Copper and Borrelia

This post is a continuation of the Cu⁺ binding protein BicA in Borrelia killing.

[1] The Bondarczuk and Piotrowska-Sege Review

These Polish authors introduce their review by telling us that copper is an essential cofactor for enzymes like cytochrome C oxidase and Cu/Zn superoxide dismutase. The we are told that too much copper can be toxic. What may be new to some of our readers on this site is the concept of an operon and environmental regulation of gene expression. Something happens and the bug not only starts transcribing blue prints for one protein but a whole bloody lot of related proteins to solve a certain environmental challenge. Let’s take ourselves a little tour of this concept that might be new to some of us. Lactose is a disaccharide of glucose and galactose. Bacteria have to produce enzymes to properly digest it.

LacI, the inducer, is always expressed. It is waiting to bind lactose. The LacI gene sits upstream of the promoter, the place where RNA polymerase attaches and starts transcribing messenger RNAs for the genes LacZ, LacY and LacA. When these mRNA are translated into proteins the cell can bring in lactose. LacI binds to lactose and undergoes a shape change that allows it to bind to the operator. This blocks transcription of the lactose utilization genes so that the bug does not waste energy making more proteins to bring in lactose that it already has enough of.

The Cus operon, dealing with copper

The cus system codes for four proteins.

• CusA is thought to transport Cu⁺ from the periplasm.
• CusB is thought to be an adapter protein that interacts with CusA.
• CusC, a member of is anchored into the outer membrane
• CusF is a periplasmic chaperone that may interact with CusB. It may be able to bind Cu⁺ and Cu²⁺.

These are some images of the Cus proteins from the rcsb.org database. The interesting thing to note is that CusF has a small domain that can bind mono and divalent metal ions.

the cop operon of E coli

The authors introduced the CueR regulates the expression of two genes: cueO and copA . CueO is a periplasmic multi copper oxidase. This enzyme oxidizes Cu (I) to a less toxic Cu (II) and reduces dioxygen to water through four single-electron transfer steps. The CueO activity is oxygen dependent. This post is going to skip the Cue family of proteins and go directly to the Cop proteins.

• CopA transports Cu⁺ out of the cytoplasm at the cost of ATP.
• CopB is an outer membrane protein with dubious copper binding properties.
• CopC is a soluble periplasmic chaperone folded into a Greek key β barrel with two distinct but interdependent binding sites for Cu⁺ and Cu²⁺.
• CopD may transport may with CopC and deliver essential copper through the inner membrane to the cytoplasm.
• CopS is the copper sensor. Like LacI it is constantly expressed regardless of the copper in the environment. CopS may interact with CopA or CopC.
• CopR interacts with the copper sensor CopS and controls the transcription of genes of the cop operon.

So many of these E coli proteins are partial to Cu⁺. Do similar operons exist in Borrelia? The sequence of the E coli CopC protein was used to search the Borrelia database on ncbi and nothing came up.

Copper operons in Borrelia, they are not the same

BmtA getting metals in the spirochete [2]

We have a focus on Borrelia and Lyme disease. A recent review discusses all of the moving parts of transporting the metals needed for growth while keeping them from becoming toxic in the life cycle of Borrelia.

This review just did not seem to be that concerned with copper. There are some interesting philosophical points in going from a rod to a spirochete.

E coli vs B burgdorferi [3]

The Borrelia burgdorferi Fur homologue, also known as Borrelia oxidative stress regulator (BosR), promotes spirochetal adaptation to the mammalian host by directly repressing the lipoproteins required for tick colonization and indirectly activating those required for establishing infection in the mammal. Here, we examined whether the DNA-binding activity of BosR was regulated by any of the four most prevalent transition metal ions in B. burgdorferi, Mn, Fe, Cu, and Zn.

Fur acts as a dimer transcription repressor in the presence of iron. The supplemental data shows the family tree of Fur transcription repressors along with the Borellia homologue BosA. BosA is proposed to bind metals via for conserved cysteines. A twist to the dominant paradigm of Fur is that i may also act as a transcription activator of very different genes whose transcription is repressed. [3] BosA has a “structural” Zn²⁺ that does not contribute to turning on or off its transcription repression. It does bind about four iron or coppers per dimer. [3] The addition of up to 10 μM Fe²⁺ and Mn²⁺ had no effect on the ability of BosA to bind to DNA. Cu²⁺ and Zn²⁺ caused a dose dependent decrease in DNA binding. When the reducing agent TCEP was added to the binding reactions, Cu⁺ failed to inhibit binding. These cations could also displace BosA already bound to DNA. Cu²⁺ was found to bind to BosA with higher affinity than Zn²⁺

Outer surface protein A, OspA, is thought to promote survival in the tick between blood meals. Simultaneous with the disappearance of OspA, the spirochete population in the midgut begins to express an OspC and migrates to the salivary gland. Upregulation of OspC begins during the first day of feeding and peaks 48 hours after attachment.

In the above image OspA is high in the midgut while OspC expression is low. Note that copper is taking OspA and OspC in opposite directions of what happens to them when they jump to the mammalian host.

Cellular Cu level is much higher in B. burgdorferi (Bb) than in the model organism E. coli (Ec). The Mn, Fe, Cu, and Zn levels in the B. burgdorferi type strain B31 and the E. coli K-12 strain TOP10 were determined by ICP-SFMS. Both were cultivated microaerobically at 33°C to early stationary phase in the BSK-H medium (Sigma-Aldrich). In addition, TOP10 was cultivated aerobically at 37°C in the BSK-H medium and in LB. Data represent means (plus SD) of two or three biological replicates. P values are derived from a two-way ANOVA, followed by Bonferroni post test. For clarity, only P values for comparisons between B. burgdorferi and E. coli are shown: **, P < 0.01; ***, P < 0.001. [3]

Conclusions

We covered the Lac Operon, the textbook example of how gene transcription responds to environmental changes. We’ve covered two operons in E coli that help this bug respond to potentially toxic amounts of copper. Even though Borrelia is also a Gram Negative bacterium, it seems to have slightly different ways of dealing with copper as it moves from tick to mammal.

References

1. Bondarczuk K, Piotrowska-Seget Z. Molecular basis of active copper resistance mechanisms in Gram-negative bacteria. Cell Biol Toxicol. 2013 Dec;29(6):397-405. PMC free article
2. Troxell B, Yang XF. Metal-dependent gene regulation in the causative agent of Lyme disease. Front Cell Infect Microbiol. 2013 Nov 15;3:79. PMC free article
3. Wang P, Yu Z, Santangelo TJ, Olesik J, Wang Y, Heldwein E, Li X. BosR Is A Novel Fur Family Member Responsive to Copper and Regulating Copper Homeostasis in Borrelia burgdorferi. J Bacteriol. 2017 Jul 25;199(16):e00276-17. PMC free article
4. Neubert MJ, Dahlmann EA, Ambrose A, Johnson MDL. Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon. mSphere. 2017 Oct 18;2(5):e00372-17. PMC free article