Tejas Dharmaraj

All Publications

• Bacteriophage therapy for multidrug-resistant infections: current technologies and therapeutic approaches. The Journal of clinical investigation Kim, M. K., Suh, G. A., Cullen, G. D., Perez Rodriguez, S., Dharmaraj, T., Chang, T. H., Li, Z., Chen, Q., Green, S. I., Lavigne, R., Pirnay, J. P., Bollyky, P. L., Sacher, J. C. 2025; 135 (5)

  Abstract

  Bacteriophage (phage) therapy has emerged as a promising solution to combat the growing crisis of multidrug-resistant (MDR) infections. There are several international centers actively engaged in implementation of phage therapy, and recent case series have reported encouraging success rates in patients receiving personalized, compassionate phage therapy for difficult-to-treat infections. Nonetheless, substantial hurdles remain in the way of more widespread adoption and more consistent success. This Review offers a comprehensive overview of current phage therapy technologies and therapeutic approaches. We first delineate the common steps in phage therapy development, from phage bank establishment to clinical administration, and examine the spectrum of therapeutic approaches, from personalized to fixed phage cocktails. Using the framework of a conventional drug development pipeline, we then identify critical knowledge gaps in areas such as cocktail design, formulation, pharmacology, and clinical trial design. We conclude that, while phage therapy holds promise, a structured drug development pipeline and sustained government support are crucial for widespread adoption of phage therapy for MDR infections.

  View details for DOI 10.1172/JCI187996

  View details for PubMedID 40026251

• Whole-body Bacteriophage Distribution Characterized by a Physiologically based Pharmacokinetic Model. bioRxiv : the preprint server for biology Echterhof, A., Dharmaraj, T., Blankenberg, P., Targ, B., Bollyky, P. L., Smith, N. M., Blankenberg, F. 2025

  Abstract

  In 2019 there were over 2.8 million cases of antibiotic-resistant bacterial infection in the US with gram negative organisms having up to a 6% rate of mortality. Bacteriophage (phage) therapy holds great promise to treat such infections. However, the biologic features which influence the pharmacokinetics (PK) of phage have been difficult to characterize due to a lack of standardized protocols of phage purification, tissue assay, and labeling. Here we present robust methods for ultrapure phage preparation as well as non-destructive highly stable attachment of radio-iodide to phage using the well described Sulfo-SHPP linker. We purified and radiolabeled the phage strains, PAML-31-1, OMKO1, and Luz24 lytic to drug-resistant Pseudomonas aeruginosa for biodistribution assay in normal young adult CD-1 mice injected via penile vein. Groups of 5 mice were euthanized and tissues/organs removed for weighing and scintillation well counting of I-125 activity at 30 min, 1h, 2h, 4h, 8h, and 24h. A physiologically based PK (PBPK) model was then constructed focusing on compartments describing blood, lung, muscle, bone, liver, stomach, spleen, small intestines, large intestines, and kidney. Model permeability coefficient (PS) was estimated across all organs as being 0.0227. Tissue partition coefficients (KP) were estimated for high perfusion organs (lung and kidney) as 0.000138, GI organs (liver, spleen, and stomach) as 0.627, and all other organs as 0.220. Elimination was governed by MPS-mediated elimination (TMPS,deg) and active secretion at epithelial barriers (CLActive), which were estimated as 0.00301 h and 0.0145 L/h/kg, respectively. Monte Caro simulations showed that the rapid elimination phage in humans is expected, resulting in phage blood concentrations being lower than 102 PFU/mL (limit of quantification by plaque assay) by 12 hours. As such, multi-dose regimens and continuous infusion regimens were the only strategies that allowed continuously detectible phage concentrations. Evaluation of different dose levels showed that at a maximum dose of 1012 PFU, phage concentrations are expected to be approximately 107 PFU/g. Our physiologically based PK model of phage represents the first rigorous pre-clinical assessment of phage PK utilizing contemporary pharmacometric approaches amenable to both pre-clinical and clinical study design.

  View details for DOI 10.1101/2025.02.06.636931

  View details for PubMedID 39975270

  View details for PubMedCentralID PMC11839030

• Bacteriophage purification using CIMmultus monolithic OH-column chromatography for therapeutic purposes. bioRxiv : the preprint server for biology Echterhof, A., Dharmaraj, T., Blankenberg, P., Hajfathalian, M., Blankenberg, F., Sacher, J., Bollyky, P. L. 2025

  Abstract

  Bacteriophages, viruses that selectively infect bacteria, are increasingly explored as potential therapies to combat antibiotic-resistant infections. Effective phage purification is vital for therapeutic use, particularly to reduce endotoxin (lipopolysaccharide, LPS) and host protein contamination from production processes in gram-negative bacterial lysates. This study investigates the purification efficiency of CIMmultus OH-monolithic chromatography for seven similarly sized tailed phages (Myoviridae and Siphoviridae families) infecting Pseudomonas aeruginosa. Using preferential exclusion chromatography with a potassium phosphate gradient, we achieved significant reductions in host proteins and endotoxins, yielding therapeutically compliant samples suitable for human intravenous injection. We observed recovery rates of 85-95% for smaller phages (e.g., PAML-31-1, LPS5), with lower recoveries for larger "giant" phages (e.g., OMKO1, PhiKZ). All purified samples met endotoxin limits, with average reductions of 98.5% for proteins and up to 6.30 log reductions for endotoxins. Despite increased DNA content post-purification in most samples, the approach shows promise for clinical-grade phage purification. This chromatographic strategy is scalable, reproducible, and free from hazardous chemicals, making it suitable for industrial phage production.

  View details for DOI 10.1101/2025.02.03.636326

  View details for PubMedID 39975350

  View details for PubMedCentralID PMC11838533

• The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo. JCI insight Echterhof, A., Dharmaraj, T., Khosravi, A., McBride, R., Miesel, L., Chia, J., Blankenberg, P. M., Lin, K., Shen, C., Lee, Y., Yeh, Y., Liao, W. T., Blankenberg, F. G., Dabrowska, K., Amanatullah, D. F., Frymoyer, A. R., Bollyky, P. L. 2024; 9 (20)

  Abstract

  With the increasing prevalence of antimicrobial-resistant bacterial infections, there is interest in using bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of i.v. administered bacteriophage in uninfected mice. A single dose of LPS-5, a bacteriophage recently used in human clinical trials to treat drug-resistant Pseudomonas aeruginosa, was administered i.v. to both immunocompetent BALB/c and neutropenic CD1 mice. Phage concentrations were assessed in peripheral blood and spleen at 0.25, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance was only minimally affected by neutropenia. Indeed, the half-lives of phages in blood in BALB/c and CD1 mice were 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that significant inactivation of circulating phages occurs over time. These data indicate that alternative factors, but not neutrophils, inactivate i.v. administered phages.

  View details for DOI 10.1172/jci.insight.181309

  View details for PubMedID 39435664

• Hydrogels for Local and Sustained Delivery of Bacteriophages to Treat Multidrug-Resistant Wound Infections. bioRxiv : the preprint server for biology Lin, Y. H., Dharmaraj, T., Chen, Q., Echterhof, A., Manasherob, R., Zheng, L. J., de Leeuw, C., Peterson, N. A., Stannard, W., Li, Z., Hajfathalian, M., Hargil, A., Martinez, H. A., Chang, T. H., Blankenberg, F. B., Amanatullah, D., Chaudhuri, O., Bollyky, P. 2024

  Abstract

  Bacteriophages (phages), viruses that specifically target and kill bacteria, represent a promising strategy to combat multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa (Pa). However, delivering sufficient concentrations of active phages directly to the infection site remains challenging, with current methods having variable success. Here we present "HydroPhage", an innovative hydrogel system for the sustained release of high-titer phages to effectively treat infections caused by MDR pathogens. Our injectable hydrogels, featuring dual-crosslinking of hyaluronic acid and PEG-based hydrogels through static covalent thioether bonds and dynamic covalent hemithioacetal crosslinks (DCC), encapsulate phages at concentration up to 1011 PFU/mL, and achieves controlled release of 109 PFU daily over a week, surpassing levels of current clinical dosages, with more than 60% total phage recovery. In a preclinical mouse model of extended wound infection, compared to intravenous treatment, we demonstrate enhanced bacterial clearance by localized, high-dose, and repeated phage dosing despite the emergence of bacterial resistance to phages. This work advances the development of clinically practical wound dressings tailored for resistant infections.

  View details for DOI 10.1101/2024.05.07.593005

  View details for PubMedID 38766200

  View details for PubMedCentralID PMC11100690

• Rapid assessment of changes in phage bioactivity using dynamic light scattering. PNAS nexus Dharmaraj, T., Kratochvil, M. J., Pourtois, J. D., Chen, Q., Hajfathalian, M., Hargil, A., Lin, Y. H., Evans, Z., Oromí-Bosch, A., Berry, J. D., McBride, R., Haddock, N. L., Holman, D. R., van Belleghem, J. D., Chang, T. H., Barr, J. J., Lavigne, R., Heilshorn, S. C., Blankenberg, F. G., Bollyky, P. L. 2023; 2 (12): pgad406

  Abstract

  Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

  View details for DOI 10.1093/pnasnexus/pgad406

  View details for PubMedID 38111822

  View details for PubMedCentralID PMC10726995

• Native lamin A/C proteomes and novel partners from heart and skeletal muscle in a mouse chronic inflammation model of human frailty FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Elzamzami, F. D., Samal, A., Arun, A. S., Dharmaraj, T., Prasad, N. R., Rendon-Jonguitud, A., Devine, L., Walston, J. D., Cole, R. N., Wilson, K. L. 2023; 11: 1240285

  Abstract

  Clinical frailty affects ∼10% of people over age 65 and is studied in a chronically inflamed (Interleukin-10 knockout; "IL10-KO") mouse model. Frailty phenotypes overlap the spectrum of diseases ("laminopathies") caused by mutations in LMNA. LMNA encodes nuclear intermediate filament proteins lamin A and lamin C ("lamin A/C"), important for tissue-specific signaling, metabolism and chromatin regulation. We hypothesized that wildtype lamin A/C associations with tissue-specific partners are perturbed by chronic inflammation, potentially contributing to dysfunction in frailty. To test this idea we immunoprecipitated native lamin A/C and associated proteins from skeletal muscle, hearts and brains of old (21-22 months) IL10-KO versus control C57Bl/6 female mice, and labeled with Tandem Mass Tags for identification and quantitation by mass spectrometry. We identified 502 candidate lamin-binding proteins from skeletal muscle, and 340 from heart, including 62 proteins identified in both tissues. Candidates included frailty phenotype-relevant proteins Perm1 and Fam210a, and nuclear membrane protein Tmem38a, required for muscle-specific genome organization. These and most other candidates were unaffected by IL10-KO, but still important as potential lamin A/C-binding proteins in native heart or muscle. A subset of candidates (21 in skeletal muscle, 30 in heart) showed significantly different lamin A/C-association in an IL10-KO tissue (p < 0.05), including AldoA and Gins3 affected in heart, and Lmcd1 and Fabp4 affected in skeletal muscle. To screen for binding, eleven candidates plus prelamin A and emerin controls were arrayed as synthetic 20-mer peptides (7-residue stagger) and incubated with recombinant purified lamin A "tail" residues 385-646 under relatively stringent conditions. We detected strong lamin A binding to peptides solvent exposed in Lmcd1, AldoA, Perm1, and Tmem38a, and plausible binding to Csrp3 (muscle LIM protein). These results validated both proteomes as sources for native lamin A/C-binding proteins in heart and muscle, identified four candidate genes for Emery-Dreifuss muscular dystrophy (CSRP3, LMCD1, ALDOA, and PERM1), support a lamin A-interactive molecular role for Tmem38A, and supported the hypothesis that lamin A/C interactions with at least two partners (AldoA in heart, transcription factor Lmcd1 in muscle) are altered in the IL10-KO model of frailty.

  View details for DOI 10.3389/fcell.2023.1240285

  View details for Web of Science ID 001098802800001

  View details for PubMedID 37936983

  View details for PubMedCentralID PMC10626543

• Bacteriophage and Bacterial Susceptibility, Resistance, and Tolerance to Antibiotics. Pharmaceutics Chen, Q., Dharmaraj, T., Cai, P. C., Burgener, E. B., Haddock, N. L., Spakowitz, A. J., Bollyky, P. L. 2022; 14 (7)

  Abstract

  Bacteriophages, viruses that infect and replicate within bacteria, impact bacterial responses to antibiotics in complex ways. Recent studies using lytic bacteriophages to treat bacterial infections (phage therapy) demonstrate that phages can promote susceptibility to chemical antibiotics and that phage/antibiotic synergy is possible. However, both lytic and lysogenic bacteriophages can contribute to antimicrobial resistance. In particular, some phages mediate the horizontal transfer of antibiotic resistance genes between bacteria via transduction and other mechanisms. In addition, chronic infection filamentous phages can promote antimicrobial tolerance, the ability of bacteria to persist in the face of antibiotics. In particular, filamentous phages serve as structural elements in bacterial biofilms and prevent the penetration of antibiotics. Over time, these contributions to antibiotic tolerance favor the selection of resistance clones. Here, we review recent insights into bacteriophage contributions to antibiotic susceptibility, resistance, and tolerance. We discuss the mechanisms involved in these effects and address their impact on bacterial fitness.

  View details for DOI 10.3390/pharmaceutics14071425

  View details for PubMedID 35890320

• Filamentous bacteriophage delays healing of Pseudomonas-infected wounds. Cell reports. Medicine Bach, M. S., de Vries, C. R., Khosravi, A., Sweere, J. M., Popescu, M. C., Chen, Q., Demirdjian, S., Hargil, A., Van Belleghem, J. D., Kaber, G., Hajfathalian, M., Burgener, E. B., Liu, D., Tran, Q., Dharmaraj, T., Birukova, M., Sunkari, V., Balaji, S., Ghosh, N., Mathew-Steiner, S. S., El Masry, M. S., Keswani, S. G., Banaei, N., Nedelec, L., Sen, C. K., Chandra, V., Secor, P. R., Suh, G. A., Bollyky, P. L. 2022; 3 (6): 100656

  Abstract

  Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.

  View details for DOI 10.1016/j.xcrm.2022.100656

  View details for PubMedID 35732145

• Filamentous Bacteriophage Delay Healing Of Pseudomonas-Infected Wounds Khosravi, A., Bach, M. S., de Vries, C. R., Sweere, J. M., Popescu, M., Chen, Q., Hargil, A., Van Belleghem, J. D., Kaber, G., Burgener, E. B., Liu, D., Quynh-Lam Tran, Dharmaraj, T., Sunkari, V., Balaji, S., Ghosh, N., Mathew-Steiner, S. S., Keswani, S. G., Banaei, N., Nedelec, L., Sen, C. K., Chandra, V., Secor, P. R., Suh, G., Bollyky, P. WILEY. 2022: A27

  View details for Web of Science ID 000763583000063

• Rare BANF1 Alleles and Relatively Frequent EMD Alleles Including 'Healthy Lipid' Emerin p.D149H in the ExAC Cohort FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY Dharmaraj, T., Guan, Y., Liu, J., Badens, C., Gaborit, B., Wilson, K. L. 2019; 7: 48

  Abstract

  Emerin (EMD) and barrier to autointegration factor 1 (BANF1) each bind A-type lamins (LMNA) as fundamental components of nuclear lamina structure. Mutations in LMNA, EMD and BANF1 are genetically linked to many tissue-specific disorders including Emery-Dreifuss muscular dystrophy and cardiomyopathy (LMNA, EMD), lipodystrophy, insulin resistance and type 2 diabetes (LMNA) and progeria (LMNA, BANF1). To explore human genetic variation in these genes, we analyzed EMD and BANF1 alleles in the Exome Aggregation Consortium (ExAC) cohort of 60,706 unrelated individuals. We identified 13 rare heterozygous BANF1 missense variants (p.T2S, p.H7Y, p.D9N, p.S22R, p.G25E, p.D55N, p.D57Y, p.L63P, p.N70T, p.K72R, p.R75W, p.R75Q, p.G79R), and one homozygous variant (p.D9H). Several variants are known (p.G25E) or predicted (e.g., p.D9H, p.D9N, p.L63P) to perturb BANF1 and warrant further study. Analysis of EMD revealed two previously identified variants associated with adult-onset cardiomyopathy (p.K37del, p.E35K) and one deemed 'benign' in an Emery-Dreifuss patient (p.D149H). Interestingly p.D149H was the most frequent emerin variant in ExAC, identified in 58 individuals (overall allele frequency 0.06645%), of whom 55 were East Asian (allele frequency 0.8297%). Furthermore, p.D149H associated with four 'healthy' traits: reduced triglycerides (-0.336; p = 0.0368), reduced waist circumference (-0.321; p = 0.0486), reduced cholesterol (-0.572; p = 0.000346) and reduced LDL cholesterol (-0.599; p = 0.000272). These traits are distinct from LMNA-associated metabolic disorders and provide the first insight that emerin influences metabolism. We also identified one novel in-frame deletion (p.F39del) and 62 novel emerin missense variants, many of which were relatively frequent and potentially disruptive including p.N91S and p.S143F (∼0.041% and ∼0.034% of non-Finnish Europeans, respectively), p.G156S (∼0.39% of Africans), p.R204G (∼0.18% of Latinx), p.R207P (∼0.08% of South Asians) and p.R221L (∼0.15% of Latinx). Many novel BANF1 variants are predicted to disrupt dimerization or binding to DNA, histones, emerin or A-type lamins. Many novel emerin variants are predicted to disrupt emerin filament dynamics or binding to BANF1, HDAC3, A-type lamins or other partners. These new human variants provide a foundational resource for future studies to test the molecular mechanisms of BANF1 and emerin function, and to understand the link between emerin variant p.D149H and a 'healthy' lipid profile.

  View details for DOI 10.3389/fcell.2019.00048

  View details for Web of Science ID 000467203500001

  View details for PubMedID 31024910

  View details for PubMedCentralID PMC6459885

• LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes. Frontiers in genetics Florwick, A., Dharmaraj, T., Jurgens, J., Valle, D., Wilson, K. L. 2017; 8: 79

  Abstract

  Mutations in LMNA, encoding nuclear intermediate filament proteins lamins A and C, cause multiple diseases ('laminopathies') including muscular dystrophy, dilated cardiomyopathy, familial partial lipodystrophy (FPLD2), insulin resistance syndrome and progeria. To assess the prevalence of LMNA missense mutations ('variants') in a broad, ethnically diverse population, we compared missense alleles found among 60,706 unrelated individuals in the ExAC cohort to those identified in 1,404 individuals in the laminopathy database (UMD-LMNA). We identified 169 variants in the ExAC cohort, of which 37 (∼22%) are disease-associated including p.I299V (allele frequency 0.0402%), p.G602S (allele frequency 0.0262%) and p.R644C (allele frequency 0.124%), suggesting certain LMNA mutations are more common than previously recognized. Independent analysis of LMNA variants via the type 2 diabetes (T2D) Knowledge Portal showed that variant p.G602S associated significantly with type 2 diabetes (p = 0.02; odds ratio = 4.58), and was more frequent in African Americans (allele frequency 0.297%). The FPLD2-associated variant I299V was most prevalent in Latinos (allele frequency 0.347%). The ExAC cohort also revealed 132 novel LMNA missense variants including p.K108E (limited to individuals with psychiatric disease; predicted to perturb coil-1B), p.R397C and p.R427C (predicted to perturb filament biogenesis), p.G638R and p.N660D (predicted to perturb prelamin A processing), and numerous Ig-fold variants predicted to perturb phenotypically characteristic protein-protein interactions. Overall, this two-pronged strategy- mining a large database for missense variants in a single gene (LMNA), coupled to knowledge about the structure, biogenesis and functions of A-type lamins- revealed an unexpected number of LMNA variants, including novel variants predicted to perturb lamin assembly or function. Interestingly, this study also correlated novel variant p.K108E with psychiatric disease, identified known variant p.I299V as a potential risk factor for metabolic disease in Latinos, linked variant p.G602 with type 2 diabetes, and identified p.G602S as a predictor of diabetes risk in African Americans.

  View details for DOI 10.3389/fgene.2017.00079

  View details for PubMedID 28663758

  View details for PubMedCentralID PMC5471320

• How chromosomes unite Nature Dharmaraj, T., Wilson, K. 2017; 551 (7682): 568-569

  View details for DOI 10.1038/d41586-017-07439-7