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KINETIC MODEL FRAMEWORKS OF ANIMAL CELL CULTURES FOR CONTROL AND OPTIMIZATION
Title
KINETIC MODEL FRAMEWORKS OF ANIMAL CELL CULTURES FOR CONTROL AND OPTIMIZATION
Creator
Yilmaz, Denizhan
Date
2019
Description
This dissertation proposes four different kinetic model frameworks that havebeen developed for optimization and control of monoclonal antibody...
Show moreThis dissertation proposes four different kinetic model frameworks that havebeen developed for optimization and control of monoclonal antibody producing mammalian cell cultures to improve biopharmaceutical production by decreasing the costof trial and error experimentation. The developed models mainly describe the transient metabolic behavior of mammalian cell culture under different culture conditionsand predicts cell growth and death, cell metabolism, and monoclonal antibody synthesis, and production. These models are developed via ordinary differential equationsbased on the assumption of well-mixing reactor. All developed models were calibrated, and their predictive capabilities were tested with experimental reports published in the literature. Good agreement was obtained between model predictions and experimental data. The presented results illustrate that the developed models successfully describe and predict the transient behavior of mammalian cell cultures and can be a useful tool for biopharmaceutical production.
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DEVELOPING FUSION BACTERIOCINS FOR ERADICATING PSEUDOMONAS AERUGINOSA BIOFILMS
Title
DEVELOPING FUSION BACTERIOCINS FOR ERADICATING PSEUDOMONAS AERUGINOSA BIOFILMS
Creator
An, Sungjun
Date
2022
Description
The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients and...
Show moreThe opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Due to its remarkable ability to resist antibiotics, eradicating P. aeruginosa has become increasingly difficult. As previously reported, we have successfully engineered a colicin-secretion system that kills target biofilm cells rapidly and selectively in multispecies biofilms as well as demonstrated the potential of using live microorganisms engineered to produce antimicrobial colicin protein to treat biofilm-associated infections. In this study,we constructed a fusion colicin-pyocin that could target P. aeruginosa by DNase activity of colicin E2. The newly engineered bacteriocin-secretion system upon the shift in target, maintained biofilm inhibition capacity. Both during biofilm formation and after its development, the system was able to suppress the P. aeruginosa biofilm. This result opened up the possibility that it could be used for novel live biotherapeutics. A further study was conducted to overcome the challenge of requiring an exogenous inducer. We applied the concept of Quorum-Sensing signal that recognize autoinducer as a trigger of fusion colicin-pyocin producing genetic circuit so that it automates the production and secretion of fusion colicin-pyocin as soon as the genetic circuit senses the target population growing. This study demonstrated that combining the domains of colicin and pyocin could broaden the genetic circuit target range, maintaining strain specificity, while employing the QS system could remove the fundamental problem of diffusion or degradation of extra compounds as they approach engineered cells.
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Investigating anti-biofilm and anti-persister activities of natural compounds and antimicrobial proteins
Title
Investigating anti-biofilm and anti-persister activities of natural compounds and antimicrobial proteins
Creator
Jin, Xing
Date
2020
Description
Bacterial biofilm formation is frequently involved in the development of chronic infectious diseases. Inhibiting biofilms is challenging due...
Show moreBacterial biofilm formation is frequently involved in the development of chronic infectious diseases. Inhibiting biofilms is challenging due to their tolerance against conventional antibiotics which are not effective to penetrating biofilm matrix to kill the cells residing in biofilms. Metabolically dormant cells known as persisters are also not eradicated by antibiotic treatment. Therefore, novel antimicrobial drugs that can kill non-growing persisters or inhibit biofilms are needed urgently. Here, we investigate the anti-biofilm and anti-persister activities of new drug candidates including plant extracts, fatty acids and colicins. We firstly screened 50 different plant extracts on enterohemorrhagic E. coli and Listeria monocytogenes, and identified Cancavalia ensiformis-derived lectin Concanavalin A (ConA) inhibits biofilm formation of enterohemorrhagic E. coli and Listeria monocytogenes by binding to carbohydrates on bacterial cell surface. Biofilm results support that ConA lectin can be applied for developing anti-adherent and anti-biofilm agents to control biofilms. Also, fatty acids may be promising candidates as anti-persister or anti-biofilm agents, because some fatty acids exhibit antimicrobial effects. We screened a fatty acid library consisting of 65 different fatty acid molecules for altered persister formation. We found that undecanoic acid, lauric acid, and N-tridecanoic acid inhibited E. coli persister cell formation including enterohemorrhagic E. coli EDL933. These fatty acids were all medium chain saturated forms. Furthermore, the fatty acids repressed EHEC biofilm formation (for example, by 8-fold for lauric acid) without having antimicrobial activity. This study demonstrates that medium chain saturated fatty acids can serve as anti-persister and anti-biofilm agents that may be applied to treat bacterial infections. Colicins, a type of antimicrobial bacteriocins, are considered as a viable alternative of conventional antibiotics due to their unique cell killing mechanisms that can damage cells by pore-forming on the cell membrane, nuclease activity, and cell wall synthesis inhibition. In this study, we utilized cell-free protein synthesis to produce colicins with different modes of action. We optimized the production yield and activity of colicins in cell-free system. Also, we tested effect of cell-free produced colicins on persister cell formation and biofilm formation. We illustrated that colicins kill persister cells and biofilm cells. Moreover, colicins produced from the engineered probiotic E. coli cells, which can be used as a living medicine, specifically and significantly eradicate target biofilms without affecting other bacterial population. Colicins have great potential to be an antibiotic alternative, and engineered probiotic E. coli is a potential candidate for engineered bacterial therapeutics.
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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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