Michael D L Johnson

Interests

Research

Biochemistry, Biophysics, Microbiology, Microbial Genomics, Immunology

Teaching

microbiology, scientific communication, biochemistry of microbial systems, molecular microbiology

Courses

Scholarly Contributions

Journals/Publications

• Knox, C. J., Ab Latif, F. M., Cornejo, N. R., & Johnson, M. D. (2024). Mentoring across difference and distance: building effective virtual research opportunities for underrepresented minority undergraduate students in biological sciences. mBio, 15(1), e0145223.
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  Summer Research Experiences for Undergraduates (REUs) are established to provide platforms for interest in scientific research and as tools for eventual matriculation to scientific graduate programs. Unfortunately, the COVID-19 pandemic forced the cancellation of in-person programs for 2020 and 2021, creating the need for alternative programming. The National Summer Undergraduate Research Project (NSURP) was created to provide a virtual option to REUs in microbiology to compensate for the pandemic-initiated loss of research opportunities. Although in-person REUs have since been restored, NSURP currently remains an option for those unable to travel to in-person programs in the first place due to familial, community, and/or monetary obligations. This study examines the effects of the program's first 3 years, documenting the students' experiences, and suggests future directions and areas of study related to the impact of virtual research experiences on expanding and diversifying science, technology, engineering, and mathematics.
• Senthil Kumar, S., Gunda, V., Reinartz, D. M., Pond, K. W., Thorne, C. A., Santiago Raj, P. V., Johnson, M. D., & Wilson, J. E. (2024). Oral streptococci and induce distinct morphological, inflammatory, and metabolic signatures in macrophages. Infection and immunity, e0053623.
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  Oral streptococci, key players in oral biofilm formation, are implicated in oral dysbiosis and various clinical conditions, including dental caries, gingivitis, periodontal disease, and oral cancer. Specifically, is associated with esophageal, gastric, and pharyngeal cancers, while is linked to oral cancer. However, no study has investigated the mechanistic links between these species and cancer-related inflammatory responses. As an initial step, we probed the innate immune response triggered by and in RAW264.7 macrophages. These bacteria exerted time- and dose-dependent effects on macrophage morphology without affecting cell viability. Compared with untreated macrophages, macrophages infected with exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1β, NOS2, and COX2, accompanied by enhanced NF-κB activation. In contrast, -infected macrophages failed to elicit a robust inflammatory response. Seahorse Xfe96 analysis revealed an increased extracellular acidification rate in macrophages infected with compared with . At the 24-h time point, the presence of led to reduced extracellular itaconate, while triggered increased itaconate levels, highlighting distinct metabolic profiles in macrophages during infection in contrast to aconitate decarboxylase expression observed at the 6-h time point. This initial investigation highlights how and , two Gram-positive bacteria from the same genus, can prompt distinct immune responses and metabolic shifts in macrophages during infection.IMPORTANCEThe surge in head and neck cancer cases among individuals devoid of typical risk factors such as Human Papilloma Virus (HPV) infection and tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome's influence on physiology, the oral microbiome, notably oral streptococci, has been underappreciated during mucosal immunopathogenesis. , a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to compared with the early colonizer as an important first step toward understanding the impact of distinct oral species on the host immune response, which is an understudied determinant of OSCC development and progression.
• Akbari, M. S., Keogh, R. A., Radin, J. N., Sanchez-Rosario, Y., Johnson, M. D., Horswill, A. R., Kehl-Fie, T. E., Burcham, L. R., & Doran, K. S. (2023). The impact of nutritional immunity on Group B streptococcal pathogenesis during wound infection. mBio, 14(4), e0030423.
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  Group B (GBS) is a Gram-positive pathobiont that can cause adverse health outcomes in neonates and vulnerable adult populations. GBS is one of the most frequently isolated bacteria from diabetic (Db) wound infections but is rarely found in the non-diabetic (nDb) wound environment. Previously, RNA sequencing of wound tissue from Db wound infections in diabetic mice showed increased expression of neutrophil factors, and genes involved in GBS metal transport such as the zinc (Zn), manganese (Mn), and putative nickel (Ni) import systems. Here, we develop a Streptozotocin-induced diabetic wound model to evaluate the pathogenesis of two invasive strains of GBS, serotypes Ia and V. We observe an increase in metal chelators such as calprotectin (CP) and lipocalin-2 during diabetic wound infections compared to nDb. We find that CP limits GBS survival in wounds of non-diabetic mice but does not impact survival in diabetic wounds. Additionally, we utilize GBS metal transporter mutants and determine that the Zn, Mn, and putative Ni transporters in GBS are dispensable in diabetic wound infection but contributed to bacterial persistence in non-diabetic animals. Collectively, these data suggest that in non-diabetic mice, functional nutritional immunity mediated by CP is effective at mitigating GBS infection, whereas in diabetic mice, the presence of CP is not sufficient to control GBS wound persistence. IMPORTANCE Diabetic wound infections are difficult to treat and often become chronic due to an impaired immune response as well as the presence of bacterial species that establish persistent infections. Group B (GBS) is one of the most frequently isolated bacterial species in diabetic wound infections and, as a result, is one of the leading causes of death from skin and subcutaneous infection. However, GBS is notoriously absent in non-diabetic wounds, and little is known about why this species thrives in diabetic infection. The work herein investigates how alterations in diabetic host immunity may contribute to GBS success during diabetic wound infection.
• Dao, T. H., Iverson, A., Neville, S. L., Johnson, M. D., McDevitt, C. A., & Rosch, J. W. (2023). The role of CopA in Streptococcus pyogenes copper homeostasis and virulence. Journal of inorganic biochemistry, 240, 112122.
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  Maintenance of intracellular metal homeostasis during interaction with host niches is critical to the success of bacterial pathogens. To prevent infection, the mammalian innate immune response employs metal-withholding and metal-intoxication mechanisms to limit bacterial propagation. The first-row transition metal ion copper serves critical roles at the host-pathogen interface and has been associated with antimicrobial activity since antiquity. Despite lacking any known copper-utilizing proteins, streptococci have been reported to accumulate significant levels of copper. Here, we report that loss of CopA, a copper-specific exporter, confers increased sensitivity to copper in Streptococcus pyogenes strain HSC5, with prolonged exposure to physiological levels of copper resulting in reduced viability during stationary phase cultivation. This defect in stationary phase survival was rescued by supplementation with exogeneous amino acids, indicating the pathogen had altered nutritional requirements during exposure to copper stress. Furthermore, S. pyogenes HSC5 ΔcopA was substantially attenuated during murine soft-tissue infection, demonstrating the importance of copper efflux at the host-pathogen interface. Collectively, these data indicate that copper can severely reduce the viability of stationary phase S. pyogenes and that active efflux mechanisms are required to survive copper stress in vitro and during infection.
• Iannuzo, N., Welfley, H., Li, N. C., Johnson, M. D., Rojas-Quintero, J., Polverino, F., Guerra, S., Li, X., Cusanovich, D. A., Langlais, P. R., & Ledford, J. G. (2023). CC16 drives VLA-2-dependent SPLUNC1 expression. Frontiers in immunology, 14, 1277582.
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  CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated.
• Jaffey, J. A., Shubitz, L. F., Johnson, M. D., Bolch, C. A., da Cunha, A., Murthy, A. K., Lopez, B. S., Monasky, R., Carswell, I., Spiker, J., Neubert, M. J., & Menghani, S. V. (2023). Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis. Journal of fungi (Basel, Switzerland), 9(2).
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  The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105-310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations ( = 0.02) and serum CRP concentrations compared to controls ( < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination ( = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α ( = 0.0003), interleukin (IL)-6 ( = 0.04), interferon (IFN)-γ ( = 0.03), monocyte chemoattractant protein (MCP)-1 ( = 0.02), IL-10 ( = 0.02), and lower IL-8 ( = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis.
• Menghani, S. V., Sanchez-Rosario, Y., Pok, C., Liu, R., Gao, F., O’Brien, H., Neubert, M. J., Ochoa, K., Durckel, M., Hellinger, R. D., Hackett, N., Wang, W., & Johnson, M. (2023). Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against Streptococcal species. Frontiers in Microbiology, 13.
• O'Brien, H., Davoodian, T., & Johnson, M. D. (2023). The promise of copper ionophores as antimicrobials. Current opinion in microbiology, 75, 102355.
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  Antibiotic-resistant microbe-mediated deaths are a major worldwide health issue. Unfortunately, due to microbial adaptation to develop resistance, some antibiotics are nullified early in their usage, and worse, resistance is detected before they can even be prescribed. Copper's toxicity since antiquity against microbes at the host-pathogen interface offers a fascinating weapon to fight antimicrobial resistance. Here, we briefly review why copper is so effective, how drugs that work with copper are effective antimicrobials, and how compounds such as these could reinvigorate investment in antimicrobial development.
• Galligan, J. J., Johnson, M. D., & Cusanovich, D. A. (2022). Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei. BMC Genomics.
• Galligan, J. J., Johnson, M. D., Cusanovich, D. A., Farrera-Gaffney, D., Alvin, J. W., Rice, M. E., & Zhang, H. (2022). Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei. BMC Genomics. doi:10.1186/s12864-021-08266-x
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  Abstract Background The “Assay for Transposase Accessible Chromatin sequencing” (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics. Results In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. Conclusions We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.
• Johnson, M. D., & Knox, C. J. (2022). National Summer Undergraduate Research Project (NSURP): A Virtual Research Experience to Deliver REAL (Retention, Equity, Access, and Life-Changing) Outcomes for Underrepresented Minorities in STEM. Journal of microbiology & biology education, 23(1).
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  Summer undergraduate research experiences (SUREs) provide important onramps to secondary STEM graduate degrees and subsequent careers. Studies demonstrate that these experiences increase the likelihood of students advancing to a graduate-level STEM degree, positively impact STEM identity and confidence, and imbue a sense of professional belonging. In 2020, COVID-19 shutdowns eliminated many in-person SUREs. In response, we launched the National Summer Undergraduate Research Project (NSURP). While NSURP addressed an immediate need for a flexible research experience, we found that this model extends access to underrepresented minorities because it provides authentic research experiences for students who are unable to travel to a research location, and/or who have familial responsibilities that necessitate a flexible work model, and/or students facing financial challenges. What began as an emergency summer research program for undergraduates to address laboratory closures resulted in what we believe is a necessary and normalized addition to the undergraduate STEM training and preparation repertoire.
• Johnson, M. D., Wu, H. J., Menghani, S. V., Cutcliffe, M. P., Sanchez-Rosario, Y., Pok, C., Watson, A., Neubert, M. J., & Ochoa, K. (2022). N , N -Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance. Infection and Immunity, 90(4). doi:10.1128/iai.00597-21
• Li, Z. N., Liu, F., Jefferson, S., Horner, L., Carney, P., Johnson, M. D., King, J. P., Martin, E. T., Zimmerman, R. K., Wernli, K., Gaglani, M., Thompson, M., Flannery, B., Stevens, J., Tumpey, T., & Levine, M. Z. (2022). Multiplex Detection of Antibody Landscapes to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/Influenza/Common Human Coronaviruses Following Vaccination or Infection With SARS-CoV-2 and Influenza. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 75(Suppl 2), S271-S284.
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  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to co-circulate, representing 2 major public health threats from respiratory infections with similar clinical presentations. SARS-CoV-2 and influenza vaccines can also now be co-administered. However, data on antibody responses to SARS-CoV-2 and influenza coinfection and vaccine co-administration remain limited.
• Menghani, S. V., Cutcliffe, M. P., Sanchez-Rosario, Y., Pok, C., Watson, A., Neubert, M. J., Ochoa, K., Wu, H. J., & Johnson, M. D. (2022). ,-Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance. Infection and immunity, 90(4), e0059721.
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  Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a significant cause of disease burden in pediatric and elderly populations. The rise in unencapsulated disease-causing strains and antimicrobial resistance in S. pneumoniae has increased the need for developing new antimicrobial strategies. Recent work by our laboratory has identified ,-dimethyldithiocarbamate (DMDC) as a copper-dependent antimicrobial against bacterial, fungal, and parasitic pathogens. As a bactericidal antibiotic against S. pneumoniae, DMDC's ability to work as a copper-dependent antibiotic and its ability to work warranted further investigation. Here, our group studied the mechanisms of action of DMDC under various medium and excess-metal conditions and investigated DMDC's interactions with the innate immune system and . Of note, we found that DMDC plus copper significantly increased the internal copper concentration, hydrogen peroxide stress, nitric oxide stress, and the macrophage killing efficiency and decreased capsule. Furthermore, we found that DMDC treatment increased the quantity of innate immune cells in the lung during infection. Taken together, this study provides mechanistic insights regarding DMDC's activity as an antibiotic at the host-pathogen interface.
• Menghani, S. V., Sanchez-Rosario, Y., Pok, C., Liu, R., Gao, F., O'Brien, H., Neubert, M. J., Ochoa, K., Durckel, M., Hellinger, R. D., Hackett, N., Wang, W., & Johnson, M. D. (2022). Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against species. Frontiers in microbiology, 13, 1099330.
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  Despite the availability of several vaccines against multiple disease-causing strains of , the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including . Here, DMDCs efficacy against pathogens , , and was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives' effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against TIGR4 and tested in bactericidal and animal models. Two compounds, sodium -benzyl--methyldithiocarbamate and sodium -allyl--methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of -infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.
• Zhang, H., Rice, M. E., Alvin, J. W., Farrera-Gaffney, D., Galligan, J. J., Johnson, M. D., & Cusanovich, D. A. (2022). Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei. BMC genomics, 23(1), 214.
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  The "Assay for Transposase Accessible Chromatin sequencing" (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. With all possible combinations of components, we created 24 experimental conditions with four replicates for each (a total of 96 samples). In addition, we tested the 12 native conditions in a primary sample type (mouse lung tissue) with two different input amounts. Through these extensive comparisons, we were able to observe the effect of different ATAC-seq conditions on data quality and to examine the utility and potential redundancy of various quality metrics.
• Guerra, S., David, J. M., Younis, U. S., Whalen, M. B., Romanoski, C. E., Polverino, F., Pilon, A. L., Menghani, S. V., Martinez, F. D., Ledford, J. G., Kraft, M., Johnson, M. D., Guerra, S., David, J. M., Cress, A. E., & Addison, K. J. (2021). CC16 Binding to α4β1 Integrin Protects against Mycoplasma pneumoniae Infection.. American journal of respiratory and critical care medicine, 203(11), 1410-1418. doi:10.1164/rccm.202006-2576oc
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  Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4β1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.
• Johnson, M. D., Galgiani, J. N., Menghani, S. V., Rivera, A., Neubert, M., Hagerty, J. R., Lewis, L., Jolly, E. R., & Alvin, J. W. (2021). Demonstration of N , N -Dimethyldithiocarbamate as a Copper-Dependent Antibiotic against Multiple Upper Respiratory Tract Pathogens. Microbiology Spectrum, 9(2). doi:10.1128/spectrum.00778-21
• Menghani, S. V., Rivera, A., Neubert, M., Hagerty, J. R., Lewis, L., Galgiani, J. N., Jolly, E. R., Alvin, J. W., & Johnson, M. D. (2021). Demonstration of ,-Dimethyldithiocarbamate as a Copper-Dependent Antibiotic against Multiple Upper Respiratory Tract Pathogens. Microbiology spectrum, 9(2), e0077821.
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  Transition metals are necessary cofactors and structural elements in living systems. Exposure to high concentrations of biologically important transition metals, such as zinc and copper, results in cell toxicity. At the infection site, the immune system deploys metal sorbent proteins (e.g., lactoferrin and calprotectin) to starve pathogens of necessary metals (such as iron), while phagocytes expose engulfed pathogens to high levels of other metals, such as copper and zinc. The opportunistic pathogen Streptococcus pneumoniae (the pneumococcus) encounters macrophages during initial and protracted infections. The pneumococcus employs a copper export pathway, which improves colonization and persistent infection of the nasopharynx and the upper respiratory tract. Because copper is tightly regulated in the host, we instead sought to leverage the localized power of nutritional immunity by identifying small molecules with copper-dependent toxicity (CDT) through a targeted screen of compounds for antibiotic efficacy. We chose to include dithiocarbamates, based on the copper synergy observed in other organisms with 1-(diethylthiocarbamoyldisulfanyl)-,-diethyl-methanethioamide (tetraethylthiuram disulfide, disulfiram). We observed CDT of some dithiocarbamates in S. pneumoniae. Only ,-dimethyldithiocarbamate (DMDC) was consistently toxic across a range of concentrations with copper both and against the pneumococcus. We also observed various degrees of CDT using DMDC in Staphylococcus aureus, Coccidioides posadasii, and Schistosoma mansoni. Collectively, we demonstrate that the compound DMDC is a potent bactericidal compound against S. pneumoniae with antimicrobial efficacy against bacterial and fungal pathogens. With the rise of antibiotic resistance, approaches that add new antimicrobials to the current repertoire are vital. Here, we investigate putative and known copper ionophores in an attempt to intoxicate bacteria and use ionophore/copper synergy, and we ultimately find success with ,-dimethyldithiocarbamate (DMDC). We show that DMDC has efficacy in a copper-dependent manner and kills pathogens across three different kingdoms, Streptococcus pneumoniae, , and Schistosoma mansoni, and efficacy against S. pneumoniae. As such, dithiocarbamates represent a new potential class of antimicrobials and thus warrant further mechanistic investigation.
• Peng, T., Zong, Y., Johnson, M. D., Menghani, S. V., Lewis, M. L., & Galgiani, J. N. (2021). A quantitative enzyme-linked immunoassay (ELISA) to approximate complement-fixing antibody titers in serum from patients with coccidioidomycosis. Diagnostic microbiology and infectious disease, 99(1), 115198.
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  Coccidioidomycosis is most frequently diagnosed serologically, and the quantitative test for complement-fixing antibodies is considered prognostically useful. Because complement-fixing antibody testing is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. In this report, we restrict the complement-fixing, antibody-binding domain to a 200-amino-acid recombinant peptide of the known antigen. Over-lapping truncations of this peptide do not bind complement-fixing antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal biotin-mimic peptide tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by 1-2 logs in different sera. The newly developed ELISA shows a significant quantitative correlation with complement-fixing antibody titers. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.
• Sanchez-Rosario, Y., & Johnson, M. D. (2021). Media Matters, Examining Historical and Modern Growth Media and the Experiments They Affect. Frontiers in cellular and infection microbiology, 11, 613623.
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  While some bacteria can thrive for generations in minerals and salts, many require lavish nutrition and specific chemicals to survive to the point where they can be observed and researched. Although researchers once boiled and rendered animal flesh and bones to obtain a media that facilitated bacterial growth, we now have a plethora of formulations and manufacturers to provide dehydrated flavors of historical, modified, and modern media. The purpose of media has evolved from simple isolation to more measured study. However, in some instances, media formulated to aid the metabolic, nutritional, or physical properties of microbes may not be best suited for studying pathogen behavior or resilience as a function of host interactions. While there have been comparative studies on handfuls of these media in , this review focuses on describing both the historical and modern composition of common complex (Todd Hewitt and M17), semi-defined (Adams and Roe), and defined pneumococcal media (RPMI and Van de Rijn and Kessler), key components discovered/needed for cultivation/growth enhancement, and effects these different media have on bacterial phenotypes and experimental outcomes. While many researchers find the best conditions to grow and experiment on their bacteria of choice, the reasons for some researchers to use a specific medium is at best, not discussed, and at worst, arbitrary. As such, the goal of this review is to highlight the differences in pneumococcal media to encourage investigators to challenge their decisions on why they use a given medium, discuss the recipe, and explain their reasoning.
• Smith, A. P., Lane, L. C., van Opijnen, T., Woolard, S., Carter, R., Iverson, A., Burnham, C., Vogel, P., Roeber, D., Hochu, G., Johnson, M. D., McCullers, J. A., Rosch, J., & Smith, A. M. (2021). Dynamic Pneumococcal Genetic Adaptations Support Bacterial Growth and Inflammation during Coinfection with Influenza. Infection and immunity, 89(7), e0002321.
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  Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to postinfluenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza virus-infected hosts. The majority of the 32 genes identified are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated mutants with single-gene deletions (SGD) of five of the genes identified, SPD1414, SPD2047 (), SPD0058 (), SPD1098, and SPD0822 (), to investigate their effects on fitness, disease severity, and host immune responses. The growth of the SGD mutants was slightly attenuated and , but each still grew to high titers in the lungs of mock- and influenza virus-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed altered neutrophil distribution with reduced degeneration in the lungs of influenza virus-SGD mutant-coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.
• Edwards, M., Johnson, M. D., Fernandez, R. W., Montgomery, B. L., Adelaja, A., & Akingbade, T. (2020). Mentoring to Foster a Diverse Future. Cell, 183(3), 561-563.
• Johnson, M. D. (2020). mSphere Highlights Black In Microbiology Week. mSphere, 5(5). doi:10.1128/msphere.00966-20
• Johnson, M. D., & Johnson, M. D. (2020). Highlights Black In Microbiology Week. mSphere, 5(5).
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  The inaugural Black In Microbiology Week (#BlackInMicro) is 28 September 2020 through 4 October 2020. Its mission is to "showcase the presence and accomplishments of Black microbiologists from around the globe, connect Black microbiologists with one another and foster a sense of community among them, and provide a forum for the discussion of racial disparities in microbiology and its subfields." Participation in this event will happen primarily over Twitter through the hashtag #BlackInMicro and over Zoom through registration on the website https://blackinmicrobiology.org/ An additional mission of Black In Microbiology Week is to amplify black scientists. Today, does this by presenting two mSphere of Influence commentaries from Black In Microbiology co-lead organizers Ariangela J. Kozik ("mSphere of Influence: frameshift-a vision for human microbiome research" [mSphere 5:e00944-20, 2020, https://doi.org/10.1128/mSphere.00944-20]) and Kishana Taylor ("mSphere of Influence: that's racist-COVID-19, biological determinism, and the limits of hypotheses" [mSphere 5:e00945-20, 2020, https://doi.org/10.1128/mSphere.00945-20]).
• Johnson, M. D., Baltrus, D. A., & Gardy, J. (2020). Crowdsourcing virtual summer research opportunities to support minorities in microbiology. Nature microbiology, 5(11), 1311-1313.
• Johnson, M. D., Van Doorslaer, K., Menghani, S. V., O’Brien, H., Alvin, J. W., & Sanchez-Rosario, Y. (2020). Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors. mSphere, 5(3). doi:10.1128/msphere.00411-20
• Johnson, M. D., Younis, U. S., Menghani, S. V., Addison, K. J., Whalen, M., Pilon, A. L., Cress, A. E., Polverino, F., Romanoski, C., Kraft, M., Martinez, F. D., Guerra, S., & Ledford, J. G. (2020). CC16 Binding to α4β1 Integrin (VLA-4) Protects Against Infection. American journal of respiratory and critical care medicine.
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  Club cell secretory protein (CC16) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases: asthma and COPD. While exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating anti-inflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, Leucine-Valine-Aspartic Acid (LVD), a known integrin binding motif. Recombinant CC16 was generated with and without the putative integrin binding site. A Mycoplasma pneumoniae mouse model and a flourescent cellular adhesion assay were used to determine the impact of the LVD site in regards to CC16 function during live infection and on cellular adhesion during inflammatory conditions. CC16 bound to integrin alpha 4 and beta 1 (α4β1), also known as the adhesion molecule very late antigen-4 (VLA-4), dependent on the presence of the LVD integrin binding motif. During infection, rCC16 rescued lung function parameters both when administered to the lung and intravenously, but only when the LVD integrin binding site is intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.
• O'Brien, H., Alvin, J. W., Menghani, S. V., Sanchez-Rosario, Y., Van Doorslaer, K., & Johnson, M. D. (2020). Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors. mSphere, 5(3).
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  Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems ( operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In (the pneumococcus), CopY is the copper repressor for the operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the box. Using this motif, we sought to determine whether genes outside the operon are also regulated by the CopY repressor, which was previously shown in We found that CopY did not bind to operators upstream of these candidate genes During this process, we found that the box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant ( ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.
• Johnson, M. D. (2019). Characterization of consensus operator site for Streptococcus pneumoniae copper repressor. bioRxiv. doi:https://doi.org/10.1101/676700
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  O’Brien H*, Alvin J*, Menghani SV, Van Doorslaer K, Johnson MDL##. Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY. bioRxiv. 2019 Jun 21. doi: https://doi.org/10.1101/676700
• Johnson, M. D. (2019). Dynamic pneumococcal genetic adaptations support bacterial growth and inflammation during coinfection with influenza. bioRxiv. doi:https://doi.org/10.1101/659557
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  Smith AP, Lane LC, van Opijnen T, Woolard S, Carter R, Iverson A, Burnham C, Vogel P, Roeber D, Hochu G, Johnson MDL, McCullers JA, Rosch J, Smith A. Dynamic pneumococcal genetic adaptations support bacterial growth and inflammation during coinfection with influenza. bioRxiv. 2019 Jun 4. doi: https://doi.org/10.1101/659557
• Johnson, M. D. (2019). mSphere of Influence: Hiring of Underrepresented Minority Assistant Professors in Medical School Basic Science Departments Has a Long Way To Go. mSphere, 4(5).
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  Michael D. L. Johnson is a molecular microbiologist observing the role of metals in bacterial biology. In this mSphere of Influence article, he discusses the impact that the paper "Decoupling of the minority PhD talent pool and assistant professor hiring in medical school basic science departments in the US" by Kenneth D. Gibbs, Jr., Jacob Basson, Imam M. Xierali, and David A. Broniatowski (eLife 5:e21393, 2016, https://doi.org/10.7554/eLife.21393) had on him regarding hiring underrepresented minorities at research-intensive institutions and what he considers to be the next steps.
• Johnson, M. D. (2019). mSphere of Influence: Hiring of Underrepresented Minority Assistant Professors in Medical School Basic Science Departments Has a Long Way To Go. mSphere, 4(5). doi:10.1128/msphere.00599-19
• Cunha, L. D., Yang, M., Carter, R., Guy, C., Harris, L., Crawford, J. C., Quarato, G., Boada-Romero, E., Kalkavan, H., Johnson, M. D., Natarajan, S., Turnis, M. E., Finkelstein, D., Opferman, J. T., Gawad, C., & Green, D. R. (2018). LC3-Associated Phagocytosis in Myeloid Cells Promotes Tumor Immune Tolerance. Cell, 175(2), 429-441.e16.
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  Targeting autophagy in cancer cells and in the tumor microenvironment are current goals of cancer therapy. However, components of canonical autophagy play roles in other biological processes, adding complexity to this goal. One such alternative function of autophagy proteins is LC3-associated phagocytosis (LAP), which functions in phagosome maturation and subsequent signaling events. Here, we show that impairment of LAP in the myeloid compartment, rather than canonical autophagy, induces control of tumor growth by tumor-associated macrophages (TAM) upon phagocytosis of dying tumor cells. Single-cell RNA sequencing (RNA-seq) analysis revealed that defects in LAP induce pro-inflammatory gene expression and trigger STING-mediated type I interferon responses in TAM. We found that the anti-tumor effects of LAP impairment require tumor-infiltrating T cells, dependent upon STING and the type I interferon response. Therefore, autophagy proteins in the myeloid cells of the tumor microenvironment contribute to immune suppression of T lymphocytes by effecting LAP.
• Johnson, M. D., Ambrose, A., Dahlmann, E. A., & Neubert, M. J. (2017). Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon. mSphere, 2(5). doi:10.1128/msphere.00372-17
• Neubert, M. J., Dahlmann, E. A., Ambrose, A., & Johnson, M. D. (2017). Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon. mSphere, 2(5).
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  Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu(2+) to Cu(1+), which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu(2+) to Cu(1+). This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.
• Echlin, H., Frank, M. W., Iverson, A., Chang, T., Johnson, M. D., Rock, C. O., & Rosch, J. W. (2016). Pyruvate Oxidase as a Critical Link between Metabolism and Capsule Biosynthesis in Streptococcus pneumoniae. PLoS pathogens, 12(10), e1005951.
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  The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation.
• Johnson, M. D., & Ayers, K. A. (2016). Science Sound Bites, a Podcast for STEM Curriculum Supplementation. Journal of microbiology & biology education, 17(2), 286-7.
• Meliopoulos, V. A., Van de Velde, L., Van de Velde, N. C., Karlsson, E. A., Neale, G., Vogel, P., Guy, C., Sharma, S., Duan, S., Surman, S. L., Jones, B. G., Johnson, M. D., Bosio, C., Jolly, L., Jenkins, R. G., Hurwitz, J. L., Rosch, J. W., Sheppard, D., Thomas, P. G., , Murray, P. J., et al. (2016). An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens. PLoS pathogens, 12(8), e1005804.
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  The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival.
• Johnson, M. D., Echlin, H., Dao, T. H., & Rosch, J. W. (2015). Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae. Microbiology (Reading, England), 161(11), 2127-36.
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  NAD is a necessary cofactor present in all living cells. Some bacteria cannot de novo synthesize NAD and must use the salvage pathway to import niacin or nicotinamide riboside via substrate importers NiaX and PnuC, respectively. Although homologues of these two importers and their substrates have been identified in other organisms, limited data exist in Streptococcus pneumoniae, specifically, on its effect on overall virulence. Here, we sought to characterize the substrate specificity of NiaX and PnuC in Str. pneumoniae TIGR4 and the contribution of these proteins to virulence of the pathogen. Although binding affinity of each importer for nicotinamide mononucleotide may overlap, we found NiaX to specifically import nicotinamide and nicotinic acid, and PnuC to be primarily responsible for nicotinamide riboside import. Furthermore, a pnuC mutant is completely attenuated during both intranasal and intratracheal infections in mice. Taken together, these findings underscore the importance of substrate salvage in pneumococcal pathogenesis and indicate that PnuC could potentially be a viable small-molecule therapeutic target to alleviate disease progression in the host.
• Johnson, M. D., Kehl-Fie, T. E., & Rosch, J. W. (2015). Copper intoxication inhibits aerobic nucleotide synthesis in Streptococcus pneumoniae. Metallomics : integrated biometal science, 7(5), 786-94.
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  Copper is universally toxic in excess, a feature exploited by the human immune system to facilitate bacterial clearance. The mechanism of copper intoxication remains unknown for many bacterial species. Here, we demonstrate that copper toxicity in Streptococcus pneumoniae is independent from oxidative stress but, rather, is the result of copper inhibiting the aerobic dNTP biosynthetic pathway. Furthermore, we show that copper-intoxicated S. pneumoniae is rescued by manganese, which is an essential metal in the aerobic nucleotide synthesis pathway. These data provide insight into new targets to enhance copper-mediated toxicity during bacterial clearance.
• Johnson, M. D., Kehl-Fie, T. E., Klein, R., Kelly, J., Burnham, C., Mann, B., & Rosch, J. W. (2015). Role of copper efflux in pneumococcal pathogenesis and resistance to macrophage-mediated immune clearance. Infection and immunity, 83(4), 1684-94.
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  In bacteria, the intracellular levels of metals are mediated by tightly controlled acquisition and efflux systems. This is particularly true of copper, a trace element that is universally toxic in excess. During infection, the toxic properties of copper are exploited by the mammalian host to facilitate bacterial clearance. To better understand the role of copper during infection, we characterized the contribution of the cop operon to copper homeostasis and virulence in Streptococcus pneumoniae. Deletion of either the exporter, encoded by copA, or the chaperone, encoded by cupA, led to hypersensitivity to copper stress. We further demonstrated that loss of the copper exporter encoded by copA led to decreased virulence in pulmonary, intraperitoneal, and intravenous models of infection. Deletion of copA resulted in enhanced macrophage-mediated bacterial clearance in vitro. The attenuation phenotype of the copA mutant in the lung was found to be dependent on pulmonary macrophages, underscoring the importance of copper efflux in evading immune defenses. Overall, these data provide insight into the role of the cop operon in pneumococcal pathogenesis.
• Cheng, Y., Johnson, M. D., Burillo-Kirch, C., Mocny, J. C., Anderson, J. E., Garrett, C. K., Redinbo, M. R., & Thomas, C. E. (2013). Mutation of the conserved calcium-binding motif in Neisseria gonorrhoeae PilC1 impacts adhesion but not piliation. Infection and immunity, 81(11), 4280-9.
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  Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae.
• Honsa, E. S., Johnson, M. D., & Rosch, J. W. (2013). The roles of transition metals in the physiology and pathogenesis of Streptococcus pneumoniae. Frontiers in cellular and infection microbiology, 3, 92.
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  For bacterial pathogens whose sole environmental reservoir is the human host, the acquisition of essential nutrients, particularly transition metals, is a critical aspect of survival due to tight sequestration and limitation strategies deployed to curtail pathogen outgrowth. As such, these bacteria have developed diverse, specialized acquisition mechanisms to obtain these metals from the niches of the body in which they reside. To oppose the spread of infection, the human host has evolved multiple mechanisms to counter bacterial invasion, including sequestering essential metals away from bacteria and exposing bacteria to lethal concentrations of metals. Hence, to maintain homeostasis within the host, pathogens must be able to acquire necessary metals from host proteins and to export such metals when concentrations become detrimental. Furthermore, this acquisition and efflux equilibrium must occur in a tissue-specific manner because the concentration of metals varies greatly within the various microenvironments of the human body. In this review, we examine the functional roles of the metal import and export systems of the Gram-positive pathogen Streptococcus pneumoniae in both signaling and pathogenesis.
• Porsch, E. A., Johnson, M. D., Broadnax, A. D., Garrett, C. K., Redinbo, M. R., & St Geme, J. W. (2013). Calcium binding properties of the Kingella kingae PilC1 and PilC2 proteins have differential effects on type IV pilus-mediated adherence and twitching motility. Journal of bacteriology, 195(4), 886-95.
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  Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, which is dependent on type IV pili and is influenced by two PilC-like proteins called PilC1 and PilC2. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. In this study, we set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional 9-amino-acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12-amino-acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we showed that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.
• Johnson, M. D., Garrett, C. K., Bond, J. E., Coggan, K. A., Wolfgang, M. C., & Redinbo, M. R. (2011). Pseudomonas aeruginosa PilY1 binds integrin in an RGD- and calcium-dependent manner. PloS one, 6(12), e29629.
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  PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850-859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619-621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600-608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851-859 also impacts the protein's association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.
• Orans, J., Johnson, M. D., Coggan, K. A., Sperlazza, J. R., Heiniger, R. W., Wolfgang, M. C., & Redinbo, M. R. (2010). Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility. Proceedings of the National Academy of Sciences of the United States of America, 107(3), 1065-70.
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  Several bacterial pathogens require the "twitching" motility produced by filamentous type IV pili (T4P) to establish and maintain human infections. Two cytoplasmic ATPases function as an oscillatory motor that powers twitching motility via cycles of pilus extension and retraction. The regulation of this motor, however, has remained a mystery. We present the 2.1 A resolution crystal structure of the Pseudomonas aeruginosa pilus-biogenesis factor PilY1, and identify a single site on this protein required for bacterial translocation. The structure reveals a modified beta-propeller fold and a distinct EF-hand-like calcium-binding site conserved in pathogens with retractile T4P. We show that preventing calcium binding by PilY1 using either an exogenous calcium chelator or mutation of a single residue disrupts Pseudomonas twitching motility by eliminating surface pili. In contrast, placing a lysine in this site to mimic the charge of a bound calcium interferes with motility in the opposite manner--by producing an abundance of nonfunctional surface pili. Our data indicate that calcium binding and release by the unique loop identified in the PilY1 crystal structure controls the opposing forces of pilus extension and retraction. Thus, PilY1 is an essential, calcium-dependent regulator of bacterial twitching motility.
• Wang, H., Li, H., Moore, L. B., Johnson, M. D., Maglich, J. M., Goodwin, B., Ittoop, O. R., Wisely, B., Creech, K., Parks, D. J., Collins, J. L., Willson, T. M., Kalpana, G. V., Venkatesh, M., Xie, W., Cho, S. Y., Roboz, J., Redinbo, M., Moore, J. T., & Mani, S. (2008). The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor. Molecular endocrinology (Baltimore, Md.), 22(4), 838-57.
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  Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.
• Hess, P. R., Barnes, C., Woolard, M. D., Johnson, M. D., Cullen, J. M., Collins, E. J., & Frelinger, J. A. (2007). Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin. Blood, 109(8), 3300-7.
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  CD8+ cytotoxic T lymphocytes (CTLs) are important effector cells responsible for tissue destruction in several autoimmune and allograft-related diseases. To discover if pathogenic T cells could be selectively deleted, we investigated the ability of a toxin coupled to major histocompatibility complex (MHC) class I tetramers to kill antigen-specific CD8+ T cells. H2-D(b) tetramers were assembled using streptavidin conjugated to the ribosome-inactivating protein (RIP) saporin (SAP). These tetramers inhibited ribosome activity in vitro, retained the T-cell receptor (TCR)-binding specificity of their nontoxic counterparts, and were internalized by 100% of target cells, leading to cell death in 72 hours. Cytotoxicity was dependent on the tetramer dose and avidity for the T cell. A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.

Presentations

• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Pennsylvania State university Department of Biochemistry and Molecular Biology.
• Johnson, M. D. (2021). Copper Toxicity in Streptococcus pneumoniae. IDWEEK 2021 (Virtual).
• Johnson, M. D. (2021). Crowdsourcing research opportunities for underrepresent minorities in STEM. COM-T Research Day.
• Johnson, M. D. (2021). Metals, Media, and Microbes Matter. mSystems Thinking Series: Diversity, Equity, and Inclusion in Microbiology.
• Johnson, M. D. (2021). Metals, Microbes, Media, Music, and Me. VI4 (Vanderbilt University) Diversity Symposium "Careers in STEM: Perspectives from Black Scientists”.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Arizona State University, School of Molecular Sciences.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Brown University Department of Pathobiology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. California State University East Bay Department of Biological Sciences.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Duke University Department of Molecular Genetics and Microbiology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. The University of British Columbia Department of Microbiology and Immunology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Tufts University Molecular Biology and Microbiology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Alabama at Birmingham Department of Microbiology, Birmingham, Alabama.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Berkeley Koshland Seminar Series.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Buffalo Microbiology and Immunology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Connecticut Health Center Department of Molecular Biology and Biophysics.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Maryland College Park Department of Cell Biology, Molecular Genetics, and Microbiology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Tennessee Department of Microbiology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. University of Texas at Houston Department of Microbiology and Immunology.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Wake Forest Department of Chemistry.
• Johnson, M. D. (2021). Something Old, Something New, Something Borrowed, Copper II. Wesleyan University Department of Biophysics.
• Johnson, M. D. (2021). Swipe Right for Research: How I Made a Crowdsourced, Matchmaking, Virtual Summer Research Program Called NSURP (Plenary). ASMCUE.
• Johnson, M. D. (2021). The National Summer Undergraduate Research Project. ASM World Microbe Forum.
• Johnson, M. D. (2021). The National Summer Undergraduate Research Project. Scifoo (sponsored by Google).
• Johnson, M. D. (2021). The wonderful world of copper ionophores as antimicrobials (and other copper tales) (Keynote). Wind River Conference.
• Johnson, M. D. (2021). The wonderful world of copper ionophores as antimicrobials. American Chemical Society Rocky Mountain.
• Johnson, M. D. (2020, December). Something Old, Something New, Something Borrowed, Copper II. Uniformed Services University of the Health Sciences Bethesda, MD.
• Johnson, M. D. (2020, December). Something Old, Something New, Something Borrowed, Copper II. University of Michigan Department of Microbiology & Immunology, Ann Arbor, MI (Postdoctoral Fellow Invitee).
• Johnson, M. D. (2020, February). Something Old, Something New, Something Borrowed, Copper II. University of Maryland Baltimore Department of Pharmaceutical Sciences, Baltimore, MD.
• Johnson, M. D. (2020, March). Something Old, Something New, Something Borrowed, Copper II. University of Virginia Department Microbiology, Immunology, and Cancer Biology, Charlottesville, VA.
• Johnson, M. D. (2020, May). Metal matters at the host microbial interface. Cultivating Communities: Making Sense of Host-Microbiome Interactions Through the Lens of Genetics.
• Johnson, M. D. (2020, May). Something Old, Something New, Something Borrowed, Copper II. Virtual Streptococcal Seminar Series.
• Johnson, M. D. (2020, November). Something Old, Something New, Something Borrowed, Copper II. Case Western Reserve University, Department of Biology, Cleveland, OH.
• Johnson, M. D. (2020, November). Something Old, Something New, Something Borrowed, Copper II. University of Minnesota Department of Microbiology and Immunology, Minneapolis, MN (Graduate Student Invitee).
• Johnson, M. D. (2020, October). Metal matters at the host microbial interface. Afrobiotech Conference Online.
• Johnson, M. D. (2020, October). Microbes, Metals, Music, and Lessons in Disproving Your Hypothesis. National Institutes of General Medical Sciences Director’s Early Career Investigator Lecture, Bethesda MD.
• Johnson, M. D. (2020, October). Something Old, Something New, Something Borrowed, Copper II. University of Iowa Department of Biology, Iowa City, Iowa.
• Johnson, M. D. (2020, October). Something Old, Something New, Something Borrowed, Copper II. University of Nebraska Department of Microbiology, Lincoln, Nebraska.
• Johnson, M. D. (2020, October). Something Old, Something New, Something Borrowed, Copper II. University of Richmond Department Biology, Richmond, VA.
• Johnson, M. D. (2020, September). Something Old, Something New, Something Borrowed, Copper II. Cornell University Department of Microbiology, Ithaca, NY.
• Johnson, M. D. (2019, August). Some Old, Something New, Something Borrowed, Copper II. University of Connecticut MCB Seminar Series.
• Johnson, M. D. (2019, December). Something Old, Something New, Something Borrowed, Copper II. Infectious Diseases Grand Rounds.
• Johnson, M. D. (2019, Feburary). From Music to Micro and the Fun Road In-Between. UAHS Office of Diversity and Inclusion Black History Month Talk.
• Johnson, M. D. (2019, June). Something Old, Something New, Something Borrowed, Copper II. MARC and Minority Health Disparities Program.
• Johnson, M. D. (2019, October). Metals, Microbes, and Integrins. Asthma & Airway Disease Research Center.
• Johnson, M. D. (2018, Fall). Harnessing the Power of Copper to Kill Streptococcus pneumoniae. Talk at Southern Illinois University Medical School. Springfield, Il.
• Johnson, M. D. (2018, Summer). Copper State Killer, Using Copper Toxicity to Kill Bacteria. MARC and Minority Health Disparities Program.
• Johnson, M. D. (2017, April). Calcium Affects Pseudomonas aeruginosa Pilus Biogenesis and Adhesion. Microlunch. On Campus.
• Johnson, M. D. (2017, June). A Tale of Two Metals and AMA. MARC and Minority Health Disparities. On Campus: UROC (MARC and MHD).
• Johnson, M. D. (2017, June). Harnessing the Power of Copper in Killing Pathogenic Bacteria. Scientific Advisory Board Meeting, Keystone Symposia. Keystone, CO.
• Johnson, M. D. (2017, June). Three things you need to succeed in your scientific career. University of Arizona New Start Program. On campus: University of Arizona New Start Program.
• Johnson, M. D. (2017, March). How Is Copper Toxic in Bacteria and Why Should We Care?. Microseminar. Online: Microseminar.
  More info

  https://microseminar.wordpress.com/
• Johnson, M. D. (2017, March). Three things you need to succeed in your scientific career. ABBS SeminarABBS.
• Johnson, M. D. (2017, November). A Tale of Two Metals. ABRCMS. Phoenix, AZ: ABRCMS.
• Johnson, M. D. (2017, November). Three things you need to succeed in your scientific career. ABRCMS. Phoenix, AZ: ABRCMS.
• Johnson, M. D. (2017, October). Harnessing the Power of Copper to Kill S. pneumoniae. Infectious Diseases Grand Rounds. On Campus.
• Johnson, M. D. (2017, October). Harnessing the Power of Copper to Kill Streptococcus pneumoniae. Life Science Institute Seminar Series. University of Michigan, Ann Arbor, MI: Life Science Institute.
• Johnson, M. D. (2017, September). Using Bacterial Mechanisms of Overcoming Copper Stress to Find Therapeutic Targets. University of Arizona Bioretreat 2017. Biosphere 2.
• Johnson, M. D. (2016, November). Using Copper to Go Fishing for Bacterial Therapeutic Targets. Pharmacy Talk.
• Johnson, M. D. (2016, September). How Is Copper Toxic in Bacteria and Why Should We Care?. Microlunch Seminar. U of A.
• Johnson, M. D. (2017, December). Three things you need to succeed in your scientific career. ABBS SeminarABBS.
• Johnson, M. D. (2018, December). Three things you need to succeed in your scientific career. ABBS SeminarABBS.

Poster Presentations

• Ledford, J., Guerra, S., Langlais, P. R., & Iannuzo, N. (2022, June). CC16 DEFICIENCY IMPACTS PULMONARY EPITHELIAL-DRIVEN RESPONSES DURING MYCOPLASMA PNEUMONIAE INFECTION.. Aspen Lung Conference.
• Akram, A., Iannuzo, N., Langlais, P. R., & Ledford, J. (2021, July). CC16 Deficiency in Context of Early-life Infections and Epithelial-driven Responses. KEYS Conference.
• Johnson, M. D. (2017, November). Using Copper to Find Novel Ways to Kill Pathogenic Bacteria. Founders' Day Junior Investigator Poster Forum.

Reviews

• Mays, A., Byars-Winston, A., Hinton, A., Marshall, A. G., Kirabo, A., August, A., Marlin, B. J., Riggs, B., Tolbert, B., Wanjalla, C., Womack, C., Evans, C. S., Barnes, C., Starbird, C., Williams, C., Reynolds, C., Taabazuing, C., Cameron, C. E., Murray, D. D., , Applewhite, D., et al. (2023. Juneteenth in STEMM and the barriers to equitable science(pp 2510-2517).
  More info

  We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.