Michael D L Johnson
Assistant Professor, Immunobiology
Assistant Professor, BIO5 Institute
Primary Department
Department Affiliations
(520) 626-3779
Work Summary
Metals such as calcium and iron are essential to living organisms. Some metals in excess, like copper, are detrimental to bacteria. My laboratory studies this phenomenon in Streptococcus pneumoniae to find novels method for killing pathogenic bacteria.
Research Interest
Metals serve as vital nutrients to all biological systems. During infections, bacteria must not only acquire all metals necessary for survival from within the host, such as calcium or manganese, but must also efflux metals that are toxic or in excess such as copper. The overall goal of my laboratory is to investigate how bacteria maintain homeostasis within the metal milieu. This goal involves determining how metals are processed, the orchestrated response during metal sensing, and the role that the host plays in this process during infection. Understanding how bacteria interact with metals during infections will identify novel therapeutic strategies against bacterial infections. Keywords: Infectious Diseases, Antibiotic resistance, Bacterial Pneumonia

Publications

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.

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.

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.

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.

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.

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., 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.

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.

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.

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.