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., 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.

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.

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).

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.

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.

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.

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.
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.

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.