Allovir, and Aseptiscope, Inc. He is on speakers’ bureaus of Merck, Astellas, and Takeda and holds stock options in Aseptiscope, Inc. Joshua A. Hill reports research grants paid to his institution from Karius and consulting for Karius. He reports no other relevant conflicts of interest. Yeon Joo Lee has served as an investigator for Astellas, Karius, AiCuris, and Scynexis and has received research grant support from Merck & Co. Inc. Ghady Haidar is a recipient of research grants from Allovir, Karius, and AstraZeneca and also serves on the scientific advisory boards of Karius and AstraZeneca. Alfred Luk reports no relevant conflicts of interest. Alexander Drelick reports no relevant conflicts of interest. Peter V. Chin-Hong reports no relevant conflicts of interest. Esther Benamu reports no relevant conflicts of interest. Fareed Khawaja reports an honorarium from Medscape and research support from Eurofins-Viracor. Deepa Nanayakkara reports no relevant conflicts of interest. Genovefa A. Papanicolaou reports research support from MSD and Takeda/Shire and consulting and other fees from AlloVir, Amplyx, SLC Behring, Cidara, MSD, Takeda, and Octapharma. Catherine Butkus Small reports research grant support from NIH, GlaxoSmithKline, ViiV, Abbott, Merck, Gilead, Chimerix, Shire/Takeda, Schering-Plough, Ablynx, Janssen, Ansun Biopharma, and Karyopharm Therapeutics. Monica Fung reports research grant support from NIH. Michelle A. Barron reports no relevant conflicts of interest. Thomas Davis reports no relevant conflicts of interest. Micah T. McClain reports being a consultant for UpToDate and a co-inventor of patents on Methods to Detect and Treat Acute Respiratory Infections. Eileen K. Maziarz reports no relevant conflicts of interest. Armando D. Bedoya reports no relevant conflicts of interest. Daniel L. Gilstrap reports no relevant conflicts of interest. Jamie L. Todd reports advisory board participation for Sanofi, Natera, Theravance, and Altavant Sciences (now Enzyvant Therapeutics), in addition to grants to the institution from Boehringer-Ingelheim, AstraZeneca, and CareDx. Christina E. Barkauskas reports no relevant conflicts of interest. Madeleine R. Heldman reports receiving consulting fees from Karius and Medacorp. Robert Bigelow reports stock holdings in Merck, Johnson & Johnson, COVIDien, Pfizer, Sanofi, McKesson, and Viatris and consulting fees from Elixir. Jeffrey D. Leimberger reports no relevant conflicts of interest. Ephraim L. Tsalik reports personal fees from Biomeme, stock options in Danaher, and being an employee of Danaher Corp. Olivia Wolf reports no relevant conflicts of interest. Mona Mughar reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Constance Lau reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Nicholas Noll reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Desiree Hollemon reports being a former employee of Karius Inc., Redwood City, California. Radha Duttagupta reports being a former employee of Karius Inc., Redwood City, California. Daniel S. Lupu reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Sivan Bercovici reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Bradley A. Perkins reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Timothy A. Blauwkamp reports being an employee of Karius Inc., Redwood City, California, and may own stock options. Vance G. Fowler Jr. reports personal fees from Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny, Amphliphi Biosciences, Integrated Biotherapeutics, C3J, Armata, Valanbio, Akagera, Aridis, and Roche; grants from NIH, MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Merck, Medical Biosurfaces, Locus, Affinergy, Contrafect, Karius, Genentech, Regeneron, Deep Blue, Basilea, and Janssen; royalties from UpToDate; stock options from Valanbio and ArcBio; honoraria from the Infectious Diseases Society of America for his service as Associate Editor of Clinical Infectious Diseases; and a pending patent on sepsis diagnostics. Thomas L. Holland reports being an advisor and consultant to Aridis, Basilea Pharmaceutica, Karius, and Lysovant and royalties from UpToDate. Stephen P. Bergin reports being a consultant to C3J Therapeutics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: The 2022 global outbreak of Monkeypox virus (MPXV) highlighted challenges with polymerase chain reaction detection as divergent strains emerged and atypical presentations limited the applicability of swab sampling. Recommended testing in the United States requires a swab of lesions, which arise late in infection and may be unrecognized. We present MPXV detections using plasma microbial cell-free DNA (mcfDNA) sequencing., Methods: Fifteen plasma samples from 12 case-patients were characterized through mcfDNA sequencing. Assay performance was confirmed through in silico inclusivity and exclusivity assessments. MPXV isolates were genotyped using mcfDNA, and phylodynamic information was imputed using publicly available sequences., Results: MPXV mcfDNA was detected in 12 case-patients. Mpox was not suspected in 5, with 1 having documented resolution of mpox >6 months previously. Six had moderate to severe mpox, supported by high MPXV mcfDNA concentrations; 4 died. In 7 case-patients, mcfDNA sequencing detected coinfections. Genotyping by mcfDNA sequencing identified 22 MPXV mutations at 10 genomic loci in 9 case-patients. Consistent with variation observed in the 2022 outbreak, 21 of 22 variants were G > A/C > T. Phylogenetic analyses imputed isolates to sublineages arising at different time points and from different geographic locations., Conclusions: We demonstrate the potential of plasma mcfDNA sequencing to detect, quantify, and, for acute infections with high sequencing coverage, subtype MPXV using a single noninvasive test. Sequencing plasma mcfDNA may augment existing mpox testing in vulnerable patient populations or in patients with atypical symptoms or unrecognized mpox. Strain type information may supplement disease surveillance and facilitate tracking emerging pathogens., Competing Interests: Potential conflicts of interest. S. Y. P., M. S. L., K. B., N. N., G. B., T. A. B., F. S. N., B. A. P., and S. B. are employees of Karius, Inc. R. O. was an employee of Karius, Inc, at the time the project was conceived and executed. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Pneumonia is a common cause of morbidity and mortality, yet a causative pathogen is identified in a minority of cases. Plasma microbial cell-free DNA sequencing may improve diagnostic yield in immunocompromised patients with pneumonia., Methods: In this prospective, multicenter, observational study of immunocompromised adults undergoing bronchoscopy to establish a pneumonia etiology, plasma microbial cell-free DNA sequencing was compared to standardized usual care testing. Pneumonia etiology was adjudicated by a blinded independent committee. The primary outcome, additive diagnostic value, was assessed in the Per Protocol population (patients with complete testing results and no major protocol deviations) and defined as the percent of patients with an etiology of pneumonia exclusively identified by plasma microbial cell-free DNA sequencing. Clinical additive diagnostic value was assessed in the Per Protocol subgroup with negative usual care testing., Results: Of 257 patients, 173 met Per Protocol criteria. A pneumonia etiology was identified by usual care in 52/173 (30.1%), plasma microbial cell-free DNA sequencing in 49/173 (28.3%) and the combination of both in 73/173 (42.2%) patients. Plasma microbial cell-free DNA sequencing exclusively identified an etiology of pneumonia in 21/173 patients (additive diagnostic value 12.1%, 95% confidence interval [CI], 7.7% to 18.0%, P < .001). In the Per Protocol subgroup with negative usual care testing, plasma microbial cell-free DNA sequencing identified a pneumonia etiology in 21/121 patients (clinical additive diagnostic value 17.4%, 95% CI, 11.1% to 25.3%)., Conclusions: Non-invasive plasma microbial cell-free DNA sequencing significantly increased diagnostic yield in immunocompromised patients with pneumonia undergoing bronchoscopy and extensive microbiologic and molecular testing., Clinical Trials Registration: NCT04047719., Competing Interests: Potential conflicts of interest. S. P. B. reports being a consultant to C3J Therapeutics. R. F. C. reports being a scientific advisor or consultant for ADMA Biologics, Pulmotec, Janssen, Merck/MSD, Partner Therapeutics, Takeda, Shinoigi, Genentech, Astellas, Adagio Therapeutics, Oxford Immunotec, Qiagen, Karius, and Ansun Pharmaceuticals and received research grants paid to his institution from National Institutes of Health (NIH)/National Cancer Institute (NCI), Merck/Merck, Sharp & Dohme (MSD), Karius, AiCuris, Ansun Pharmaceuticals, Takeda, Genentech, Oxford Immunotec, Freestyle, and Eurofins-Viracor, and reports payment or honoraria as a speaker for Qiagen and Viracor-Eurofins. S. D. reports research grants paid to his institution from Karius, Merck, Gilead, Ansun Biopharma, Allovir, Geovax. He has served on advisory boards of Merck, Takeda, Allovir, and Aseptiscope, Inc. He is on speaker's bureau of Merck, Astellas and Takeda, and reports payment or honoraria from Viracor for educational event at ID week 2022 and from MJH for cytomegalovirus (CMV) symposium at American Society of Hematology (ASH) 2022. He has stock options in Aseptiscope, Inc, Matinas Biopharma, and Cidara Therapeutics. He reports consulting fees from Merck and Allovir; support for attending meetings and/or travel from American Society of Transplantation and Cellular Therapy—Tandem 2022 and 2023 (registration credit), ID week 2022—Infectious Diseases Society of America (IDSA) (partial support—airfare), and Immunocompromised Host Society (ICHS) 2022 (air fare and hotel expenses); Patent # 9416395, Assigned to City of Hope. Inventors—Markus Kalkum, Karine Bagramyan, Diana Diaz-Arevalo, James I. Ito, Sanjeet S. Dadwal (assigned to City of Hope) and US Provisional Patent Application no. 63/067,855: Inventors—Markus Kalkum, Daniel Roeth, Sanjeet S. Dadwal (assigned to City of Hope); a role as Chair of Transplant Infectious Disease Special Interest Group of American Society of Transplant and Cellular Therapy (ASTCT). J. A. H. reports research grants paid to his institution from Karius and consulting for Karius and support for travel to advisory board meeting from Karius. Y. J. L. has served as an investigator for Astellas, Karius, AiCuris, and Scynexis and has received research grant support from Merck & Co Inc. G. H. is a recipient of research grants from Allovir, Karius, and AstraZeneca and also serves on the scientific advisory boards of and receives consulting fees from Karius and AstraZeneca, and reports grants or contracts from NIH; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from MDOutlook, PeerView Medical Education, International AIDS Society. A. L. reports consulting fees from Karius. F. K. reports honorarium from Medscape and research support from Eurofins-Viracor. G. A. P. reports research support from MSD and Takeda/Shire and consulting and other fees from AlloVir, Amplyx, SLC Behring, Cidara, MSD, Takeda and Octapharma. C. B. S. reports research grant support from NIH, GlaxoSmithKline, ViiV, Abbott, Merck, Gilead, Chimerix, Shire/Takeda, Schering-Plough, Ablynx, Janssen, Ansun Biopharma, and Karyopharm Therapeutics; participation on Janssen Respiratory Viruses Advisory Board and Merck Scientific Input Engagement (SIE) Panel for Molnupirivir. M. F. reports research grant support from NIH, and research funding (UCSF PI Fung) from Scynexis and Vatic, Ltd. M. T. M. reports being a consultant for UpToDate and Karius and a co-inventor of patents on Methods to Detect and Treat Acute Respiratory Infections. E. K. M. reports consulting fees from Karius Inc as a Clinical Events Committee member/reviewer—received payments as clinical adjudicator for another Karius project. D. B. M. reports consulting fees from Karius Inc.. A. D. B . reports payment or honoraria from Novartis for lecture on Sepsis Watch, AI program developed at Duke. J. L. T. reports advisory board participation for Sanofi, Natera, Theravance and Altavant Sciences (now Enzyvant Therapeutics) in addition to grants to the institution from Boehringer-Ingelheim, AstraZeneca, Sanofi, NIH, Cystic Fibrosis Foundation and CareDx; and consulting fees from Sanofi. C. E. B. reports grants or contracts from American Lung Association and NIH (National Heart, Lung, and Blood Institute [NHLBI] and National Institute of Allergy and Infectious Diseases [NIAID] funded projects—not related to this project); participation as member of a Data and Safety Monitoring Board (DSMB) for VASSPR—an ICU-based trial with no relationship to this project. R. B. reports stock holdings in Merck, Johnson & Johnson, COVIDien, Pfizer, Sanofi, McKesson, and Viatris and consulting fees from Elixir. E. L. T. reports personal fees from Biomeme and Predigen, stock options in Danaher, and being an employee of Danaher Corp. O, W. reports support for attending meetings and/or travel from Karius (as Clinical Events Classification [CEC] Project Leader initiated and attended meetings with Karius for the PICKUP study); a role as CEC Project Leader coordinated CEC committee meetings within Duke Clinical Research Institute (DCRI) for the PICKUP trial. M. M. reports being an employee of Karius Inc, Redwood City, California, and may own stock options. D. H. reports being a former employee of Karius Inc, Redwood City, California, and a patent issued for related work while at Karius as employee (WO-2020106987-A1—Detection and Prediction of Infectious Disease). R. D. reports being a former employee of Karius Inc, Redwood City, California, and is owner of Karius stock options. D. S. L. reports being an employee of Karius Inc, Redwood City, California, and may own stock options. S. B. reports being an employee of Karius Inc, Redwood City, California, and may own stock options, and reports support for attending meetings and/or travel and patents planned, issued, or pending with Karius Inc. B, A. P. reports being an employee of Karius Inc, Redwood City, California, patents planned, issued or pending with Karius Inc, and may own stock options. T. A. B. reports being an employee of and having a leadership or fiduciary role with Karius Inc, Redwood City, California, and may own stock options. V. G. F. reports personal fees from Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny, Amphliphi Biosciences, Integrated Biotherapeutics; C3J, Armata, Valanbio; Akagera, Aridis, Roche, grants from NIH, MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Merck; Medical Biosurfaces; Locus; Affinergy; Contrafect; Karius; Genentech, Regeneron, Deep Blue, Basilea, Janssen; royalties from UpToDate, stock options from Valanbio and ArcBio, honoraria from IDSA for his service as Associate Editor of Clinical Infectious Diseases, and a patent sepsis diagnostics pending. T. L. H. reports being an advisor and consultant to Aridis, Basilea Pharmaceutica, Karius, and Lysovant; royalties from UpToDate; reports grants or contracts with Karius (adjudication committee) and with NIH; reports consulting fees from Pfizer and Affinivax; participation on DSMB for platform trial for SNAP Trial; and on advisory board for Basilea. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Plasma microbial cell-free DNA sequencing (mcfDNA-Seq) is a noninvasive test for microbial diagnosis of invasive mold infection (IMI). The utility of mcfDNA-Seq for predicting IMI onset and the clinical implications of mcfDNA concentrations are unknown., Methods: We retrospectively tested plasma from hematopoietic cell transplant (HCT) recipients with pulmonary IMI and ≥1 mold identified by mcfDNA-Seq in plasma collected within 14 days of clinical diagnosis. Samples collected from up to 4 weeks before and 4 weeks after IMI diagnosis were evaluated using mcfDNA-Seq., Results: Thirty-five HCT recipients with 39 IMIs (16 Aspergillus and 23 non-Aspergillus infections) were included. Pathogenic molds were detected in 38%, 26%, 11%, and 0% of samples collected during the first, second, third, and fourth week before clinical diagnosis, respectively. In non-Aspergillus infections, median mcfDNA concentrations in samples collected within 3 days of clinical diagnosis were higher in infections with versus without extrapulmonary spread (4.3 vs 3.3 log10 molecules per microliter [mpm], P = .02), and all patients (8/8) with mcfDNA concentrations >4.0 log10 mpm died within 42 days after clinical diagnosis., Conclusions: Plasma mcfDNA-Seq can identify pathogenic molds up to 3 weeks before clinical diagnosis of pulmonary IMI. Plasma mcfDNA concentrations may correlate with extrapulmonary spread and mortality in non-Aspergillus IMI., Competing Interests: Potential conflicts of interest . T. A. B. is an employee of Karius, and A. A. A. has stock equity in Karius and was employed by Karius at the time the project was conceived and executed. M. B. received consulting fees from Merck; and research support from Merck and Gilead Sciences. J. A. H received consulting fees from Karius and Pfizer; and research support from Karius, Amplyx, and Merck. C. e. F received research support from Karius. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Secondary infection (SI) diagnosis in severe COVID-19 remains challenging. We correlated metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) with clinical SI assessment, immune response, and outcomes. We classified 42 COVID-19 inpatients as microbiologically confirmed-SI (Micro-SI, n = 8), clinically diagnosed-SI (Clinical-SI, n = 13, i.e., empiric antimicrobials), or no-clinical-suspicion-for-SI (No-Suspected-SI, n = 21). McfDNA-Seq was successful in 73% of samples. McfDNA detection was higher in Micro-SI (94%) compared to Clinical-SI (57%, p = 0.03), and unexpectedly high in No-Suspected-SI (83%), similar to Micro-SI. We detected culture-concordant mcfDNA species in 81% of Micro-SI samples. McfDNA correlated with LRT 16S rRNA bacterial burden (r = 0.74, p = 0.02), and biomarkers (white blood cell count, IL-6, IL-8, SPD, all p < 0.05). McfDNA levels were predictive of worse 90-day survival (hazard ratio 1.30 [1.02-1.64] for each log 10 mcfDNA, p = 0.03). High mcfDNA levels in COVID-19 patients without clinical SI suspicion may suggest SI under-diagnosis. McfDNA-Seq offers a non-invasive diagnostic tool for pathogen identification, with prognostic value on clinical outcomes., Competing Interests: Drs. Duttagupta and Ahmed were employed by Karius, Inc at the time of the study but are no longer employed by Karius, Inc. Drs. Kitsios and Haidar have received research funding from Karius, Inc. Drs. Kitsios and Morris have received research funding from Pfizer, Inc. Dr. Haidar serves on the Karius, Inc scientific advisory board. Dr. Mellors is a consultant to AlloVir, Infectious Disease Connect, Inc., and Gilead Sciences, Inc., has received research funding from Gilead Sciences, Inc. to the University of Pittsburgh, receives compensation from Abound Bio, Inc. (unrelated to the current work) and holds shares options in Galapogos, Infectious Disease Connect, Inc., and MingMed Biotechnology Co. Ltd. (unrelated to the current work). Dr. McVerry has received research funding from Bayer Pharmaceuticals, Inc. and consulting fees from Boehringer Ingelheim, both unrelated to this work., (© 2023 The Authors.)

Background: Microbial cell-free DNA (mcfDNA) sequencing of plasma can identify the presence of a pathogen in a host. In this study, we evaluated the duration of pathogen detection by mcfDNA sequencing vs conventional blood culture in patients with bacteremia., Methods: Blood samples from patients with culture-confirmed bloodstream infection were collected within 24 hours of the index positive blood culture and 48 to 72 hours thereafter. mcfDNA was extracted from plasma, and next-generation sequencing was applied. Reads were aligned against a curated pathogen database. Statistical significance was defined with Bonferroni adjustment for multiple comparisons (P < .0033)., Results: A total of 175 patients with Staphylococcus aureus bacteremia (n = 66), gram-negative bacteremia (n = 74), or noninfected controls (n = 35) were enrolled. The overall sensitivity of mcfDNA sequencing compared with index blood culture was 89.3% (125 of 140), and the specificity was 74.3%. Among patients with bacteremia, pathogen-specific mcfDNA remained detectable for significantly longer than conventional blood cultures (median 15 days vs 2 days; P < .0001). Each additional day of mcfDNA detection significantly increased the odds of metastatic infection (odds ratio, 2.89; 95% confidence interval, 1.53-5.46; P = .0011)., Conclusions: Pathogen mcfDNA identified the bacterial etiology of bloodstream infection for a significantly longer interval than conventional cultures, and its duration of detection was associated with increased risk for metastatic infection. mcfDNA could play a role in the diagnosis of partially treated endovascular infections., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Standard testing fails to identify a pathogen in most patients with febrile neutropenia (FN). We evaluated the ability of the Karius microbial cell-free DNA sequencing test (KT) to identify infectious etiologies of FN and its impact on antimicrobial management., Methods: This prospective study (ClinicalTrials.gov; NCT02912117) enrolled and analyzed 55 patients with FN. Up to 5 blood samples were collected per subject within 24 hours of fever onset (T1) and every 2 to 3 days. KT results were compared with blood culture (BC) and standard microbiological testing (SMT) results., Results: Positive agreement was defined as KT identification of ≥1 isolate also detected by BC. At T1, positive and negative agreement were 90% (9/10) and 31% (14/45), respectively; 61% of KT detections were polymicrobial. Clinical adjudication by 3 independent infectious diseases specialists categorized Karius results as: unlikely to cause FN (N = 0); definite (N = 12): KT identified ≥1 organism also found by SMT within 7 days; probable (N = 19): KT result was compatible with a clinical diagnosis; possible (N = 10): KT result was consistent with infection but not considered a common cause of FN. Definite, probable, and possible cases were deemed true positives. Following adjudication, KT sensitivity and specificity were 85% (41/48) and 100% (14/14), respectively. Calculated time to diagnosis was generally shorter with KT (87%). Adjudicators determined real-time KT results could have allowed early optimization of antimicrobials in 47% of patients, by addition of antibacterials (20%) (mostly against anaerobes [12.7%]), antivirals (14.5%), and/or antifungals (3.6%); and antimicrobial narrowing in 27.3% of cases., Clinical Trials Registration: NCT02912117., Conclusion: KT shows promise in the diagnosis and treatment optimization of FN., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Noninvasive diagnostic options are limited for invasive mold infections (IMIs). We evaluated the performance of a plasma microbial cell-free DNA sequencing (mcfDNA-Seq) test for diagnosing pulmonary IMI after hematopoietic cell transplant (HCT)., Methods: We retrospectively assessed the diagnostic performance of plasma mcfDNA-Seq next-generation sequencing in 114 HCT recipients with pneumonia after HCT who had stored plasma obtained within 14 days of diagnosis of proven/probable Aspergillus IMI (n = 51), proven/probable non-Aspergillus IMI (n = 24), possible IMI (n = 20), and non-IMI controls (n = 19). Sequences were aligned to a database including >400 fungi. Organisms above a fixed significance threshold were reported., Results: Among 75 patients with proven/probable pulmonary IMI, mcfDNA-Seq detected ≥1 pathogenic mold in 38 patients (sensitivity, 51% [95% confidence interval {CI}, 39%-62%]). When restricted to samples obtained within 3 days of diagnosis, sensitivity increased to 61%. McfDNA-Seq had higher sensitivity for proven/probable non-Aspergillus IMI (sensitivity, 79% [95% CI, 56%-93%]) compared with Aspergillus IMI (sensitivity, 31% [95% CI, 19%-46%]). McfDNA-Seq also identified non-Aspergillus molds in an additional 7 patients in the Aspergillus subgroup and Aspergillus in 1 patient with possible IMI. Among 19 non-IMI pneumonia controls, mcfDNA-Seq was negative in all samples, suggesting a high specificity (95% CI, 82%-100%) and up to 100% positive predictive value (PPV) with estimated negative predictive values (NPVs) of 81%-99%. The mcfDNA-Seq assay was complementary to serum galactomannan index testing; in combination, they were positive in 84% of individuals with proven/probable pulmonary IMI., Conclusions: Noninvasive mcfDNA-Seq had moderate sensitivity and high specificity, NPV, and PPV for pulmonary IMI after HCT, particularly for non-Aspergillus species., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB., Methods: B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius' clinical test., Results: B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual's cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB., Conclusions: This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing., Methods: In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results., Results: Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen., Conclusions: Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period., Level of Evidence: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G619)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Background: Invasive fungal infection is a major source of morbidity and mortality. The usage of microbial cell-free DNA for the detection and identification of invasive fungal infection has been considered as a potential alternative to invasive procedures allowing for rapid results., Objective: This analysis aimed to assess the budget implications of using the Karius ® Test in patients suspected of invasive fungal infection in an average state in the USA from a healthcare payer perspective., Methods: The analysis used a decision tree to capture key stages of the patient pathway, from suspected invasive fungal infection to either receiving treatment for invasive fungal infection or being confirmed as having no invasive fungal infection. The analysis used published costs and resource use from a targeted review of the literature. Because of the paucity of published evidence on the reduction of diagnostic tests displaced by the Karius Test, the analysis used a 50% reduction in the use of bronchoscopy and/or bronchoalveolar lavage. The impact of this reduction was tested in a scenario analysis., Results: The results of the analysis show that the introduction of the Karius Test is associated with a cost saving of US$2277 per patient; when multiplied by the estimated number of cases per year, the cost saving is US$17,039,666. The scenario analysis showed that the Karius Test only had an incremental cost of US$87 per patient when there was no reduction in bronchoscopy and bronchoalveolar lavage., Conclusions: The Karius Test may offer a valuable and timely option for the diagnosis of invasive fungal infection through its non-invasive approach and subsequent cost savings.

Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.

Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The noninvasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

We report the case of a 60-year-old man with septic shock due to Capnocytophaga canimorsus that was diagnosed in 24 hours by a novel whole-genome next-generation sequencing assay. This technology shows great promise in identifying fastidious pathogens, and, if validated, it has profound implications for infectious disease diagnosis.

Accurate evaluation of microbial communities is essential for understanding global biogeochemical processes and can guide bioremediation and medical treatments. Metagenomics is most commonly used to analyze microbial diversity and metabolic potential, but assemblies of the short reads generated by current sequencing platforms may fail to recover heterogeneous strain populations and rare organisms. Here we used short (150-bp) and long (multi-kb) synthetic reads to evaluate strain heterogeneity and study microorganisms at low abundance in complex microbial communities from terrestrial sediments. The long-read data revealed multiple (probably dozens of) closely related species and strains from previously undescribed Deltaproteobacteria and Aminicenantes (candidate phylum OP8). Notably, these are the most abundant organisms in the communities, yet short-read assemblies achieved only partial genome coverage, mostly in the form of short scaffolds (N50 = ∼ 2200 bp). Genome architecture and metabolic potential for these lineages were reconstructed using a new synteny-based method. Analysis of long-read data also revealed thousands of species whose abundances were <0.1% in all samples. Most of the organisms in this "long tail" of rare organisms belong to phyla that are also represented by abundant organisms. Genes encoding glycosyl hydrolases are significantly more abundant than expected in rare genomes, suggesting that rare species may augment the capability for carbon turnover and confer resilience to changing environmental conditions. Overall, the study showed that a diversity of closely related strains and rare organisms account for a major portion of the communities. These are probably common features of many microbial communities and can be effectively studied using a combination of long and short reads., (© 2015 Sharon et al.; Published by Cold Spring Harbor Laboratory Press.)

High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes. Nevertheless, assembly of complex genomes remains challenging, in part due to the presence of dispersed repeats which introduce ambiguity during genome reconstruction. Transposable elements (TEs) can be particularly problematic, especially for TE families exhibiting high sequence identity, high copy number, or complex genomic arrangements. While TEs strongly affect genome function and evolution, most current de novo assembly approaches cannot resolve long, identical, and abundant families of TEs. Here, we applied a novel Illumina technology called TruSeq synthetic long-reads, which are generated through highly-parallel library preparation and local assembly of short read data and which achieve lengths of 1.5-18.5 Kbp with an extremely low error rate ([Formula: see text]0.03% per base). To test the utility of this technology, we sequenced and assembled the genome of the model organism Drosophila melanogaster (reference genome strain y; cn, bw, sp) achieving an N50 contig size of 69.7 Kbp and covering 96.9% of the euchromatic chromosome arms of the current reference genome. TruSeq synthetic long-read technology enables placement of individual TE copies in their proper genomic locations as well as accurate reconstruction of TE sequences. We entirely recovered and accurately placed 4,229 (77.8%) of the 5,434 annotated transposable elements with perfect identity to the current reference genome. As TEs are ubiquitous features of genomes of many species, TruSeq synthetic long-reads, and likely other methods that generate long-reads, offer a powerful approach to improve de novo assemblies of whole genomes.

The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA.

Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.

A goal of regenerative medicine is to identify cardiovascular progenitors from human ES cells (hESCs) that can functionally integrate into the human heart. Previous studies to evaluate the developmental potential of candidate hESC-derived progenitors have delivered these cells into murine and porcine cardiac tissue, with inconclusive evidence regarding the capacity of these human cells to physiologically engraft in xenotransplantation assays. Further, the potential of hESC-derived cardiovascular lineage cells to functionally couple to human myocardium remains untested and unknown. Here, we have prospectively identified a population of hESC-derived ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells that give rise to cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro at a clonal level. We observed rare clusters of ROR2(+) cells and diffuse expression of KDR and PDGFRα in first-trimester human fetal hearts. We then developed an in vivo transplantation model by transplanting second-trimester human fetal heart tissues s.c. into the ear pinna of a SCID mouse. ROR2(+)/CD13(+)/KDR(+)/PDGFRα(+) cells were delivered into these functioning fetal heart tissues: in contrast to traditional murine heart models for cell transplantation, we show structural and functional integration of hESC-derived cardiovascular progenitors into human heart.

The pluripotent nature of human embryonic stem cells (hESCs) makes them convenient for deriving therapeutically relevant cells. Here we show using Wnt reporter hESC lines that the cells are heterogeneous with respect to endogenous Wnt signalling activity. Moreover, the level of Wnt signalling activity in individual cells correlates with differences in clonogenic potential and lineage-specific differentiation propensity. The addition of Wnt protein or, conversely, a small-molecule Wnt inhibitor (IWP2) reduces heterogeneity, allowing stable expansion of Wnt(high) or Wnt(low) hESC populations, respectively. On differentiation, the Wnt(high) hESCs predominantly form endodermal and cardiac cells, whereas the Wnt(low) hESCs generate primarily neuroectodermal cells. Thus, heterogeneity with respect to endogenous Wnt signalling underlies much of the inefficiency in directing hESCs towards specific cell types. The relatively uniform differentiation potential of the Wnt(high) and Wnt(low) hESCs leads to faster and more efficient derivation of targeted cell types from these populations.

In the nematode Caenorhabditis elegans, sex is determined by the ratio of X chromosomes to sets of autosomes: XX animals (2X:2A=1.0) develop as hermaphrodites and XO animals (1X:2A=0.5) develop as males. TRA-1, the worm ortholog of Drosophila Cubitus interruptus and mammalian Gli (Glioma-associated homolog) proteins, is the terminal transcription factor of the C. elegans sex-determination pathway, which specifies hermaphrodite fate by repressing male-specific genes. Here we identify a consensus TRA-1 binding site in the regulatory region of xol-1, the master switch gene controlling sex determination and dosage compensation. xol-1 is normally expressed in males, where it promotes male development and prevents dosage compensation. We show that TRA-1 binds to the consensus site in the xol-1 promoter in vitro and inhibits the expression of xol-1 in XX animals in vivo. Furthermore, inactivation of tra-1 enhances, whereas hyperactivation of tra-1 suppresses, lethality in animals with elevated xol-1 activity. These data imply the existence of a regulatory feedback loop within the C. elegans sex-determination and dosage-compensation cascade that ensures the accurate dose of X-linked genes in cells destined to adopt hermaphrodite fate.

Dosage compensation equalizes X-linked gene products between the sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both X chromosomes in XX animals and halves the transcription from each. The DCC is recruited to the X chromosomes by a number of loci, rex sites, and is thought to spread from these sites by an unknown mechanism to cover the rest of the chromosome. Here we describe a novel class of DCC-binding elements that we propose serve as "way stations" for DCC binding and spreading. Both rex sites and way stations comprise strong foci of DCC binding on the native X chromosome. However, rex sites maintain their ability to bind large amounts of DCC even on X duplications detached from the native X, while way stations do not. These results suggest that two distinct classes of DCC-binding elements facilitate recruitment and spreading of the DCC along the X chromosome.

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.

Both transcriptional activation and repression have essential functions in maintaining proper spatial and temporal control of gene expression. Although Wnt signalling is often associated with gene activation, we have identified several directly repressed targets of Wnt signalling in Drosophila. Here, we explore how individual Wnt target genes are specified for signal-induced activation or repression. Similar to activation, repression required binding of Armadillo (Arm) to the N terminus of TCF. However, TCF/Arm mediated repression by binding to DNA motifs that are markedly different from typical TCF-binding sites. Conversion of the novel motifs to standard TCF-binding sites reversed the mode of regulation, resulting in Wnt-mediated activation instead of repression. A mutant form of Arm defective in activation was still functional for repression, indicating that distinct domains of the protein are required for each activity. This study suggests that the sequence of TCF-binding sites allosterically regulates the TCF/Arm complex to effect either transcriptional activation or repression.

Wingless (Wg)/Wnt signaling directs a variety of cellular processes during animal development by promoting the association of Armadillo/beta-catenin with TCFs on Wg-regulated enhancers (WREs). Split ends (Spen), a nuclear protein containing RNA recognition motifs (RRMs) and a SPOC domain, is required for optimal Wg signaling in several fly tissues. In this report, we demonstrate that Spenito (Nito), the only other fly protein containing RRMs and a SPOC domain, acts together with Spen to positively regulate Wg signaling. The partial defect in Wg signaling observed with spen RNAi was enhanced by simultaneous knockdown of nito while it was rescued by expression of nito in wing imaginal discs. In cell culture, depletion of both factors causes a greater defect in the activation of several Wg targets than RNAi of either spen or nito alone. These nuclear proteins are not required for Armadillo stabilization or the recruitment of TCF and Armadillo to a WRE. Loss of Wg target gene activation in cells depleted for spen and nito was not dependent on the transcriptional repressor Yan or Suppressor of Hairless, two previously identified targets of Spen. We propose that Spen and Nito act redundantly downstream of TCF/Armadillo to activate many Wg transcriptional targets.

Escherichia coli AmtB is a member of the MEP/Amt family of ammonia transporters found in archaea, eubacteria, fungi, plants and animals. In prokaryotes, AmtB homologues are co-transcribed with a PII paralogue, GlnK, in response to nitrogen limitation. Here, we show that AmtB antagonizes PII signalling through NRII and that co-expression of GlnK with AmtB overcomes this antagonism. In cells lacking GlnK, expression of AmtB during nitrogen starvation prevented deinduction of Ntr gene expression when a nitrogen source became available. The absence of AmtB in cells lacking GlnK allowed rapid reduction of Ntr gene expression during this transition, indicating that one function of GlnK is to prevent AmtB-mediated antagonism of PII signalling after nitrogen starvation. Other roles of GlnK in controlling Ntr gene expression and maintaining viability during nitrogen starvation were unaffected by AmtB. Expression of AmtB from a constitutive promoter under nitrogen-rich conditions induced full expression of glnALG and elevated expression of glnK in wild-type and glnK cells; thus, the ability of AmtB to raise Ntr gene expression did not require a factor found only in nitrogen-starved cells. Experiments with intact cells showed that AmtB acted downstream of a uridylyl transferase uridylyl-removing enzyme (UTase/UR) and upstream of NRII, suggesting that the target was PII. AmtB also slowed the deuridylylation of PII approximately UMP upon ammonia addition, showing that multiple PII interactions were affected by AmtB. Our data are consistent with a hypothesis that AmtB interacts with PII and GlnK, and that co-transcription of glnK and amtB prevents titration of PII when AmtB is highly expressed.

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.

Escherichia coli contains two PII-like signal trans-duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of glnALG, glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation. In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen-starved cells, and the cells did not reduce glnK and nac expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild-type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild-type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild-type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild-type phenotypes.

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI approximately P (NtrC approximately P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

Escherichia coli and related bacteria contain two paralogous PII-like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR-type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high-copy plasmids, Nac imparts a growth defect to wild-type cells in an expression level-dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. beta-Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10-fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co-ordinated manner when cells become starved for nitrogen.