Browsing by Author "Marten, Mark R."
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Item Altered secretion patterns and cell wall organization caused by loss of PodB function in the filamentous fungus Aspergillus nidulans(Springer Nature Publishing AG., 2018-07-30) Boppidi, Karthik R.; Ribeiro, Liliane Fraga Costa; Iambamrung, Sirasa; Nelson, Sidney M.; Wang, Yan; Momany, Michelle; Richardson, Elizabeth A.; Lincoln, Stephen; Srivastava, Ranjan; Harris, Steven D.; Marten, Mark R.Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.Item Altered secretion patterns and cell wall organization caused by loss of PodB function in the filamentous fungus Aspergillus nidulans(Nature, 2018-07-30) Boppidi, Karthik R.; Ribeiro, Liliane FragaCosta; Iambamrung, Sirasa; Nelson, Sidney M.; Wang, Yan; Momany, Michelle; Richardson, Elizabeth A.; Lincoln, Stephen; Srivastava, Ranjan; Harris, Steven D.; Marten, Mark R.Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.Item The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance(American Society for Microbiology, 2020-02-04) Mattos, Eliciane Cevolani; Silva, Lilian Pereira; Valero, Clara; Castro, Patrícia Alves de; Reis, Thaila Fernanda dos; Ribeiro, Liliane F. C.; Marten, Mark R.; Silva-Rocha, Rafael; Westmann, Cauã; Silva, Carlos Henrique Tomich de Paula da; Taft, Carlton Anthony; Al-Furaiji, Narjes; Bromley, Michael; Mortensen, Uffe H.; Benz, J. Philipp; Brown, Neil Andrew; Goldman, Gustavo H.The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation.Item Assessment of Elasticity and Topography of Aspergillus nidulans Spores via Atomic Force Microscopy(American Society for Microbiology, 2004-09-27) Zhao, Liming; Schaefer, David; Marten, Mark R.Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA⁺ and rodA⁻ strains. Tapping-mode AFM images of wild-type and rodA⁺ spores in air showed characteristic “rodlet” protein structures covering a granular spore surface. In comparison, rodA spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA⁺ spores. Rodlets were removed from rodA⁺ spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 ± 10, 120 ± 10, and 300 ± 20 N/m and the elastic moduli to be 6.6 ± 0.4, 7.0 ± 0.7, and 22 ± 2 GPa for wild-type, rodA⁺ and rodA⁻ spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.Item Blood Serum Affects Polysaccharide Production and Surface Protein Expression in S. Aureus(2017-03-03) Islam, Nazrul; Hossain, Khwaja G.; Ross, Julia M.; Marten, Mark R.Background: S. aureus biofilm serves a major role in pathogenesis. Two of the major components of bacterial biofilm are Polysaccharides intercellular adhesions (PIA) and surface proteins. It is not known how PIA and surface proteins expressions are affected in presence of blood serum. Analyses of surface proteins expressions will provide more effective biomarker discovery that might lead to development of antimicrobial therapeutics to meet the challenges of biofilm-related infections. Method: Secondary cultures of S. aureus Philips, a biofilm-forming bacterium, were generated by inoculating 1 ml of overnight culture into 50 ml of TSB. Bacteria were cultured at several concentrations of blood serum and found that 12.5% supplemented blood serum provide s similar growth curve as normal TSB (100%). One and 2 D SASPAGE were used to separate proteins and the differentially expressed proteins were identified by nano-LC/MS. Results: Polysaccharide intercellular adhesions production was significantly increased due to the addition of blood serum in the media. We also identified two serum proteins, apolipoprotein and globulin (Fc and Fab), that remained attached with the membrane fraction of bacterial proteins. Conclusion: These results have strongly demonstrated that blood serum influences the exopolysaccharide expression in S. aureus.Item Blood Serum Affects Polysaccharide Production and Surface Protein Expression in S. Aureus(Juniper, 2017) Islam, Nazrul; Hossain, Khwaja G.; Ross, Julia M.; Marten, Mark R.Background: S. aureus biofilm serves a major role in pathogenesis. Two of the major components of bacterial biofilm are Polysaccharides intercellular adhesions (PIA) and surface proteins. It is not known how PIA and surface proteins expressions are affected in presence of blood serum. Analyses of surface proteins expressions will provide more effective biomarker discovery that might lead to development of antimicrobial therapeutics to meet the challenges of biofilm-related infections. Method: Secondary cultures of S. aureus Philips, a biofilm-forming bacterium, were generated by inoculating 1 ml of overnight culture into 50 ml of TSB. Bacteria were cultured at several concentrations of blood serum and found that 12.5% supplemented blood serum provide s similar growth curve as normal TSB (100%). One and 2 D SASPAGE were used to separate proteins and the differentially expressed proteins were identified by nano-LC/MS. Results: Polysaccharide intercellular adhesions production was significantly increased due to the addition of blood serum in the media. We also identified two serum proteins, apolipoprotein and globulin (Fc and Fab), that remained attached with the membrane fraction of bacterial proteins. Conclusion: These results have strongly demonstrated that blood serum influences the exopolysaccharide expression in S. aureus.Item Comprehensive Analysis of Aspergillus nidulans PKA Phosphorylome Identifies a Novel Mode of CreA Regulation(American Society for Microbiology, 2019-04-30) Ribeiro, Liliane F. C.; Chelius, Cynthia; Boppidi, Karthik R.; Naik, Nisha S.; Hossain, Simin; Ramsey, Jessica J. J.; Kumar, Jyothi; Ribeiro, Lucas F.; Ostermeier, Marc; Tran, Bao; Goo, Young Ah; Assis, Leandro J. de; Ulas, Mevlut; Bayram, Ozgur; Goldman, Gustavo H.; Lincoln, Stephen; Srivastava, Ranjan; Harris, Steven D.; Marten, Mark R.The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a β-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8–4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.Item Discovery of treatment for nerve agents targeting a new metabolic pathway(Springer Nature Switzerland AG., 2020-07-27) Glaros, Trevor; Dhummakupt, Elizabeth S.; Rizzo, Gabrielle M.; McBride, Ethan; Carmany, Daniel O.; Wright, Linnzi K. M.; Forster, Jeffry S.; Renner, Julie A.; Moretz, Ruth W.; Dorsey, Russell; Marten, Mark R.; Huso, Walker; Doan, Alexander; Dorsey, Carrie D.; Phillips, Christopher; Benton, Bernard; Mach, Phillip M.The inhibition of acetylcholinesterase is regarded as the primary toxic mechanism of action for chemical warfare agents. Recently, there have been numerous reports suggesting that metabolic processes could significantly contribute to toxicity. As such, we applied a multi-omics pipeline to generate a detailed cascade of molecular events temporally occurring in guinea pigs exposed to VX. Proteomic and metabolomic profiling resulted in the identification of several enzymes and metabolic precursors involved in glycolysis and the TCA cycle. All lines of experimental evidence indicated that there was a blockade of the TCA cycle at isocitrate dehydrogenase 2, which converts isocitrate to α-ketoglutarate. Using a primary beating cardiomyocyte cell model, we were able to determine that the supplementation of α-ketoglutarate subsequently rescued cells from the acute effects of VX poisoning. This study highlights the broad impacts that VX has and how understanding these mechanisms could result in new therapeutics such as α-ketoglutarate.Item Dynamic transcriptomic and phosphoproteomic analysis during cell wall stress in Aspergillus nidulans(ASBMB Publications, 2020-08-01) Chelius, Cynthia; Huso, Walker; Reese, Samantha; Doan, Alexander; Lincoln, Stephen; Lawson, Kelsi; Tran, Bao; Purohit, Raj; Glaros, Trevor; Srivastava, Ranjan; Harris, Steven D.; Marten, Mark R.The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a β-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8–4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.Item A fast and simple method to estimate relative, hyphal tensile‐strength of filamentous fungi used to assess the effect of autophagy(John Wiley & Sons, 2017-11-14) Quintanilla, Daniela; Chelius, Cynthia; Iambamrung, Sirasa; Nelson, Sidney; Thomas, Donnel; Gernaey, Krist V.; Marten, Mark R.Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. Until now, there is not an efficient method which allows its characterization. Currently available methods are very time consuming, thus, compromising their applicability in strain selection and process development. To overcome this issue, a method for fast and easy, statistically verified quantification of relative hyphal tensile strength was developed. It involves off‐line fragmentation in a high shear mixer followed by quantification of fragment size using laser diffraction. Particle size distribution (PSD) is determined, with analysis time on the order of minutes. Plots of PSD 90th percentile versus time allow estimation of the specific fragmentation rate. This novel method is demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin‐induced autophagy for Aspergillus nidulans (parental strain) and a mutant strain (ΔAnatg8) lacking an important autophagy gene. Both strains were grown in shake flasks and relative hyphal tensile strength was compared. The mutant strain grown in control conditions appears to be weaker than the parental strain, suggesting that Anatg8 may play a role in other processes involving cell wall biosynthesis. Furthermore, rapamycin‐induced autophagy resulted in apparently weaker cells even for the mutant strain. These findings confirm the utility of the developed method in strain selection and process development.Item Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors(BioMed Central, 2009-01-15) Garcia, Jose R.; Cha, Hyung J.; Rao, Govind; Marten, Mark R.; Bentley, William E.Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cellbased reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency – one verses four impellers. Results: In both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen (DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space. Conclusion: Thus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.Item Protein Image Alignment via Piecewise Affine Transformations(Mary Ann Liebert, Inc., publishers) Potra, Florian A.; Liu, Xing; Seillier-Moiseiwitsch, Françoise; Roy, Anindya; Hang, Yaming; Marten, Mark R.; Raman, Babu; Whisnant, CarolWe present a new approach for aligning families of 2D gels. Instead of choosing one of the gels as reference and performing a pairwise alignment, we construct an ideal gel that is representative of the entire family and obtain a set of piecewise affine transformations that optimally align each gel of the family to the ideal gel. The coefficients defining the transformations as well as the ideal landmarks are obtained as the solution of a large-scale quadratic programming problem that can be solved efficiently by interior-point methods.Item Proteome Analysis to Assess Physiological Changes in Escherichia coli Grown Under Glucose-Limited Fed-Batch Conditions(Wiley Periodicals, 2005-09-22) Raman, Babu; Nandakumar, M.P.; Muthuvijayan, Vignesh; Marten, Mark R.Proteome analysis was used to compare global protein expression changes in Escherichia coli fermentation between exponential and glucose-limited fed-batch phase. Two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry were used to separate and identify 49 proteins showing>2-fold difference in expression. Proteins up regulated during exponential phase include ribonucleotide biosynthesis enzymes and ribosomal recycling factor. Proteins up regulated during fed-batch phase include those involved in high-affinity glucose uptake, transport and degradation of alternate carbon sources and TCA cycle, suggesting an enhanced role of the cycle under glucose- and energy-limited conditions. We report the up regulation of several putative proteins (ytfQ,ygiS,ynaF,yggX,yfeX), not identified in any previous study under carbon-limited conditions.Item Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions(BioMed Central Ltd, 2014-04-30) Islam, Nazrul; Kim, Yonghyun; Ross, Julia M.; Marten, Mark R.Background: The biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s−1) to identify proteins that are associated with biofilm. Results: Global protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins. Conclusions: The reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections.Item Rheological Characterization and Biological and Mass Transfer Kinetics of Cellulase-Producing T. reesei Viscous Suspensions(1995) Marten, Mark R.; Velkovska, Svetlana; Khan, Saad A.; Ollis, David F.Steady and dynamic shear measurements are utilized to characterize the rheological behavior of Trichoderma reesi RUT-C30 fungal suspensions during batch growth on xylose (soluble substrate) or cellulose (particulate solid substrate) at three different fermentor impeller speeds (250, 400 and 550 rpm). Biomass concentrations versus time were unimodal on xylose and bimodal on cellulose. This behavior is consistent with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second, slower growth phase due to hydrolysis of recalcitrant cellulose by increasing cellulase concentrations. Critical dissolved oxygen (DO) concentration for T. ressei growth on cellulose was found to be 0.073 mmol/L. The DO was kept above this level by supplementing air feed with pure oxygen, implying that mass transfer limitations were not the cause of bimodal cell growth. Steady shear rheological data showed shear thinning behavior and a yield stress for all broth samples regardless of substrate. Casson and Herschel-Bulkley constitutive equations fit steady shear data well. Dynamic shear measurements on broth suspensions indicated a "gel-like" behavior at low strains, with micro structural breakdown at larger displacements. Time variations of the Casson model parameters (yield stress and Casson viscosity) and dynamic moduli (elastic and vscous modulus) strongly followed biomass profiles: a single maximum in all rheological variables resulted when cells were grown on xylose, or on cellulose at impeller speeds of 400 or 550 rpm, and dual maxima were observed for cellulose - grown cells at 250 rpm.