Nicholas M. Eakley
University of Wisconsin-Madison
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Nicholas M. Eakley.
Microbial Pathogenesis | 2011
Daniel C. Shippy; Nicholas M. Eakley; Philip N. Bochsler; Amin A. Fadl
Salmonella enterica serovar Typhimurium is a frequent cause of enteric disease due to the consumption of contaminated food. Identification and characterization of bacterial factors involved in Salmonella pathogenesis would help develop effective strategies for controlling salmonellosis. To investigate the role of glucose-inhibited division gene (gidA) in Salmonella virulence, we constructed a Salmonella mutant strain in which gidA was deleted. Deletion of gidA rendered Salmonella deficient in the invasion of intestinal epithelial cells, bacterial motility, intracellular survival, and induction of cytotoxicity in host cells. Deletion of gidA rendered the organism to display a filamentous morphology compared to the normal rod-shaped nature of Salmonella. Furthermore, a significant attenuation in the induction of inflammatory cytokines and chemokines, histopathological lesions, and systemic infection was observed in mice infected with the gidA mutant. Most importantly, a significant increase in LD(50) was observed in mice infected with the gidA mutant, and mice immunized with the gidA mutant were able to survive a lethal dose of wild-type Salmonella. Additionally, deletion of gidA significantly altered the expression of several bacterial factors associated with pathogenesis as indicated by global transcriptional and proteomic profiling. Taken together, our data indicate GidA as a potential regulator of Salmonella virulence genes.
Archives of Microbiology | 2012
Daniel C. Shippy; Joseph A. Heintz; Ralph M. Albrecht; Nicholas M. Eakley; Ashok K. Chopra; Amin A. Fadl
Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.
Current Microbiology | 2014
Daniel C. Shippy; Nicholas M. Eakley; Dareen M. Mikheil; Amin A. Fadl
Salmonellaenterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ∆flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ∆flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.
The Journal of Antibiotics | 2012
Dareen M. Mikheil; Daniel C. Shippy; Nicholas M. Eakley; Ogi Okwumabua; Amin A. Fadl
The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutants motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.
Current Microbiology | 2013
Jaclyn M. Rehl; Daniel C. Shippy; Nicholas M. Eakley; Megan D. Brevik; Jordan M. Sand; Mark E. Cook; Amin A. Fadl
Glucose-inhibited division (GidA) protein is widely distributed in nature, and is highly conserved among bacteria and eukarya. In our previous study, a gidA mutant was attenuated in both in vitro and in vivo models of Salmonella infection. Furthermore, deletion of gidA resulted in a marked reduction in the expression of many virulence genes and proteins, suggesting a role for GidA in the regulation of Salmonella virulence. In this study, the effect of different environmental conditions (glucose, EDTA, and pH 5) on GidA expression in Salmonella was examined. Transcriptional analysis using real-time RT-PCR and a β-galactosidase assay, displayed no differences in gidA transcription and promoter activity in different environmental conditions. Conversely, semiquantitative Western blot analysis revealed a significant increase in the GidA expression in Salmonella when grown under different environmental conditions. Salmonella in vitro virulence assays showed an increased virulence potential in the environmental conditions correlating to the increase in GidA expression. Together, our data indicate that GidA expression is modulated under different environmental conditions which correlate to increased Salmonella in vitro virulence.
Gut Pathogens | 2013
Daniel C. Shippy; Nicholas M. Eakley; Dareen M. Mikheil; Amin A. Fadl
BackgroundSalmonella is often implicated in foodborne outbreaks, and is a major public health concern in the United States and throughout the world. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Adhesion to epithelial cells in the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified stdA as a potential adhesion mutant of SE. Therefore, we hypothesize StdA plays a significant role in adhesion of SE to the intestinal mucosa of poultry.Methods and resultsTo test our hypothesis, we created a mutant of SE in which stdA was deleted. Growth and motility were assayed along with the in vitro and in vivo adhesion ability of the ∆stdA when compared to the wild-type SE strain. Our data showed a significant decrease in motility in ∆stdA when compared to the wild-type and complemented strain. A decrease in adhesion to intestinal epithelial cells as well as in the small intestine and cecum of poultry was observed in ∆stdA. Furthermore, the lack of adhesion correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens.ConclusionsThese studies suggest StdA is a major contributor to the adhesion of Salmonella to the intestinal mucosa of poultry.
Archives of Microbiology | 2016
Alexis M. Bennett; Daniel C. Shippy; Nicholas M. Eakley; Ogi Okwumabua; Amin A. Fadl
Salmonella is a threat to public health due to consumption of contaminated food. Screening of a transposon library identified a unique mutant that was growth and host cell binding deficient. The objective of this study was to determine the functional role of glucosamine-6-phosphate synthase (GlmS) in the biology and pathogenesis of Salmonella. To examine this, we created a glmS mutant (ΔglmS) of Salmonella and examined the effect on cell envelope integrity, growth, metabolism, and pathogenesis. Our data indicated ΔglmS was defective in growth unless media were supplemented with d-glucosamine (d-GlcN). Examination of the bacterial cell envelope revealed that ΔglmS was highly sensitive to detergents, hydrophobic antibiotics, and bile salts compared to the wild type (WT). A release assay indicated that ΔglmS secreted higher amounts of β-lactamase than the WT in culture supernatant fractions. Furthermore, ΔglmS was attenuated in cell culture models of Salmonella infection. Taken together, this study determined an important role for GlmS in the pathogenesis and biology of Salmonella.
Microbiology and Immunology | 2011
Nicholas M. Eakley; Philip N. Bochsler; P. Gopal Reddy; Amin A. Fadl
Previous work by the present authors indicated a murein lipoprotein mutant of Salmonella shows a marked down‐regulation in expression of yqhC. Because YqhC is a putative DNA‐binding protein, it is likely involved in modulation of Salmonella genes. Deletion of yqhC renders Salmonella defective in invasion of intestinal epithelial cells, motility, and induction of cytotoxicity. In the present study, further attenuation in induction of inflammatory cytokines/chemokines and histopathological lesions was seen in mice infected with the yqhC mutant. On the other hand, deletion of yqhC did not significantly affect the LD50 in mice or the ability of Salmonella to survive and replicate in vivo. To better understand how YqhC affects Salmonella virulence and to identify factors potentially modulated by YqhC, comparative transcriptome and proteome analysis of the yqhC mutant and the WT Salmonella was performed. Data from these experiments indicate that deletion of yqhC significantly alters the transcription of several genes associated with the SPI‐1 encoded T3SS and flagellar regulons, correlating with the yqhC mutant phenotype. Overall, this study indicates that deletion of the yqhC gene causes a number of virulence‐related defects in vitro, but has a modest effect in vivo, despite affecting induction of inflammatory cytokines and histopathology.
Annals of Microbiology | 2015
Daniel C. Shippy; Nicholas M. Eakley; Dareen M. Mikheil; Anna De La Cotera; Amin A. Fadl
Salmonella is a major public health concern due to the consumption of contaminated food. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. In this study, transposon mutagenesis identified SEN3800 as a potential binding mutant of SE. Therefore, we hypothesize that SEN3800 plays a role in the colonization ability of SE in the gastrointestinal tract of poultry. To test our hypothesis, we created a mutant of SE in which SEN3800 was deleted. We then tested the in-vitro and in-vivo binding ability of ∆SEN3800 when compared to the wild-type and complemented SE strains. Our data showed a significant decrease in the binding ability of ∆SEN3800 to T84 intestinal epithelial cells, as well as in the small intestine and cecum of poultry. Furthermore, this binding defect correlated to a defect in invasion, as evidenced by a cell culture model using T84 intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate that SEN3800 contributes to the colonization ability of Salmonella in the gastrointestinal tract of poultry.
Microbial Pathogenesis | 2013
Daniel C. Shippy; Nicholas M. Eakley; Charles T. Lauhon; Philip N. Bochsler; Amin A. Fadl