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Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Title
Characterization of the Pseudomonas aeruginosa NQR Complex, a Novel Form of Bacterial Proton Pump, and the Ubiquinone Binding Site
Creator
Raba, Daniel Alexander
Date
2019
Description
The proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of...
Show moreThe proton/sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) plays a key role in the energy metabolism of a diverse range of bacteria, including pathogenic species such as Vibrio cholera, Pseudomonas aeruginosa, Chlamydia trachomatis, as well as others. Residing in the cytoplasmic membrane of these bacteria, the enzyme couples the transfer of electrons to the pumping of cations across the cell membrane. In all previously studied homologues, the enzyme generates a sodium gradient through its pumping activity that can be utilized by the cell to power essential homeostatic processes. Furthermore, the electrochemical gradient generated by this enzyme has been shown to regulate the production of virulent factors and the efficacy of antibiotic extrusion and elimination. Although certain homologues have been investigated, particularly that of V. cholerae (Vc-NQR), the NQR homologues belonging to important pathogenic species have not been well studied. In the research detailed in this thesis, the first characterization of the NQR of P. aeruginosa (Pa-NQR) is described which identified this homologue as a new form of bacterial proton pump, differentiating it from all other studied homologues of NQR. Additionally, as part of this study our research group characterized the mechanism of inhibition of Pa-NQR by the molecule HQNO which is produced by P. aeruginosa and is known to be a strong inhibitor of Vc-NQR. Our results show that Pa-NQR possesses resistance to inhibition by this molecule compared to Vc-NQR, pinpointing residue F155 of subunit D as being important to resistance and the type of inhibition to be partial-mixed. Moreover, in further developing the understanding of the NQR of V. cholerae, we investigated the binding site of ubiquinone, the final electron acceptor of NQR’s electron transfer process, determining residues P185, L190, and F193 to be important for maintaining the structural composition of the ubiquinone pocket, ensuring efficient substrate binding and catalysis.
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SODIUM-BASED ENERGY METABOLISM AND DYNAMIC ENERGY DEPENDENCY OF CHLAMYDIA TRACHOMATIS
Title
SODIUM-BASED ENERGY METABOLISM AND DYNAMIC ENERGY DEPENDENCY OF CHLAMYDIA TRACHOMATIS
Creator
Liang, Pingdong
Date
2019
Description
Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for various human diseases including trachoma, genital tract...
Show moreChlamydia trachomatis is an obligate intracellular bacterium that is responsible for various human diseases including trachoma, genital tract infections, and lymphogranuloma venereum. Energy metabolism consists many essential pathways to generate energy for every organism. However, it remains much unknown in C. trachomatis. For decades, C. trachomatis has been considered as an “energy parasite”, which needs the energy supply from the host cells entirely. In contrast, genomic studies show that this bacterium is capable of encoding enzymes that involve energy metabolism. However, no experimental data were provided to support the genomic information due to the peculiar developmental cycle of C. trachomatis inside the host cells. In this project, the oxidative phosphorylation pathway of C. trachomatis is first identified with experimental results. This pathway starts with the sodium pumping NADH:Ubiquinone oxidoreductase enzyme complex (NQR) transferring the electrons along the respiratory chain and generating a sodium gradient across the membrane. C. trachomatis contains an A-type ATPase that can utilize this sodium gradient to generate ATP. In vitro experiments in mammalian cells with different respiratory inhibitors show that C. trachomatis is not an obligate energy parasite. Instead, it has a dynamic energy dependency on the host metabolism that the bacterium switches from entirely to partially relying on the host energy metabolism for its energy requirement. The sodium gradient established by NQR and/or other transporters is of great importance to chlamydial metabolism. Further, the respiratory inhibitors test on interferon-γ-induced persistence of C. trachomatis in mammalian cell cultures shows that an inhibited energy metabolism prevents and eliminates the persistent form. This study provides new insights about antibiotics development and therapeutic methods against C. trachomatis infections.
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Establishing Bisphenol A Degradation and Enhancing Microbial Fuel Cell Performance by Biofilm Optimization of Shewanella Oneidensis MR1
Title
Establishing Bisphenol A Degradation and Enhancing Microbial Fuel Cell Performance by Biofilm Optimization of Shewanella Oneidensis MR1
Creator
Zhou, Jiacheng
Date
2023
Description
Bisphenol A (BPA) has been widely used as a plasticizer in the production of synthetic polymers, such as those used in food storage containers...
Show moreBisphenol A (BPA) has been widely used as a plasticizer in the production of synthetic polymers, such as those used in food storage containers and bottles. However, BPA interferes with endocrine systems, causing carcinogenicity, immunotoxicity, and embryotoxicity. Biological water treatment processes scarcely remove BPA, owing to the poor BPA degradability and efficiency of the applied microorganisms. Shewanella oneidensis has been studied and used for the biodegradation process in wastewater treatment because of its excellent extracellular electron transfer properties. In this work, we engineered S. oneidensis MR1 to enable BPA degradation by producing ferredoxin (Fdbisd) and cytochrome P450 (P450bisd) originating from Sphingomonas bisphenolicum AO1. The engineered S. oneidensis exhibited a higher BPA degradation efficiency than that of Escherichia coli producing the same enzymes. The endogenous ferredoxin and ferredoxin reductase of S. oneidensis participated in BPA degradation, and overexpression of mtrC, omcA, and So0521, which encode S. oneidensis cytochromes, decreased BPA. We developed BPA-degrading S. oneidensis biofilms. We measured these optimized BPA-degrading S. oneidensis biofilm in a single chamber microbial fuel cell formed on different carbon electrodes by morphology. Cyclic voltammetry and electrochemical impedance spectroscopy were measured to analyze the biofilm-electrode performance. The biofilm colonization was also measured by confocal laser scanning microscope and scanning electron microscope. And the developed microbial fuel cell was used to degrade BPA and the biofilm developed on different type of carbon anodes was identified. This study provides insights into biocatalyst utilization for the biological degradation of toxic organic compounds.
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Population dynamics and pathogens of the western bean cutworm (Striacosta albicosta)
Title
Population dynamics and pathogens of the western bean cutworm (Striacosta albicosta)
Creator
Bunn, Dakota C.
Date
2022
Description
Understanding an herbivorous pest’s population dynamics is necessary to ensure proper integrated pest management strategies are being...
Show moreUnderstanding an herbivorous pest’s population dynamics is necessary to ensure proper integrated pest management strategies are being developed and used. The western bean cutworm is a pest of both corn and dry beans that is understudied and difficult to manage due to its nocturnal lifestyle, adaptation to current management techniques and a general lack of understanding regarding its population structure. Our studies focused on the effects of host plant and pathogens on western bean cutworm population structure and found that mainly adults which developed on corn are contributing to the next generation of western bean cutworm in Michigan, making corn and dry beans unsuitable as co-refuges in insecticide resistance management strategies.We also found a 100% prevalence of the Nosema sp. in the adult population of western bean cutworm in Michigan. This prevalence, when paired with the consistent crop damage caused annually by the western bean cutworm, which confirms an abundance of cutworms are present, suggests the infection is slow acting and non-lethal to its host. Following sequencing, assembly, and annotation of the Nosema sp. genome, we were unable to provide a reason for the Nosema sp.’s low virulence, however, we were able to confirm the presence of all 6 polar tube proteins. Upon further examination of the Nosema sp. genome we were able to determine that it is a true Nosema with genome size of ~9.57 Mbp (~20% of which are transposable elements), which is within the typical range for this genus.
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UTILIZING BACTERIAL INTERACTIONS TO CONTROL PATHOGENIC BIOFILM FORMATION
Title
UTILIZING BACTERIAL INTERACTIONS TO CONTROL PATHOGENIC BIOFILM FORMATION
Creator
Fang, Kuili
Date
2020
Description
Many chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm...
Show moreMany chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm formation is a result of dynamic intra- or inter-species interactions. However, the nature of molecular interactions between bacteria in multi-species biofilms are not well understood compared to those in mono-species biofilms. The first project (Chapter 3) investigated the ability of probiotic Escherichia coli Nissle 1917 (EcN) to outcompete the biofilm formation of pathogens including enterohemorrhagic E. coli (EHEC), Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis. When dual-species biofilms were formed, EcN inhibited the EHEC biofilm population by 14-fold compared to EHEC mono-species biofilms. This figure was 1,100-fold for S. aureus and 8,300-fold for S. epidermidis; however, EcN did not inhibit P. aeruginosa biofilms. In contrast, commensal E. coli did not exhibit any inhibitory effect toward other bacterial biofilms. We identified that EcN secretes DegP, a bifunctional (protease and chaperone) periplasmic protein, outside the cells and controls other biofilms. Although three E. coli strains tested in this study expressed degP, only the EcN strain secreted DegP outside the cells. The deletion of degP disabled the activity of EcN in inhibiting EHEC biofilms, and purified DegP directly repressed EHEC biofilm formation. Hence, probiotic E. coli outcompetes pathogenic biofilms via extracellular DegP activity during dual-species biofilm formation. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a pathogen causing the outbreaks of hemorrhagic colitis. Conventional antibiotics treatment is not recommended for EHEC infection as antibiotics trigger Shiga toxin production of EHEC and aggravate hemolytic-uremic syndrome. EHEC biofilm formation is closely associated with its virulence expression. Previously, we identified that probiotic E. coli Nissle 1917 (EcN) secretes DegP resulting in the inhibition of EHEC biofilm formation in a dual culture. DegP is a serine protease exhibiting both proteolytic and chaperone functions and binds to outer membrane proteins (OMPs) of target cells. However, the extracellular function of DegP is not clear. We hypothesized that binding of DegP to OMPs of EHEC might inhibit EHEC biofilm formation by affecting the adhesion ability or changing biofilm-related gene regulations of EHEC. We constructed EHEC mutants lacking ompA, ompC, or ompF individually and in combination and assessed their biofilm formation in the presence of DegP-secreting EcN in the co-culture or by adding purified DegP. It was found that both ompA and ompC double deletion decreased EHEC single species biofilm, and also caused that DegP inhibited more EHEC biofilm (about 25 fold inhibition) than DegP inhibited EHEC wt biofilm (about 10 fold), indicating that OmpA and OmpC are more related to EHEC biofilm than OmpF, and OmpA and OmpC might deplete DegP inhibitory functions. On the other hand, DegP S210A, a DegP mutant lacking protease function, inhibited EHEC wt biofilm, indicating that DegP’s biofilm inhibition function is not from its protease activity. Additionally, EHEC transcription profiles in the presence of DegP showed that DegP up-regulated expressions of cellulose production related genes (csgD and bcsA) and motility related genes (flhD, qseB), which were all involved in EHEC biofilm inhibition, and down-regulated Shiga toxin 2 virulence gene (stx2). Besdies, DegP promoted EHEC cellulose production and motility, which is consistent with transcription profile, and Shiga toxin 2 production will be further tested. This study reveals a new function of DegP secreted by EcN in controlling biofilms and leads us to develop an alternative strategy to control biofilm-related infections. Foodborne pathogen Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the Chapter 5 study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 times 10^6 CFU/cm2 L. monocytogenes ATCC19115 in single-species biofilms to 1.1 times 10^5 CFU/cm2 in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (> 30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.
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Development of validation guidelines for high pressure processing to inactivate pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in treated juices
Title
Development of validation guidelines for high pressure processing to inactivate pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in treated juices
Creator
Rolfe, Catherine
Date
2020
Description
The fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the functional...
Show moreThe fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the functional juice division is cold pressure, which refers to the application of high pressure processing (HPP) at low temperatures for a mild treatment to inactivate foodborne pathogens instead of thermal pasteurization. HPP juice manufacturers are required to demonstrate a 5-log reduction of the pertinent microorganism to comply with FDA Juice HACCP. The effectiveness of HPP on pathogen inactivation is determinant on processing parameters, juice composition, packaging application, as well as the bacterial strains included for validation studies. Unlike thermal pasteurization, there is currently no consensus on validation study approaches for bacterial strain selection or preparation and no agreement on which HPP process parameters contribute to overall process efficacy.The purpose of this study was to develop validation guidelines for HPP inactivation and post-HPP recovery of pressure resistant and matrix-adapted Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in juice systems. Ten strains of each microorganism were prepared in three growth conditions (neutral, cold-adapted, or acid-adapted) and assessed for barotolerance or sensitivity. Pressure resistant and sensitive strains from each were used to evaluate HPP inactivation with increasing pressure levels (200 – 600 MPa) in two juice matrices (apple and orange). A 75-day shelf-life analysis was conducted on HPP-treated juices inoculated with acid-adapted resistant strains for each pathogen and examined for inactivation and recovery. Individual strains of E. coli O157:H7, Salmonella spp., and L. monocytogenes demonstrated significant (p <0.05) differences in reduction levels in response to pressure treatment in high acid environments. E. coli O157:H7 was the most barotolerant of the three microorganism in multiple matrices. Bacterial screening resulted in identification of pressure resistant strains E. coli O157:H7 TW14359, Salmonella Cubana, and L. monocytogenes MAD328, and pressure sensitive strains E. coli O15:H7 SEA13B88, S. Anatum, and L. monocytogenes CDC. HPP inactivation in juice matrices (apple and orange) confirmed acid adaptation as the most advantageous of the growth conditions. Shelf-life analyses reached the required 5-log reduction in HPP-treated juices immediately following pressure treatment, after 24 h in cold storage, and after 4 days of cold storage for L. monocytogenes MAD328, S. Cubana, and E. coli O157:H7 TW14359, respectively. Recovery of L. monocytogenes in orange juice was observed with prolonged cold storage time. These results suggest the preferred inoculum preparation for HPP validation studies is the use of acid-adapted, pressure resistant strains. At 586 – 600 MPa, critical inactivation (5-log reduction) was achieved during post-HPP cold storage, suggesting sufficient HPP lethality is reached at elevated pressure levels with a subsequent cold holding duration.
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