Two-Dimensional Fluorescence Difference Gel Electrophoretic Analysis ofStreptococcusmutansBiofilms

Rathsam, Catherine; Eaton, Ruth E.; Simpson, Christine L.; Browne, Gina V.; Valova, Valentina A.; Harty, D. W. S.; Jacques, Nicholas A.

doi:10.1021/pr0502471

Cited by 41 publications

(35 citation statements)

References 73 publications

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“…In that proteome analysis, performed by Len et al (39) and using conditions of acid stress, several genes involved in branched-chain amino acid biosynthesis were upregulated at pH 5.0, including ilvC, encoding a keto acid reductoisomerase, and ilvE, encoding a branched-chain amino acid aminotransferase. Two-dimensional fluorescence gel analysis of S. mutans biofilms also identified ilvE as being expressed differentially (48). Taken together, these findings suggest a role for amino acid biosynthetic genes in the acid stress response and could represent a pathway for the modulation of acid end products of fermentation.…”

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confidence: 67%

The Branched-Chain Amino Acid Aminotransferase Encoded by ilvE Is Involved in Acid Tolerance in Streptococcus mutans

et al. 2012

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Here we constructed and characterized an ilvE deletion mutant of S. mutans UA159. Growth experiments revealed that the ilvE mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of ilvE causes a decrease in acid tolerance. The ilvE strain exhibits a defect in F 1 -F o ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the ilvE promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acidadaptive repertoire of S. mutans.

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confidence: 67%

The Branched-Chain Amino Acid Aminotransferase Encoded by ilvE Is Involved in Acid Tolerance in Streptococcus mutans

et al. 2012

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“…2D-DIGE was used to identify significant changes in 179 protein spots for E. coli in response to treatment with benzoic acid, a common food preservative that reduces the proton-motive force across the membrane (52). By comparing biofilm bacteria to planktonic cells, it was determined that Streptococcus mutans upregulates the synthesis of glucan (a component of plaque) and competence proteins while downregulating nucleotide biosynthesis, glycolysis, and central metabolism during biofilm growth (30). 2D-DIGE was also used to identify novel Helicobacter pylori Cag-associated proteins by comparing the proteomic profiles of wild-type Cag ϩ H. pylori and a cag pathogenicity island deletion strain (4).…”

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confidence: 99%

Quantitative Profile of the Uropathogenic Escherichia coli Outer Membrane Proteome during Growth in Human Urine

Alteri

Mobley

2007

Infection and Immunity

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Outer membrane proteins (OMPs) of microbial pathogens are critical components that mediate direct interactions between microbes and their surrounding environment. Consequently, the study of OMPs is integral to furthering the understanding of host-pathogen interactions and to identifying key targets for development of improved antimicrobial agents and vaccines. In this study, we used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry to characterize the uropathogenic Escherichia coli (UPEC) outer membrane subproteome; 30 individual OMPs present on the bacterial surface during growth in human urine were identified. Fluorescence difference gel electrophoresis was used to identify quantitative changes in levels of UPEC strain CFT073 OMPs during growth in urine; six known receptors for iron compounds were induced in this environment, i.e., ChuA, IutA, FhuA, IroN, IreA, and Iha. A seventh putative iron compound receptor, encoded by CFT073 open reading frame (ORF) c2482, was also identified and found to be induced in urine. Further, the induction of these seven iron receptors in human urine and during defined iron limitation was verified by using quantitative real-time PCR (qPCR). An eighth iron receptor, fepA, displayed similar induction levels under these conditions as measured by qPCR but was not identified by 2D-PAGE. Addition of 10 M FeCl 2 to human urine repressed the transcription of all eight iron receptor genes. A number of fecal-commensal, intestinal pathogenic, and uropathogenic E. coli strains all displayed similar growth rates in human urine, showing that the ability to grow in urine per se is not a urovirulence trait. Thus, human urine is an iron-limiting environment and UPEC enriches its outer membrane with iron receptors to contend with this iron limitation.The bacterial outer membrane represents the critical interface between the microorganism and its environment. For pathogenic bacteria, the proteins localized to the outer membrane directly interact with components of the host and can act as adhesins or receptors to facilitate colonization and mediate acquisition of nutrients and macromolecules from this restrictive niche (6,26). Because these proteins are surface exposed, they are attractive targets for the development of improved antimicrobial agents and vaccines to eradicate infecting microorganisms or stimulate protective immune responses such as neutralizing or opsonizing immunoglobulins (10, 21). Elucidating the composition and profile of bacterial outer membrane proteins can help to better understand how specific bacterial pathogens interact with the host and to identify potential therapeutic targets.Uropathogenic Escherichia coli (UPEC) bacteria are extraintestinal pathogens that cause the majority of uncomplicated urinary tract infections (UTI) (49). It has been determined that these infections impact public health by exacting more than two billion dollars of health care costs annually (7,19). It is well known that UPEC bacteria utilize type 1 fimbriae (...

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“…In this regard, Rathsam et al [9] showed that compared with 2D-GE proteome analysis, DIGE analysis resulted in a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression in biofilms formed by Streptococcus mutans. Additionally, we used an EDTA-based extraction method which resulted in higher protein yield than the vortexing method, which also likely contributed to the differences in protein profiles between the two studies.…”

Section: Discussionmentioning

confidence: 99%

“…Technological advances in proteomics have allowed the identification of large-scale changes in protein profiles of biological systems, including microbial biofilms [9][10][11][12][13]. Recently, we performed proteomic analysis of cell walls of mature biofilms formed on catheter discs using two-dimensional difference-in-gel electrophoresis (DIGE), and showed that biofilm cell walls (as compared to planktonic cells) express differential levels of various proteins, including alcohol dehydrogenase (Adh1p) [14].…”

Section: Introductionmentioning

confidence: 99%

Proteomics and Pathway Mapping Analyses Reveal Phase-Dependent Over-Expression of Proteins Associated with Carbohydrate Metabolic Pathways in Candida albicans Biofilms

Lattif¹,

Chandra²,

Chang³

et al. 2008

TOPROTJ

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Candida albicans is the most commonly isolated fungus associated with biofilms, which are extracellular matrix (ECM)-encased, drug-resistant microbial communities formed on indwelling medical devices. Protein profiles of fungal biofilms have not been investigated in detail, although such profiles are believed to play critical roles in fungal biofilm formation. In this study, we used two-dimensional difference-in-gel electrophoresis (DIGE)-based proteomics to identify differentially expressed proteins in C. albicans biofilms grown to early and mature phases, compared to planktonic cells. The resulting proteomic data set was subjected to pathway mapping to reveal phase-specific pathways that were differentially expressed in biofilm cell walls and extracellular matrix (ECM). Our analyses showed 107 proteins to be differentially expressed in ECM, while 44 were differentially expressed in cell walls during biofilm formation, compared to planktonic controls. Furthermore, 95% (102/107) and 68% (30/44) of these differentially expressed proteins were upregulated in ECM and cell walls of biofilms, respectively. These proteins were mapped to cellular pathways, which revealed that these differentially expressed proteins were associated with several metabolic pathways, in a phase-dependent manner. For example, among ECM-associated proteins, proteins within 18 pathways were differentially expressed, with two pathways (glutamate and nitrogen metabolism) unique to early phase, and four pathways (purine, Gly/Ser/Thr, and inositol metabolism, and carbon fixation) unique to mature phase biofilms. Such differences were also observed in cell wallassociated proteins, where proteins associated with 14 specific pathways were differentially regulated. We also found glycolytic enzymes including the key enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were overexpressed in biofilms at both early and mature phases, compared to planktonic controls. Iodoacetate-mediated inhibition of this enzyme completely abrogated the ability of C. albicans to form biofilms, indicating the role of glycolysis/gluconeogenesis pathways in biofilm formation. Taken together, we demonstrate that ECM and cell walls of C. albicans biofilms express increased levels of specific proteins within pathways in a phase-dependent manner, suggesting that these pathways, especially glycolysis/gluconeogenesis, play critical roles in fungal biofilm formation and maintenance.

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Two-Dimensional Fluorescence Difference Gel Electrophoretic Analysis ofStreptococcusmutansBiofilms

Cited by 41 publications

References 73 publications

The Branched-Chain Amino Acid Aminotransferase Encoded by ilvE Is Involved in Acid Tolerance in Streptococcus mutans

The Branched-Chain Amino Acid Aminotransferase Encoded by ilvE Is Involved in Acid Tolerance in Streptococcus mutans

Quantitative Profile of the Uropathogenic Escherichia coli Outer Membrane Proteome during Growth in Human Urine

Proteomics and Pathway Mapping Analyses Reveal Phase-Dependent Over-Expression of Proteins Associated with Carbohydrate Metabolic Pathways in Candida albicans Biofilms

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