Bacterial Stress Response Proteins and Their Influence on the Immune Response Amy Thees

How do bacteria adapt to changes in their environment? Types of stress –Physicochemical and chemical –Nutritional deprivation –Toxic compounds –Interactions with other cells Strategies (Adaptation) –Eliminate stressor –Repair damage –Escape

Examples of Bacterial Stress Responses Heat and cold shock Envelope and oxidative stress Oxygen and amino acid scarcity Osmotic and pH stress SOS response to DNA damage Metalloregulation between bacterial homeostasis and resistance Sporulation DNA uptake

Infection and Colonization Adapt metabolism to host environment Combat innate antimicrobial host defense mechanisms –Iron limitation Structure and catalytic cofactor –Production of toxic reactive oxygen (ROS) and nitrogen species (RNS) Ex. Superoxide anion (O 2 -), hydroxyl radical (OH- ), peroxynitrite (ONOO-), hydrogen peroxide (H 2 O 2 ), nitric oxide (NO)

Bacterial Stress Proteins OxyR –LysR regulator –Transcriptional activator in oxidizing conditions PerR –Ferric uptake regulator –Peroxide-responsive repressor –Gram positive Both regulate expression of: –Catalase peroxidase (kat) –Alkyl hydroperoxide reductase (ahp)

Electron shuttles to relieve excess reducing equivalents and redox-active compounds due to ROS damage and oxidative stress Electron carrier flavoprotein (Fld) Mobile electron shuttle with flavin mononucleotide (FMN) prostetic group Back up for ferredoxin (Fd) Long chain- Anabaena (IsiB) and Escherichia coli (FldA) Short chain- Pseudomonas aeruginosa and Pseudomonas putida Annotated as mioC (ortholog in Escherichia coli) Pseudomonas aeruginosa Opportunistic human pathogen Wide metabolic diversity Large flexible genome- adaptable FldP- encoded by PA14_22540 gene and 184 amino acids

Figure 1. Primary and secondary structures of FldP from P. aeruginosa. Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen

Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen FldP from P. aeruginosa is a functional flavodoxin Figure 2

Role of FldP in cell survival and ROS accumulation upon exposure to H2O2 Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen Figure 3

Role of FldP in cell survival and ROS accumulation upon exposure to H2O2 Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen Figure 3

Figure 4. FldP mitigates H2O2-induced cell death in a mutT-deficient P. aeruginosa. Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen Figure 5. FldP decreases the H2O2-induced mutation frequency in mutT-deficient P. aeruginosa.

Figure 6. Induction and regulation of fldP expression. Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen Figure 7. FldP enhances P. aeruginosa survival within mammalian macrophages and during in vivo infection of Drosophila melanogaster.

Figure 8. fldP is a component of the P. aeruginosa variable genome. Moyano AJ, Tobares RA, Rizzi YS, Krapp AR, Mondotte JA, et al. (2014) A Long-Chain Flavodoxin Protects Pseudomonas aeruginosa from Oxidative Stress and Host Bacterial Clearance. PLoS Genet 10(2): e doi: /journal.pgen Figure 9. Transcriptional organization of RGP32.

FldP Protection in Oxidative Stress Strong induction of fldP gene in response to H 2 O 2 –Role in oxidant response Enhanced ROS build-up and lower survival of fldP null mutants exposed to H 2 O 2 –Defense against ROS Partial protection by FldP overexpression to P.aeruginosa cells deficient in mutT against harmful effects and increased mutational burden caused by H 2 O 2 –Contributes to bacterial tolerance to oxidative response of host immune system

Mycobacterium avium ssp. paratuberculosis (MAP) Intracellular macrophage persister Heat resistant- pasteurization Johne’s disease in cattle and other ruminants Controversal causative agent in Crohn’s disease Slow growing FurA Iron homeostasis- binds Fe 2+ in E. coli Potential regulatory component of stress response Located upstream of katG (catalase peroxidase)

Eckelt, E., Meißner, T., Meens, J., Laarmann, K., Nerlich, A., Jarek, M., Goethe, R. (2015). FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis. Frontiers in Microbiology, 6, 16. doi: /fmicb Figure 1 Characterization of MAPΔfurA Deletion Mutant

Determination of the FurA Regulon of MAP RNA deep sequencing –Transcriptome- sequence and frequency of RNA molecules at particular time in a specific cell type Count number of mRNAs encoded by individual genes Results –48 genes differentially expressed in mutant strain compared to wt 13 higher- Table 1 35 lower- Table 2 – Orthologous groups 40% metabolism 20.75% cellular processes and signaling 39.62% poorly characterized or uncharacterized ***No genes related to iron homeostasis***

Eckelt, E., Meißner, T., Meens, J., Laarmann, K., Nerlich, A., Jarek, M., Goethe, R. (2015). FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis. Frontiers in Microbiology, 6, 16. doi: /fmicb Figure 3: Response of FurA Regulated AhpD to Oxidative Stress Figure 2: Regulatory Influence of FurA

Eckelt, E., Meißner, T., Meens, J., Laarmann, K., Nerlich, A., Jarek, M., Goethe, R. (2015). FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis. Frontiers in Microbiology, 6, 16. doi: /fmicb Induction of Oxidative Burst in Macrophages

Eckelt, E., Meißner, T., Meens, J., Laarmann, K., Nerlich, A., Jarek, M., Goethe, R. (2015). FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis. Frontiers in Microbiology, 6, 16. doi: /fmicb Survival of MAP Strains in Macrophages C

Eckelt, E., Meißner, T., Meens, J., Laarmann, K., Nerlich, A., Jarek, M., Goethe, R. (2015). FurA contributes to the oxidative stress response regulation of Mycobacterium avium ssp. paratuberculosis. Frontiers in Microbiology, 6, 16. doi: /fmicb Role of FurA for Survival in the Host

Conclusion FurA acts as repressor for a selective group of genes involved in the response to oxidative stress –Iron-based sensor of ROS Closely related to PerR FurA contributes to activation of second group of genes –Macrophage survival

My Grant Proposal Specific Aim: Characterize the role of PmtA, a bacterial metallothionein protein in Pseudomonas aeruginosa, in macrophage survival and determine whether the mechnism is diminished in PmtA deficient mutant, PW4670. Hypothesis:

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