Project 1: Thioredoxin System Targeted Inhibition of Staphylococcuus Aureus
Principal Investigator: Beth Burgwyn Fuchs, PhD
The major goals of the project were to validate thioredoxin reductase (TrxR) as an antimicrobial target and to develop new inhibitors. Using auranofin as a proof-of-concept compound, we found that it was an effective inhibitor of Gram-positive bacteria that included Enterococcus faecium and Staphylococcus aureus. Minimal inhibitory concentrations were 1 g/mL or lower when testing laboratory reference strains or bacterial clinical isolates. Over 500 clinical isolates of drug-sensitive and drug-resistant strains demonstrated sensitivity to auranofin suggesting that existing resistance mechanisms are not useful to prevent auranofin antimicrobial activity. Further, exposing a drug-resistant strain of S. aureus to auranofin for 25 days did not promote resistance, suggesting that resistance is not easily developed upon extended drug exposure.
To test the hypothesized target, S. aureus TrxR was expressed, isolated, and used in an assay to gauge enzyme activity. The thioredoxin system is comprised of NADPH, TrxR, and thioredoxin (Trx), which acts as an antioxidant by facilitating the reduction of reactive oxygen species via the transfer of electrons from the NADPH donor. TrxR is a catalyst within the system, transferring electrons from NADPH to Trx. Inhibition of TrxR results in ROS accumulation, leading to apoptosis. The enzymatic assay used the S. aureus TrxR to convert 5,5'-dithio-bis(2-dinitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) providing a colorimetric readout. We saw that auranofin reduced TrxR enzymatic activity in a dose-dependent manner.
Using a target-based screen, Bay11-7085 was identified as another TrxR inhibitor. Using structural information from Bay11-7085 and auranofin, structural hybrids were designed and synthesized. Two lead compounds, AU1 and AU5, were identified that demonstrated anti-S. aureus activity with low minimal inhibitory concentrations at 0.25 g/mL and 0.5 g/mL, respectively. Both AU1 and AU5 inhibited TrxR enzymatic activity. Further, the compounds caused a decrease in intracellular free thiols. Lead compounds were examined for cytotoxic liability in human erythrocytes, primary hepatocytes, and renal proximal tubule cells. For all tested cell types, AU1 and AU5 lethal dose (LD50) concentrations exceeded the minimal inhibitory concentration against S. aureus. Thus, the bacterial inhibitory concentration was low enough that bacteria were susceptible without adverse damage to human cells. The lead compounds AU1 and AU5 present important information for the development of a new antimicrobial compound that specifically inhibits drug-resistant strains of S. aureus. Further, the evidence collected indicates that TrxR is a valid antimicrobial target.
Felix L, Mylonakis E, Fuchs BB. Thioredoxin Reductase Is a Valid Target for Antimicrobial Therapeutic Development Against Gram-Positive Bacteria. Front Microbiol. 2021 Apr 16;12:663481. doi: 10.3389/fmicb.2021.663481. PMID: 33936021; PMCID: PMC8085250.
Mishra B, Khader R, Felix LO, Frate M, Mylonakis E, Meschwitz S, Fuchs BB. A Substituted Diphenyl Amide Based Novel Scaffold Inhibits Staphylococcus aureus Virulence in a Galleria mellonella Infection Model. Front Microbiol. 2021 Oct 5;12:723133. doi: 10.3389/fmicb.2021.723133. PMID: 34675898; PMCID: PMC8524085.
Rossoni RD, de Barros PP, Mendonça IDC, Medina RP, Silva DHS, Fuchs BB, Junqueira JC, Mylonakis E. The Postbiotic Activity of Lactobacillus paracasei 28.4 Against Candida auris. Front Cell Infect Microbiol. 2020 Aug 4;10:397. doi: 10.3389/fcimb.2020.00397. PMID: 32850495; PMCID: PMC7417517.
Tharmalingam N, Ribeiro NQ, da Silva DL, Naik MT, Cruz LI, Kim W, Shen S, Dos Santos JD, Ezikovich K, D'Agata EM, Mylonakis E, Fuchs BB. Auranofin is an effective agent against clinical isolates of Staphylococcus aureus. Future Med Chem. 2019 Jun;11(12):1417-1425. doi: 10.4155/fmc-2018-0544. Epub 2019 Jul 12. PMID: 31298580; PMCID: PMC7202268.
Liu H, Shukla S, Vera-González N, Tharmalingam N, Mylonakis E, Fuchs BB, Shukla A. Auranofin Releasing Antibacterial and Antibiofilm Polyurethane Intravascular Catheter Coatings. Front Cell Infect Microbiol. 2019 Feb 28;9:37. doi: 10.3389/fcimb.2019.00037. PMID: 30873389; PMCID: PMC6403144.
Project 2: Neutrophil Integrins in Defense Against Drug-Resistant C. Albicans
Principal Investigator: Brian W. LeBlanc, PhD
Systemic fungal infections such as those caused by Candida sp. are a frequent cause of nosocomial infections and are particularly problematic in patients maintained in the surgical ICU for extended periods of time. Invasive measures such as intravenous catheters and hyperalimentation contribute to risk of acquiring candidiasis. Management of candidiasis patients is complicated by the increasing frequency of Candida isolates that have developed resistance to current anti-fungal drugs, making the development of alternative treatments a priority. Neutrophils are the primary line of host defense against tissue infection with Candida. Therefore, better insight into mechanisms of neutrophil defense against Candida is needed to design immune strategies that can teach neutrophils to destroy persistent Candida and decrease reliance on anti-fungal antibiotics. The therapeutic targeting of the neutrophil integrin receptor, CR3, to improve survival and outcomes to challenge with drug-resistant, clinical isolates of Candida is proposed.
This proposal will establish the significance of beta2-to-beta1 integrin cross-talk in controlling anti-Candida effector functions of neutrophils. A novel role for beta1 integrins, particularly VLA3, in mediating the anti-Candida response will be examined both in vitro and in vivo using conditional VLA3 knockout mice. VLA3 is presented as a therapeutic target to dampen an excessive inflammatory component of the response to Candida infection while preserving the protective NET response. Additionally, the targeting of CR3 with soluble β-glucan to improve the neutrophil anti-fungal host response to drug-sensitive Candida, as well as fluconazole-resistant strains and clinical isolates resistant to the echinocandin class of anti-fungals, will be evaluated as a therapeutic treatment. Alternative treatments that target and support host defense would reduce the usage of anti-fungal antibiotics and in so doing lessen the frequency of drug resistance.
AIM 1: To test the hypothesis that CR3 cross-talk up-regulates VLA3 and down-regulates VLA5.
AIM 1.B: Is VLA3-induced neutrophil clustering dispensable for Candida albicans killing?
AIM 1.C: Is VLA5 activation necessary and sufficient for NETosis and Candida killing?
AIM 2: The neutrophil response to drugresistant C. albicans.
AIM 2.A: Anti-fungal response of Drug-Resistant Candida in vitro. AIM 2.B. Drug-Resistant Candida virulence in vivo.
AIM 3: To determine the relevance of netosis and swarming/clustering on host defense in vivo.
AIM 3.A. Effect of genetic depletion of PAD4 on morbidity, mortality and host defense to C. albicans.
AIM 3.B: To determine the role of VLA3 in the neutrophil response to infection and injury.
AIM 4: To determine the ability of soluble β-glucan to improve host defense against challenge with C. albicans.
Outcomes from this project have been published in a paper titled “Chitotriosidase Activity Is Counterproductive in a Mouse Model of Systemic Candidiasis.”
Project 3: Changes in Microbiome Structure and Functional Responses During Antibiotic
Principal Investigator: Peter A. Belenky, PhD (awarded R01)
The development and spread of antibiotic resistance is jeopardizing the efficacy of our current antibiotic arsenal. To formulate targeted therapies that make better use of existing antibiotics and reduce the development of resistance, we must understand how antibiotics impact both the pathogenic and beneficial members of the human microbiome. This is particularly important because the disruption of microbiome homeostasis by antibiotics is associated with multiple microbiome-related diseases, such as Clostridium difficile-associated diarrhea and inflammatory bowel disease.
Current descriptive microbiome research, focused on identifying taxonomic changes, has not addressed the mechanistic question of why specific bacteria within a microbial community are negatively impacted by antibiotics while others are not. The work proposed here will move past this limitation by transcriptionally profiling the impacts of antibiotics on the total microbial community in vivo to provide functional and mechanistic insight into the action of antibiotics in the microbiome. The central hypothesis of this study is that the induction of tolerance and resistance mechanisms mediates toxicity to antibiotic exposure in susceptible members of the microbiome.
This proposal is focused on tolerance mechanisms related to the metabolic state of bacteria before and during treatment. The microbiome consists of many metabolic microenvironments and within these communities metabolically active bacteria are likely to be more susceptible than less active species. Total transcriptional profiles of the microbiome can be used to study the roles of well-defined tolerance and resistance mechanisms within the microbiome in vivo.
This project will test our central hypothesis in three aims:
Aim 1) Determine the impacts of broad-spectrum antibiotics on the structure of the salivary microbiome derived from clinical samples.
Aim 2) Determine the total transcriptional response of the microbiome to bactericidal and bacteriostatic antibiotic therapy.
Aim 3) Profile the impacts of ciprofloxacin on the structure and function of the murine microbiome in conjunction with host metabolic perturbation.
In addition to testing this hypothesis, a key goal of this exploratory proposal is to implement a novel outpatient-based methodology to study the response of the human microbiome to antibiotic therapy. The ultimate goal of this work is to provide information about the impact of antibiotic therapy on the structure and function of the microbiome, in order to allow clinicians to select therapies that minimize microbiome-related complications and the transfer and development of resistance.
This basic knowledge will help clinicians to improve antibiotic therapy by promoting evidence-based, targeted treatments to safeguard our current arsenal of antibiotics.
- Determine the impacts of broad-spectrum antibiotics on the structure of the salivary microbiome.
- Determine the total transcriptional response of the microbiome to bactericidal and bacteriostatic antibiotic therapy.
- Profile the impacts of the host’s metabolic state on the structural and functional responses of the murine gut and salivary microbiomes to ciprofloxacin.
The phase 1 project funding was instrumental in providing the Belenky lab with key preliminary data and time to develop new techniques related to the metagenomic and metatranscriptomic analysis of the microbiome undergoing antibiotics. This, in turn, supported successful R21, R01, and USDA applications and multiple high-impact publications. This support was instrumental in allowing the PI to reach independence. Dr. Belenky has also been able to support local microbiome research by starting the RI microbiome consortium.
The initial goal of this project was to determine how and why microbial metabolism impacts bacterial susceptibility to antibiotics within the microbiome. The overall hypothesis was that active metabolic activity contributes to toxicity, whereas less efficient metabolism contributed to resistance within bacteria in this community. To solve this problem, we need to apply a systems-biology perspective to study the impacts of bacterial metabolism and the host environment on microbiome structure and function after antibiotics.
Funding from our phase 1 project allowed us to develop a unique metagenomic and metatranscriptomic approach to analyze microbiome structure and function in response to antibiotics under different dietary conditions. In our 2019 Cell Metabolism paper, we show that metabolism plays a key role alongside host diet in modifying the extent of microbiome disruption in response to antibiotics. We conducted an in-depth metagenomic and metatranscriptomic analysis of the murine microbiome under antibiotic stress and found that antibiotics profoundly change the transcriptional landscape of this community and induce significant metabolic dysfunction. We have expanded on this work and demonstrated that the consumption of a Western-style diet impacts the murine microbiome and promotes elevated antibiotic-induced disruption (mSystems). This indicates that host diet likely plays a key role in the regulation of antibiotic-induced dysbiosis via the modulation of microbiome metabolism. To expand on this concept further, we used metabolomics and metatranscriptomics to profile how changing host metabolism via induced hyperglycemia changed the response of the microbiome to antibiotics. We found that induced hyperglycemia changed the metabolome of the microbiome; promoting amino acid metabolism and respiratory activity. This elevated respiratory active activity is associated with higher levels of antibiotic-induced microbiome disruption (Cell Reports).
Cabral D. J., Penumutchu S., Reinhart E. M., Zhang C., Korry B. J., Wurster J. I., Nilson R., Guang A., Sano W. H., Rowan-Nash A. D., Li H., Belenky P. “Microbial Metabolism Modulates Antibiotic Susceptibility within the Murine Gut Microbiome”. Cell Metabolism, 30(4); 800-823.e7 (2019) PMCID: PMC4648786
Rowan-Nash AD, Araos R, D'Agata EMC, Belenky P. “Antimicrobial Resistance Gene Prevalence in a Population of Patients with Advanced Dementia Is Related to Specific Pathobionts”. iScience (cell press). 2020 Mar 27;23(3):100905. doi: 10.1016/j.isci.2020.100905. Epub 2020 Feb 13. PMCID: PMC7044522
Cabral DJ, Wurster JI, Korry BJ, Penumutchu S, Belenky P. Consumption of a Western-Style Diet Modulates the Response of the Murine Gut Microbiome to Ciprofloxacin. mSystems. 2020 Jul 28;5(4):e00317-20. doi: 10.1128/mSystems.00317-20., PMID: 32723789; PMCID: PMC7394352
Wurster J.I.G, Peterson R.L., Brown C.E. U, Penumutchu S. G, Guzior D.V., Neugebauer K., Sano W.H., Sebastian M.M., Quinn R.A., Belenky P. “Streptozotocin-induced hyperglycemia alters the cecal metabolome and exacerbates antibiotic-induced dysbiosis”. Cell Reports. 2021 Dec 14;37(11):110113. doi: 10.1016/j.celrep.2021.110113. PubMed PMID: 34910917
Korry B.J. G, Lee SYE U, Chakrabarti AK U, Choi AH U, Ganser C. G, Machan J.T., Belenky P. “Genotoxic agents produce stressor-specific spectra of spectinomycin resistance mutations based on mechanism of action and selection in Bacillus subtilis”. Antimicrob Agents Chemother (ASM) (2021) Aug 2:AAC0089121. doi: 10.1128/AAC.00891-21. PMID: 34339280
Project 4: Development of Antimicrobial Peptides as Effective Antimicrobials against ESKAPE Pathogens
Principal Investigator: Biswajit Mishra, PhD
Current difficulties in discovering and developing novel antibiotics is becoming a major health care crisis. This situation is even more challenging with the emergence of antimicrobial resistance. The underlying hypothesis of the proposed project is that the design of α-helical peptides in silico offers excellent possibilities for identifying novel antimicrobial compounds with respect to time efficiency and cost reduction. The long-term goal of this proposal is to utilize structure-guided principles to design novel antimicrobial peptides (AMPs) in silico that will be effective against systemic bacterial infection caused by both Gram-negative bacteria and Gram-positive bacteria. We aim to engineer short α-helical AMPs with suitable potency, selectivity, stability, and appropriate PK/PD properties. We will test our central hypothesis with three specific aims: Aim 1) In silico design of bacteria-specific AMPs that includes manual design based on a classic 3.613 α-helix core (Pauling–Corey–Branson α-helix) containing 12 amino acid residues with varying charge and hydrophobicity balance followed by molecular dynamics modeling and structure predictions using AlphaFold2. Aim 2) Determine the activity, toxicity, stability, and mechanism of action of designed peptides. Aim 3) In vivo efficacy evaluation of peptides in a Galleria mellonella wax moth model and subsequently in a systemic mouse infection model. In addition, we will also evaluate nanoparticle formulations of selected, designed AMPs for potential future in vivo use in mice using a poly(lactic-co-glycolic acid) (PLGA) and/or chitosan nanocarrier. Upon completion of the proposed objectives, novel candidate AMPs will be identified that will help mitigate the current antimicrobial crisis.
We proposed the in silico designing and screening of peptides obtained from a model 3.613 α-helix. Incorporating selective charge and hydrophobic amino acids in the peptide sequence in a specific ratio will ensure that the AMPs target specific bacteria with a unique membrane phospholipid composition. We designed peptides based on a 12 amino acid core α-helical fragment with 3 turns (4 amino acids per turn). We interchanged lys and arg as the charged amino acids at the specific position (in all 1-4) positions and incorporated all other hydrophobic amino acids at the rest of the positions. In the resultant peptide we increased the hydrophobicities to ~60% optimal to target Staphylococcus aureus. The peptides noted as 1D were found to be efficacious against S. aureus and worked on the bacterial membrane (detailed in previous progress reports). The RNA seq data was also evident for its membrane targeting mechanism. At least 1771 genes were regulated in S. aureus in the presence of the peptide 1D. Out of which, 888 were down regulated and 883 were upregulated. Interestingly more than 200 genes were found to be associated with membrane function. Significant down regulated genes include spa, the staphylococcal protein A; norB, a multidrug efflux pump; fnbB, fibronectin-binding protein; sel, enterotoxin typeL and finally, a clp protease. While the most upregulated genes involved cap8B, type 8 capsular polysaccharide protein and its variants like cap8D, cap8C and capA.
Ganesan N, Mishra B, Felix L, Mylonakis E. Antimicrobial Peptides and Small Molecules Targeting the Cell Membrane of Staphylococcus aureus. Microbiol Mol Biol Rev. 2023 Jun 28;87(2):e0003722. doi: 10.1128/mmbr.00037-22. Epub 2023 Apr 27. PMID: 37129495; PMCID: PMC10304793.
Mishra B, Felix L, Basu A, Kollala SS, Chhonker YS, Ganesan N, Murry DJ, Mylonakis E. Design and Evaluation of Short Bovine Lactoferrin-Derived Antimicrobial Peptides against Multidrug-Resistant Enterococcus faecium. Antibiotics (Basel). 2022 Aug 10;11(8):1085. doi: 10.3390/antibiotics11081085. PMID: 36009954; PMCID: PMC9404989.
Project 5: Using Deep RNA Sequencing to Develop Novel Antimicrobial Treatment Strategies (Project Under NIH/NIGMS Review)
Principal Investigator: Alger Fredericks, PhD (Project under NIH/NIGMS Review)
Sepsis is a global health problem and is defined as organ dysfunction as a result of an altered immune response to an infection. With such a large burden of mortality worldwide, better diagnostics and treatments are necessary for this disease. This application will focus on monitoring the immune response during severe infections that lead to sepsis. Current clinical practices rely on traditional blood culture to assess infections and immunoglobulin activity. Here we propose to use deep RNA sequencing as a means to evaluate the mechanism of immunosuppression in sepsis patients as a result of severe infection over time to better characterize immunosuppression in sepsis patients, allowing clinicians to more effectively treat antimicrobial resistant infections.
Pilot 1: Mechanism of Synergy Between β-lactams & Aminoglycosides in Gram-positive Bacteria
Pilot 1: Mechanism of Synergy Between β-lactams & Aminoglycosides in Gram-positive Bacteria
Pilot Project Leader: Mónica García-Solache, MD, PhD
The main goal of this pilot project is to understand how beta-lactam exposure induces increased aminoglycoside uptake in gram-positive bacteria leading to antibiotic synergism. Longer-term goals are to identify new strategies for drug development and improvement of the activity of existing agents based on our project results.
Specific aim 1: Determine the effect of beta-lactam binding on the membrane proton motive force of gram-positive bacteria. As an additional sub-aim, I will analyze the role of cell wall damage induced by beta-lactams and the metabolic changes associated with it on aminoglycoside internalization. Specific aim 2: Analyze the role of specific Class A and Class B PBPs in beta-lactam/aminoglycoside synergism utilizing E. faecium and S. aureus PBP mutants.
The results obtained so far show a complex picture about the mechanism behind beta-lactam /aminoglycoside synergism. The results obtained with S. aureus indicate that cell wall damage alone is not sufficient to induce increased aminoglycoside killing (as a proxy for increased aminoglycoside uptake), but cell wall alterations such as those caused by the deletion of some of the pbp’s favors synergism between beta-lactams and aminoglycosides. In enterococci, at least E. faecalis the treatment with either beta-lactams, vancomycin or lysozyme is enough to induce increased killing by aminoglycosides, which suggest that cell wall damage triggers increased uptake.
Pilot 2: Development of Antimicrobial Peptides as Effective Antimicrobials against ESKAPE Pathogens
Pilot Project Leader: Biswajit Mishra, PhD
The development of new antibiotic options is critical for counteracting the infection caused by multidrug resistant (MDR) bacteria. Possible solutions include the discovery or repurposing of compounds. More recently, with the development of novel α-helical peptide sequences with systemic efficacy, the antimicrobial peptides (AMPs) have been provided a potential alternative against MDR bacteria. Our long-term goal is to develop short α-helical AMPs with efficacy against a clinically relevant group of bacteria. The objective of this grant is to design the shortest 3.613-helix with only 12 amino acids containing AMPs that target a wide range of human infectious bacteria. The peptide design principles based on charge hydrophobicity balance will be used to create a pool of most probable active AMPs. Aided with the recently discovered synthetic retinoids and bithionol as a new antibiotic and repurposing counterpart, it increases the application strategies during combinations approaches. These discoveries will facilitate new avenues of therapeutic opportunities. Combining the peptides with retinoids, bithionol, and conventional antibiotics will enhance the chances of development of a new regime of antibiotic therapy for drug-resistant infections. Our specific aims include: (Aim 1) Rational design of potent antimicrobial peptides designed in silico could help cut the cost for chemical synthesis; (Aim 2) Lead peptides will be prioritize based on in vitro, and in vivo antimicrobial efficacy, reduced toxicity and mechanism of action; (Aim 3) New combinations of the peptides with retinoids, bithionol, and traditional antibiotics with in vivo efficacy will be established. These studies are expected to help us in establishing quick testing platforms for new AMP sequences and their combinations against a variety of pathogens. The proposed research is innovative because we will investigate the possibilities of novel AMPs sequences and combinations that have not been discovered earlier.
Pilot 3: Antimicrobial Susceptibility Testing and Whole Genome Sequencing of Neisseria Gonorrhoeae in Rhode Island: Connecting Isolate Profiles with Patient Geography and Behavior
Principal Investigator: Sara Vargas, PhD
Pilot Project Leader: Sara Vargas, PhD
Gonorrhea is the second most diagnosed sexually transmitted infection in the United States and, though curable, quickly develops resistance to antimicrobial treatments. Monitoring the spread of gonorrhea, and associated patterns of sexual behavior, is needed to prevent outbreaks and slow the emergence of antimicrobial resistance.
This pilot project brought together Sara Vargas, PhD (PI), a behavioral scientist and sexual health researchers, at the Miriam Hospital Center for Behavioral and Preventive Medicine, Philip Chan, MD (mentor), an infectious disease expert, at the Miriam Hospital STD Clinic, and the CARTD biorepository core. The main goal of this pilot was to examine associations between demographics, sexual behavior, and antimicrobial susceptibility/resistance at an outpatient STI clinic in an urban area (Providence, Rhode Island) to better understand how gonorrhea – and specifically gonorrhea with reduced susceptibility to antibiotic treatment – is locally transmitted. A secondary goal of this pilot was to provide Dr. Vargas and the CARTD Biorepository Core training in collecting and analyzing gonorrhea specimens to assess and improve feasibility for subsequent work. In addition to data collected from this pilot project, Drs. Vargas and Chan looked at clinic-level data to further describe demographics and behaviors associated with gonorrhea diagnosis.
In looking at clinic data from every encounter from 2015 to 2021, we found increasing rates of gonorrhea infection overtime at all sites (oral, genital, rectal), and identified specific demographics (i.e., race and gender) as well as specific sexual behaviors (i.e., condom non-use) that are tracking with increased rates of infection. We also found distinct profiles among individuals who tested positive for gonorrhea as compared to chlamydia or gonorrhea and chlamydia co-infection. These findings are currently under peer review for publication in high-impact sexual health journals and results will be made publicly available in line with NIH public access policy.
In-depth sexual behavior data was collected via survey from 20 participants who were diagnosed with gonorrhea in the past year. This data includes demographics, identities, zip code where they believe to have been infected, types of partners, type of sexual encounters, condom use/non-use, and other risk behaviors. This data is intended to be hypothesis generating for the next phase of the project and will be disseminated as a conference proceeding and/or peer-reviewed publication this year.
Finally, over the course of this pilot study, the study team has refined a protocol to allow for the successful procurement, storage, and analysis of gonorrhea samples that will be used in future iterations of this work.
Pilot 4: Investigating the Role of Ribosome Heterogeneity in a Bacterial Pathogen
Principal Investigator: Kathryn Ramsey, PhD (awarded R35)
Pilot Project Leader: Kathryn Ramsey, PhD
As antibiotic resistant bacteria become more common, the need for new antibiotics continues to increase. The work proposed here would investigate a novel target for antibiotic development that could lead to multiple drugs for the treatment of specific bacterial infections in the future.
Aim 1: Identify the molecular mechanisms by which bS21-2 influences translation of a specific T6SS protein
Aim 2: Initiate experiments to determine how bS21 influences global gene expression.
The cellular machine that produces proteins through the process of translation, the ribosome, is often thought of as monolithic. Yet ribosome composition within a species and even within a cell can be heterogenous. Our research has focused on the role(s) of heterogenous ribosomes in bacterial gene regulation and their potential as a novel antibiotic target. We had already determined that in the human pathogen and potential bioweapon Francisella tularensis, ribosomes incorporating different versions (homologs) of the ribosomal protein bS21 influence expression of virulence genes. In particular, we found that one ribosomal protein homolog, bS21-2, leads to increased production of certain key virulence proteins. These results are consistent with a model in which ribosomes with distinct compositions have altered specificity for translation and that this is important for virulence.
In this project, we found that a particular section of an mRNA, the beginning or 5´ end, is sufficient to lead to increased protein production in cells containing ribosomes with bS21-2 compared to cells that do not have ribosomes containing bS21-2. Only the 5´ end of mRNAs for certain genes exhibit this behavior, which we call bS21-2-responsive. We also found that modifying this section of the mRNA so that translation initiation is extremely efficient prevents responsiveness to bS21-2. Finally, we identified a particular sequence in the 5´ end of the mRNA of one gene that is essential for responsiveness to bS21-2. In addition to these findings, we are developing protocols to determine if these findings are reproducible outside of a cell (in vitro) using purified components and have examined how a particular bS21 homolog controls its own production.
We have also made progress towards being able to use a global approach to identify which mRNAs are translated by ribosomes containing homologs of bS21. This approach relies on using an affinity-based method to isolate ribosomes with a particular bS21 homolog. We have demonstrated that this technique is feasible and are continuing to validate specific protocols.
Pilot 5: Developing an Inhibitor of Molecular Evolution to Combat Antimicrobial Resistance
Principal Investigator: Alexandra Deaconescu, PhD (awarded R35)
The increased use (and misuse) of antibiotics in the last decades has led to the rapid development of pathogenic strains that are now resistant to these agents, leading to a global public health crisis. The objective of this proposal is to develop broad-spectrum inhibitors of molecular evolution to combat antimicrobial resistance in Gram-negative and Gram-positive pathogens. We will target the transcription-repair coupling factor Mfd, a large multi-functional ATPase widely conserved in most bacteria, but absent in most eukaryotes. Mfd mediates transcription-coupled DNA repair and resolves conflicts between replication and transcription due to its ability to dissociate stalled/paused transcription elongation complexes, and in the case of transcription-coupled DNA repair, to recruit the UvrA subunit of the nucleotide excision repair Uvr(A)BC excinuclease. Paradoxically, under certain conditions, Mfd also leads to hypermutation. The precise mechanisms behind this pro-mutagenic role have remained not well understood, but recent studies implicate Mfd-dependent formation of R-loops. To aid in structure-guided drug design, we have at our disposal a series of high-resolution crystal structures of Mfd, which together with rich functional information, enable us to screen in silico for small molecules that selectively target specific Mfd activities (ATPase function, DNA binding, DNA translocation, RNAP binding, UvrA binding). We will combine in silico screening with in vitro and in vivo functional studies as well as structure determination of inhibitor-bound Mfd using X-ray crystallography or, if deemed necessary, electron cryo-microscopy. Due to their ability to inhibit Mfd, thus hypermutation, these compounds would essentially function as broad-spectrum inhibitors of molecular evolution, and would ideally be administered in combination with well-characterized, narrow-spectrum antibiotics to prevent the development of antibiotic resistance mutations and tailor treatment to infection by various pathogens.
The major focus has been the generation of a series of reagents and the development of in vitro assays of Mfd functions and interactions with binding partners that could be deployed in the high-throughput screening of small molecule inhibitors of Mfd. In addition, we have finalized a series of four high-resolution crystal structures of Mfd in alternative functional states, which can now be added to the already existing structural information for drug screening purposes.
Pilot 6: Using Deep RNA Sequencing to Develop Novel Antimicrobial Treatment Strategies
Principal Investigator: Alger Fredericks, PhD
Sepsis is defined by altered physiology leading to organ and immune system dysfunction due to an infection and typically results in intensive care unit admission. Sepsis is responsible for one in five deaths worldwide. Previous sepsis research has focused on changes in gene and protein expression in blood.
Recent advances in next generation sequencing technologies have allowed for transcriptomics to become a valuable tool in gene expression analyses. Deep RNA sequencing produces hundreds of millions of data points that can detect both host gene expression as well gene expression on the level of specifics pathogens within the host.
Here, we use Methicillin Resistant Staphylococcus aureus (MRSA) infection in sepsis patients as a model to evaluate whether changes in MRSA resistance gene expression and changes in the host immune response (antibody and T cell receptors) can be correlated with disease state. In this study we evaluate resistance gene expression in S. aureus both in vitro in culture conditions mimicking those of sepsis patients such as hypoxia, acidosis, and high temperature, and in vivo to measure changes in resistance gene expression. We then evaluate the host immune response, as identified by production of novel antibodies and T cell receptors in sepsis patients with MRSA. In addition, based upon the antibodies and T cell receptors identified we hope to design and administer personalized compounds to treat patients based on their individual molecular profile. This study has the potential to make a significant contribution to the field of personalized medicine and improve the care of patients with sepsis due to MRSA.
Fredericks AM, East KW, Shi Y, Liu J, Maschietto F, Ayala A, Cioffi WG, Cohen M, Fairbrother WG, Lefort CT, Nau GJ, Levy MM, Wang J, Batista VS, Lisi GP, Monaghan SF. Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations. Front Mol Biosci. 2022 Dec 16;9:1080964. doi: 10.3389/fmolb.2022.1080964. PMID: 36589229; PMCID: PMC9800910.
Fredericks AM, Jentzsch MS, Cioffi WG, Cohen M, Fairbrother WG, Gandhi SJ, Harrington EO, Nau GJ, Reichner JS, Ventetuolo CE, Levy MM, Ayala A, Monaghan SF. Deep RNA sequencing of intensive care unit patients with COVID-19. Sci Rep. 2022 Sep 21;12(1):15755. doi: 10.1038/s41598-022-20139-1. PMID: 36130991; PMCID: PMC9491252.
Pilot 7: Acquisition of Anti-Microbial Resistance Caused by Lung Infection-Induced Hypoxia
Principal Investigator: Amanda Jamieson, PhD (awarded R01)
Pilot Project Leader: Amanda Jamieson, PhD
Pneumonia, either viral or bacterial, is the leading cause of death among children under 5 years of age. Influenza A virus (IAV) leads to an estimated 500,000 deaths annually, in addition to the hospitalizations and loss of productivity from infected people. In many cases, IAV infection is lethal due to coinfections with opportunistic bacterial pathogens such as S. pneumoniae, S. aureus, H. Influenzae, and S. pyogenes. Many fatal infections during the 1918 influenza pandemic had a bacterial coinfection. In addition, a survey of 35 adult intensive care units in the United States during the 2009 IAV pandemic found that 34% of patients with influenza-like-illness suffered from bacterial coinfections. Bacterial resistance to antibiotics impairs treatment of secondary bacterial pneumonia after IAV infection. Multidrug resistant bacteria have started to erode the gains that were made in battling infectious disease with the introduction of antibiotics in the last century, raising the possibility of a post-antibiotic era. Therefore, it is of utmost importance that we understand contributing factors to antibiotic resistance, how to prevent antibiotic resistance, and to develop novel therapeutics for multidrug resistant bacteria.
Infection with specific respiratory viruses including IAV and SARS-CoV-2 causes hypoxic conditions. I have a longstanding interest in secondary bacterial pneumonia that follows from IAV infection, and how the lung microbiome changes in response to infection and injury. Recently my lab has started to explore how bacterial pathogens adapt to the altered environment created by pulmonary IAV infection. Our analysis of this coinfection model demonstrates that it recapitulates many aspects of what is seen in vivo during coinfection, but with a tractable reductionist approach.
Using a novel in vitro system of coinfection using human bronchial epithelial cells (HBECs) grown in the air liquid interface (ALI) we have performed triple RNA-Seq that revealed distinct transcriptional changes in the host, virus, and bacteria during coinfection. Hypoxia is also a known risk factor for formation of biofilms and selection for bacteria that express antibiotic resistance genes. These are both known factors that allow for bacteria to evade killing by many classes of antibiotics. The limited previous work looking at antibiotic resistance of bacteria after IAV infection have come to several different conclusions have suggested that there are more antibiotic resistant bacteria after IAV infection than before.
With this proposal we will test our hypothesis that the hypoxic environment created by IAV infection contributes to biofilm formation and antibiotic resistance in bacterial pneumonia that develops after IAV infection. These studies will lay the foundation for future studies on understanding the impact of viral respiratory infections on the development of antibiotic resistant bacteria in the respiratory tract.
Specific Aim 1: Analyze clinical samples from pneumonia patients to test the hypothesis that the hypoxic environment found during viral infection with IAV triggers antibiotic resistance.
Specific Aim 2: Use air liquid interface model to further test the hypothesis that the lung epithelial environment caused by IAV infection can influence the development of antibiotic resistance.
Pilot 8: Characterization of the Antimicrobial Mechanisms of Silver Carboxylate (AgCar) Micro Pilot
Principal Investigator: Dioscaris R. Garcia, PhD
Pilot Project Leader: Dioscaris R. Garcia, PhD
This study focuses on the characterization of the mechanism of action of an organometallic silver-based antimicrobial with predictable pharmacokinetics. This project is aligned with the mission of the CARTD COBRE towards finding novel therapeutics, and the broader impact of the need for novel antimicrobials in the fight against the evolving threat of antibiotic resistance.
Aim 1: Demonstrate the Ability of AgCar to Trigger Release of Reactive Oxygen Species (ROS), Catalase (CAT), Peroxidase (POD), and Superoxide Dismutase (SOD).
Aim 2: Demonstrate Ability of AgCar to Induce Apoptosis-Like Effect in Gram Positive and Gram Negative Bacterial Pathogens Relative to Nanoparticle Silver
This study aimed to explore the antimicrobial mechanisms of silver carboxylate within the context of common nosocomial pathogens encountered in orthopedics: Staphylococcus epidermidis, and Serratia marcescens) and persister cells generated from Methicillin-Resistant S. aureus (MRSA) clinical strains VRS1 and MW2, and to determine if silver carboxylate was able to elicit apoptotic-like antimicrobial activity on gram positive Methicillin-Sensitive Staphylococcus aureus (MSSA) and gram negative Multi-Drug Resistant Serratia marcescens.
Our data shows that silver carboxylate was able to elicit a dose-dependent reactive oxygen species release on all pathogens tested at the 1x minimal effective concentration MIC on S. epidermidis, S. marcescens, and MRSA VRS1 and MW2-derived persister cells within a 6hr exposure period, with a doubling in the relative release at 10x MIC. All pathogens tested were very sensitive to silver carboxylate at the same concentration within a 24hr exposure. This data is consistent with previous data which showed that silver carboxylate is as potent or more potent than last resort antibiotics tobramycin, linezolid, polymyxin e, and vancomycin within a 24hr exposure. Data also showed that no pathogen was able to elicit an increase in the activity of peroxidase, catalase, or superoxide dismutase within the same time exposure. Additionally, the data suggests that silver carboxylate is also able to elicit apoptotic-like antimicrobial activity on gram positive Methicillin-Sensitive Staphylococcus aureus (MSSA) and gram negative Multi-Drug Resistant Serratia marcescens.
This data aligns with prior data which also showed that silver carboxylate is cytotoxic on primary-derived human cell lines involved in wound healing, while also eliciting apoptotic-cell death. Future studies will focus on further characterizing silver carboxylate’s apoptotic mechanisms of eukaryotic and apoptotic-like mechanisms of antimicrobial activities in pathogens of clinical importance and exploration of other antimicrobial mechanisms of action attributed to other silver formulations.
Pilot 9: Structure-Based Design of Beta-Lactamase Inhibitors (Micro Pilot)
Principal Investigator: Mandar Naik, PhD
Pilot Project Leader: Mandar Naik, PhD
A sharp and widespread increase in antimicrobial resistance (AMR) over the past three decades has seriously threatened our capability to treat bacterial infections. Of particular concern is the emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of pathogens that resist even the last-resort drugs like carbapenems, cephalosporins, and polymyxins. World Health Organization (WHO) has given highest priority to AMR research on the Gram-negative bacteria Acinetobacter, Pseudomonas and species of Enterobacterales that are increasingly emerging as XDR strains. The resistant strains cause systemic infection in the absence of appropriate immune response and do not respond to known antibiotic drugs that are destroyed by specialized enzymes. The goal of this project is to develop structure-based rational methodology to devise inhibitors against bacterial serine β-lactamase enzymes that are a leading cause of microbial resistance. These new inhibitors can be co-administered to increase efficacy of the β-lactam antibiotics.
Aim 1: NMR fragment-based screening of TEM-1
Aim 2: Study fragment binding to a set of representative β-lactamases
Aim 3: Synthesis and evaluation of new β-lactamase inhibitors
Our project aimed to devise new beta-lactamase inhibitors using structure-based knowledge. This was done using NMR spectroscopy which can generate atomic resolution information in a protein structure. We used TEM-1 beta-lactamase as a model and studied changes in its active site on the binding of a commercially available inhibitor, Avibactam. NMR spectroscopy was successful in identifying the avibactam-binding interface on TEM-1. We then screened one thousand chemical fragments library by NMR to identify fragments that bind to TEM-1. Our search found 69 chemical entities that interact with TEM-1. We then performed computational modeling to localize the fragment binding interfaces. In the next stage of this project, we will use NMR to study the simultaneous binding of avibactam and these fragments to TEM-1. We will then synthesize new inhibitors that will link these fragments to the drug scaffold and study them by enzyme kinetics and antimicrobial activity assays.
We also used NMR spectroscopy to study three other clinically important beta-lactamase enzymes representing diverse classes. These proteins were labeled with 13C and 15N isotopes and purified to homogeneity. 3D- NMR experiments were then performed to assign observed resonances to the individual residue in that specific enzyme.
Our main findings include:
- Localization of avibactam binding interface on TEM-1 by NMR spectroscopy
- Identification of 69 fragments that bind to TEM-1
- Computational docking experiments to predict binding of these fragments to TEM-1.
- >80% complete backbone NMR assignments of beta-lactamase SHV-1.
- 35% complete assignments of beta-lactamase OXA-40.
- NMR relaxation experiments to probe motional features and allosteric mechanism in beta-lactamases.
Pilot 10: Repurposing Aztreonam and Disulfiram to Combat Helicobacter Pylori
Principal Invetigator: Nagendran Tharmalingam, PhD
The emergence of antibiotic-resistant bacteria is now a serious threat to the global community. Fatalities caused by AMR bacteria are estimated to surpass the cancer-related casualties in 2050. World Health Organization (WHO) published a list of critical pathogens and rushed scientists to study the need for novel antibiotics. We focus on repurposing FDA-approved drugs against clarithromycin-resistant H. pylori. Hence the novel antibacterial discovery, either synthetic or biotic sources, is the savior in crisis, and pathogen-specific antibiotics can overcome the AMR.
Repurposing clinical drugs can be a savior and time-conserving strategy against antibiotic-resistant bacterial pathogens to explore the novel structural moieties and their target against pathogens. We did a pilot screen of clinical molecules against H. pylori and found that the intravenous monobactam antibacterial aztreonam and the anti-alcoholic agent disulfiram are active against H. pylori, specifically at acidic pH. Our preliminary data suggest that aztreonam and disulfiram show down-regulated virulence, motility in H. pylori, and hindered vacuolation in H. pylori-infected gastric epithelial cells. Aztreonam and disulfiram control H. pylori-induced upregulation of inflammatory and cancer progressing genes in H. pylori-infected gastric epithelial cells. We found that aztreonam or disulfiram could rescue the H. pylori-infected host (p<0.001) using an insect host model: Galleria mellonella. We utilized a murine gastric infection model using C57BL/6 mouse show that the oral administration of aztreonam (p<0.0001) or disulfiram (p=0.0004) decrease the inflammation and eliminate the H. pylori adherence to gastric mucosal or epithelial cells.
This proposal focus on two aims to investigate the detailed antibacterial activity of aztreonam and disulfiram in vitro coupled with their mechanism of action as aim one and in vivo efficacy of aztreonam and disulfiram in the presence of a proton pump inhibitor (PPI) as aim two.
Aim one will use state-of-the-art technologies such as RNA-sequencing to unveil the H. pylori responses to the aztreonam or disulfiram. The RNA studies will be carried out in H. pylori, H. pylori-infected gastric cells, and gastric tissue of infected mice. The whole-genome sequencing of drug-resistant H. pylori DNA will be utilized to understand the molecular target of aztreonam or disulfiram. We will also utilize fluorescence probes and metabolic profiling via LC-MS to understand the drug interaction with H. pylori.
Aim two will focus on the in vivo efficacy of aztreonam or disulfiram combined with PPI and its role in reducing inflammation and bacterial clearance from the gastric environment in mouse stomach.
The proposal's aim falls under the goal of drug development against antibiotic drug resistance and the purpose of the COBRE-CARTD center's scope. However, we will use the preliminary results to apply for NIH grants such as R01 to continue this study in more detail.
We are preparing the publication “Repurposing aztreonam and disulfiram to combat Helicobacter pylori” to understand and develop the drug for H. pylori therapy.