IU Indianapolis ScholarWorks :: Browsing by Author "Anderson, Gregory G."

Browsing by Author "Anderson, Gregory G."

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Analysis of Polysaccharide and Biofilm Production of the Emerging Cystic Fibrosis Pathogen Stenotrophomonas maltophilia
(Office of the Vice Chancellor for Research, 2016-04-08) Casiano-Rivera, Félix M.; Anderson, Gregory G.
Stenotrophomonas maltophilia is an emerging Cystic Fibrosis (CF) lung pathogen, which displays high intrinsic resistance to a number of different antibiotics. Additionally, S. maltophilia is thought to increase antibiotic resistance by forming biofilms during infection. Biofilm disruption could promote clearance of the microorganism from CF-infected lungs. However, the genetic, biochemical and immunological mechanisms underlying S. maltophilia biofilm formation are not well understood. Secreted polysaccharides have been proven to form a vital component of the matrix that surrounds and protects biofilm bacteria. Hundreds of S. maltophilia mutants were generated by transposon mutagenesis and screened for strains with reduced polysaccharide secretion.. These strains were screened through the use of agar plates containing the polysaccharide-binding dye, Congo red. Experimental controls included a S. maltophilia wild-type strain (positive control) and a confirmed polysaccharide deficient mutant (negative control). A Congo red liquid binding assay was utilized to identify the amount of Congo red bound in the samples, which confirmed the amount of polysaccharide present in them. A total of 1,728 mutants were screened with 61 mutants showing reduced polysaccharide production. The mutants were further narrowed down to 8 samples showing the most consistent phenotype. Arbitrary-primed polymerase chain reactions (AP-PCR), followed by sequencing, will be performed on the selected samples in an effort to identify the genes mutated in polysaccharide deficient strains. We will also perform immunological assays in an effort to understand underlying immune responses to S. maltophilia. In order to determine the effects of the deletion of gpmA in S. maltophilia, we will be performing an in-vitro co-culture assay using the wild-type strain and the gpmA mutant strain. These studies will yield evidence to the molecular process involved in polysaccharide production, which could lead to mechanisms to disrupt biofilm formation in CF patients.
Antibiotic Treatment of Pseudomonas aeruginosa Biofilms Stimulates Expression of mgtE, a Virulence Modulator
(2012-08-07) Redelman, Carly Virginia; Anderson, Gregory G.; Blazer-Yost, Bonnie.; Bauer, Margaret.
Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.
Detection and Quantification of Taste and Odor Producing Bacteria in Eagle Creek Reservoir
(2019-08) Koltsidou, Ioanna; Picard, Christine J.; Druschel, Gregory K.; Anderson, Gregory G.
The accelerated growth of algal blooms in water bodies has caused the increased occurrence of taste and odor (T&O) episodes worldwide. Even though T&O compounds have not been associated with adverse health effects, their presence can have extensive socio-economic impacts in contaminated waters. Eagle Creek Reservoir, a eutrophic water body, which supplies about 80% of Indianapolis drinking water, experiences frequent and sometimes severe odorous outbreaks. The terpenoid bacterial metabolites, 2-methylisoborneol (2-MIB) and geosmin, have been identified as the main compounds contributing to those T&O problems, which occur seasonally when the reservoir receives most of its water and nutrient loads from discharge events. In this study, ECR’s microbial community composition was assessed by a 16S next generation sequencing approach, confirming the presence of the major bacterial phyla of Cyanobacteria, Proteobacteria, Actinobacteria and Bacteroidetes, which are commonly found in freshwater environments. The relative abundance of Cyanobacteria, which are regarded as the main T&O producers in freshwater, followed the fluctuation of 2-MIB and geosmin concentrations closely. Mapping sequence analysis of a metagenomic dataset, successfully recovered the genes responsible for the synthesis of geosmin and 2-MIB, demonstrating the microbial ability for odorous compound production in ECR. Quantification of the geoA and MIBS genes in Cyanobacteria was achieved by the development and application of qPCR assays on water samples collected from the reservoir. A statistically significant positive correlation was found between MIBS gene quantity and MIB concentration for all sampling locations, implying that this assay could potentially be used as a tool for the early prediction of upcoming T&O episodes. The geoA gene detection assay, did not correlate well with geosmin concentrations, suggesting that even though the gene might be present, this does not necessarily mean that it is metabolically active.
Development of a New in vitro System for Cystic Fibrosis Research
(Office of the Vice Chancellor for Research, 2013-04-05) Coffey, Barbara M.; Anderson, Gregory G.
Individuals with cystic fibrosis (CF) have a life expectancy of 40 years and require daily treatments to mitigate the effects of the disease. CF impacts organs throughout the body, especially the lungs, where thick mucus builds up, impairs breathing, and provides an environment for bacterial growth. Chronic lung infection is the leading cause of mortality in CF. The majority of CF lung infections are caused by Pseudomonas aeruginosa, a common bacterium which typically does not cause disease in healthy individuals. In the CF lung, however, P. aeruginosa burrows into the thick mucus layer, evades the immune system, and resists antibiotic therapy by encasing itself in a protective matrix called a biofilm. Laboratory methods for studying biofilm are not true replicas of the CF lung environment, leaving a knowledge gap between how bacteria grow in a test tube (in vitro) and how they grow in the lungs of a person with CF. The focus of this work is to develop an improved laboratory model which combines artificial sputum (as a surrogate for mucus in the CF lung) and cultured CF airway epithelial cells. To assess the potential of this model, we have performed experiments to compare P. aeruginosa in artificial sputum versus standard laboratory media. Results demonstrate that P. aeruginosa in artificial sputum exhibits differences in growth, biofilm formation, toxin production, cytotoxicity, and protein expression, compared to results in standard media. These data suggest that our model system can contribute new information to the understanding of CF airway infection. The aim of future studies is to use this system to identify sputum components and bacterial proteins which have not been recognized previously by standard methods. It is our ultimate goal to contribute knowledge leading to improved longevity and quality of life for people with CF.
DNA alternate polymerase PolB mediates inhibition of type III secretion in Pseudomonas aeruginosa
(Elsevier, 2021) Chakravarty, Shubham; Ramos-Hegazy, Layla; Gasparovic, Abigail; Anderson, Gregory G.; Biology, School of Science
Opportunistic pathogen Pseudomonas aeruginosa uses a variety of virulence factors to cause acute and chronic infections. We previously found that alternate DNA polymerase gene polB inhibits P. aeruginosa pyocyanin production. We investigated whether polB also affects T3SS expression. polB overexpression significantly reduced T3SS transcription and repressed translation of the master T3SS regulator ExsA, while not affecting exsA mRNA transcript abundance. Further, polB does not act through previously described genetic pathways that post-transcriptionally regulate ExsA. Our results show a novel T3SS regulatory component which may lead to development of future drugs to target this mechanism.
DOES LOW MAGNESIUM IN CYSTIC FIBROSIS CONTRIBUTE TO BACTERIAL PATHOGENICITY?
(2012-04-13) Coffey, Barbara M.; Anderson, Gregory G.
Cystic fibrosis (CF) is a genetic disease for which there is currently no cure. Individuals with CF are plagued by myriad symptoms, including chronic pneumonia, which diminishes quality of life and reduces life expectancy to 40 years. The most common bacterium in CF patients’ lungs is Pseudomonas aeruginosa, a highly adaptable organism capable of surviving robust antibi-otic treatment. At the heart of developing improved treatments for CF pa-tients is the need to better understand P. aeruginosa pathogenicity. To this end, we have been studying the role of magnesium, which is often found at below normal levels in CF patients. Magnesium is an essential element in numerous cellular functions in both bacteria and humans. In previous re-search, we developed a P. aeruginosa strain with a deletion of the magnesi-um transport protein MgtE, as well as 16 plasmids carrying different muta-tions of the mgtE gene. Experiments with these constructs demonstrated a relationship between magnesium transport and bacterial toxin production. In the research presented here, we hypothesize that lower levels of magnesium may trigger a bacterial response, causing a change in P. aeruginosa patho-genicity. Changes may include differential growth, toxin release, and for-mation of biofilms, which are surface-adhered, antibiotic tolerant bacterial communities in a protective polysaccharide matrix. Using various magnesi-um levels, we have measured P. aeruginosa growth rates, motility, biofilm formation, and cytotoxicity toward cultured cells derived from the CF bron-chial epithelium. Preliminary results suggest that lower magnesium contrib-utes to changes in the bacterium that favor persistence in the CF lung. On-going studies include the effect of long-term growth of P. aeruginosa in low magnesium and how this impacts a number of virulence factors. We antici-pate that our research will elucidate the relationship between magnesium and P. aeruginosa pathogenicity and potentially lead to improved treatments for CF patients.
Does Magnesium Transport Protein (MgtE) Contribute to the Antibiotic Resistance of Pseudomonas aeruginosa?
(Office of the Vice Chancellor for Research, 2013-04-05) Umwali, Audrey; Anderson, Gregory G.
Pseudomonas aeruginosa is an environmental and opportunistic bacterial pathogen that is resistant to antibiotic treatment when it forms biofilms in the lungs of patients with cystic fibrosis. Biofilms are densely packed communities of bacteria embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS is a polymeric cluster composed of extracellular DNA, proteins and polysaccharides. Based on previous studies, in a low Mg2+ environment, P. aeruginosa wild-type is less or non-resistant to antibiotics and in a high Mg2+ environment, P. aeruginosa is more resistant to antibiotics. The purpose of this project was to find out if the magnesium transport protein (MgtE) is a contributor to the antibiotic resistance of P. aeruginosa .This was accomplished by using two different strains of P.aeruginosa; PA14 wild-type and GGA52 mutant (without the magnesium transporting protein). Four antibiotics were used; gentamicin, tobramycin, ciproflaxin and imipenem. The minimum inhibitory concentration (MIC) of each antibiotic was determined by culturing the bacteria strains on LB agar plates and use Etest strips to observe growth. N-minimal media supplemented with varying magnesium concentration was used to test if Mg2+ increased or reduced the antibiotic resistance at the MIC of P. aeruginosa as well as counting bacterial colonies. The mutant strain (GGA52) is expected to be less resistant than the wild type strain (PA14) because it does not have MgtE. If these predictions are true, then MgtE is an important contributor to the antibiotic resistance of P. aeruginosa. These results can be helpful in understanding the mechanism of antibiotic resistance of P. aeruginosa in patients with cystic fibrosis.
Dual Functions of the Protein MgtE in Pseudomonas aeruginosa
(2012-07-03) Coffey, Barbara M.; Anderson, Gregory G.; Marrs, James A.; Randall, Stephen K.
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen which readily establishes itself in the lungs of people with cystic fibrosis (CF). Most CF patients have life-long P. aeruginosa infections. By modulating its own virulence and forming biofilms, P. aeruginosa is able to evade both host immune responses and antibiotic treatments. Previous studies have shown that the magnesium transporter MgtE plays a role in virulence modulation by inhibiting transcription of the type III secretion system, a mechanism by which bacteria inject toxins directly into the eukaryotic host cell. MgtE had already been identified as a magnesium transporter, and thus its role in regulating cytotoxicity was indicative of dual functions for this protein. This research focused on a structure-function analysis of MgtE, with the hypothesis that the magnesium transport and cytotoxicity functions could be exerted independently. Cytotoxicity assays were conducted using a co-culture model system of cystic fibrosis bronchial epithelial cells and a ∆mgtE strain of P. aeruginosa transformed with plasmids carrying wild type or mutated mgtE. Magnesium transport was assessed using the same mgtE plasmids in a Salmonella strain deficient in all magnesium transporters. Through analysis of a number of mgtE mutants, we found two constructs – a mutation in a putative magnesium binding site, and an N-terminal truncation – which demonstrated a separation of functions. We further demonstrated the uncoupling of functions by showing that different mgtE mutants vary widely in their ability to regulate cytotoxicity, whether or not they are able to transport magnesium. Overall, these results support the hypothesis of MgtE as a dual function protein and may lead to a better understanding of the mechanisms underlying P. aeruginosa virulence. By understanding virulence mechanisms, we may be able to develop treatments to reduce infections and pave the way to better health for people with cystic fibrosis.
THE EFFECTS OF QUORUM SENSING ANTAGONISTS ON BIOFILM
(Office of the Vice Chancellor for Research, 2012-04-13) Guzman, Juan M.; Anderson, Gregory G.
Cystic fibrosis (CF) is a recessive genetic disorder that causes the for-mation of thick mucus plugs in the lungs of approximately 30,000 people in the United States and 60,000 individuals world-wide. Pseudomonas aeruginosa, an opportunistic bacterial pathogen, is able to colonize the mu-cus plugs and form antibiotic resistant biofilms. These microbial colonies, known as biofilms, cause serious problems for individuals living with CF. P. aeruginosa biofilms are able to cause chronic infections in the lungs of CF patients leading to increased morbidity and mortality. Using a modified bio-film assay, we tested the effects of modified chemical compounds and amino acids on P. aeruginosa biofilm dispersion. A previous study performed on P. aeruginosa, found that treatment of d- and l- amino acids resulted in biofilm dissemination. Through additional experiments, we will identify modified chemical compounds that induce biofilm dispersion. This research will in-crease our knowledge of P. aeruginosa biofilm dispersion, and allow us to explore new forms of treatment and therapy for CF patients with chronic in-fections that could be life threatening.
Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination
(Wiley Blackwell (Blackwell Publishing), 2013-02) Anderson, Gregory G.; Kenney, Thomas F.; Macleod, David L.; Henig, Noreen R.; O'Toole, George A.; Department of Biology, School of Science
Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4:1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L(-1) , while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L(-1) FTI and 16 mg L(-1) tobramycin. Fosfomycin (1024 mg L(-1)) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L(-1)) and tobramycin (4 mg L(-1)) and high concentrations of fosfomycin (1024 mg L(-1)) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides.

Browsing by Author "Anderson, Gregory G."

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