Changes in the proteome of Candida albicans in response to azole, polyene, and echinocandin antifungal agents.

The yeast Candida albicans is an opportunistic human fungal pathogen and the cause of superficial and systemic infections in immunocompromised patients. The classes of antifungal agents most commonly used to treat Candida infections are the azoles, polyenes, and echinocandins. In the present study, we identified changes in C. albicans protein abundance using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectroscopy following exposure to representatives of the azole (ketoconazole), polyene (amphotericin B), and echinocandin (caspofungin) antifungals in an effort to elucidate the adaptive responses to these classes of antifungal agents. We identified 39 proteins whose abundance changed in response to ketoconazole exposure. Some of these proteins are involved in ergosterol biosynthesis and are associated with azole resistance. Exposure to amphotericin B altered the abundance of 43 proteins, including those associated with oxidative stress and osmotic tolerance. We identified 50 proteins whose abundance changed after exposure to caspofungin, including enzymes involved in cell wall biosynthesis and integrity, as well as the regulator of beta-1,3-glucan synthase activity, Rho1p. Exposure to caspofungin also increased the abundance of the proteins involved in oxidative and osmotic stress. The common adaptive responses shared by all three antifungal agents included proteins involved in carbohydrate metabolism. Some of these antifungal-responsive proteins may represent potential targets for the development of novel therapeutics that could enhance the antifungal activities of these drugs.

Upc2p-associated differential protein expression in Candida albicans.

The gain-of-function mutation G648D in UPC2 causes ERG11 up-regulation and increased fluconazole resistance in Candida albicans. In this study, we performed 2-DE and PMF to identify proteomic alterations in an ERG11-overexpressing fluconazole-resistant C. albicans clinical isolate compared with its fluconazole-susceptible parent strain. We identified 23 differentially expressed proteins, and among them, seven became differentially expressed in a C. albicans wild-type strain after the introduction of a UPC2 allele carrying this mutation. These Upc2p-regulated proteins may contribute to fluconazole resistance in C. albicans.

Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells.

Adhesion to epithelial cells and flagella-mediated motility are critical virulence traits for many Gram-negative pathogens, including enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhoea in travellers and children in developing countries. Many flagellated pathogens export putative adhesins belonging to the two-partner secretion (TPS) family. However, the actual function of these adhesins remains largely undefined. Here we demonstrate that EtpA, a TPS exoprotein adhesin of enterotoxigenic E. coli, mimics and interacts with highly conserved regions of flagellin, the major subunit of flagella, and that these interactions are critical for adherence and intestinal colonization. Although conserved regions of flagellin are mostly buried in the flagellar shaft, our results suggest that they are at least transiently exposed at the tips of flagella where they capture EtpA adhesin molecules for presentation to eukaryotic receptors. Similarity of EtpA to molecules encoded by other motile pathogens suggests a potential common pattern for bacterial adhesion, whereas participation of conserved regions of flagellin in adherence has implications for development of vaccines for Gram-negative pathogens.

Proteomic analysis of Mrr1p- and Tac1p-associated differential protein expression in azole-resistant clinical isolates of Candida albicans.

Azole resistance in Candida albicans is frequently caused by the overexpression of multi-drug efflux pump genes MDR1, CDR1, and CDR2 due to gain-of-function mutations in the zinc cluster transcription factors Mrr1p and Tac1p. In this study, we performed a comparative proteomic analysis to identify proteins whose expression level is influenced by these transcription factors. Both 2-DE and PMF were used to examine the expression profiles of six pairs of matched C. albicans isolates carrying gain-of-function mutations in either MRR1 or TAC1 resulting in the overexpression of either MDR1 or CDR1 and CDR2. Using this approach, 17 differentially expressed proteins were identified in the MDR1-overexpressing isolates, while 14 were identified in the isolates that overexpress CDR1 and CDR2. Furthermore, we found that the expression of many of these proteins was increased in a wild-type strain of C. albicans after the introduction of a gain-of-function allele of MRR1 or TAC1. Moreover, disruption of MRR1 and TAC1 in isolates carrying gain-of-function mutations resulted in decreased expression of these proteins, confirming their regulation by Mrr1p or Tac1p. Several proteins involved in heat shock and carbohydrate metabolism were differentially expressed in all clinical isolate sets, but these proteins were not dependent upon either Tac1p or Mrr1p.

Proteome cataloging and relative quantification of Francisella tularensis tularensis strain Schu4 in 2D PAGE using preparative isoelectric focusing.

The protein complement of whole cell extract of the bacterium Francisella tularensis tularensis was analyzed using two-dimensional electrophoresis with preparative isoelectric focusing in the first dimension. The format allows the quantification of relative protein abundance by linear densitometry and extends the potential dynamic range of protein detection by as much as an order of magnitude. The relative abundance and rank order of 136 unique proteins identified in F. tularensis tularensis were established. It is estimated that 16% of the moderately to highly expressed proteins and 8% of all predicted non-pseudogenes were identified by comparing this proteome information with the relative abundance of mRNA as measured by microarray. This rank-ordered proteome list provides an important resource for understanding the pathogenesis of F. tularensis and is a tool for the selection and design of synthetic vaccines. This method represents a useful additional technique to improve whole proteome analyses of simple organisms.

High-throughput proteomics processing of proteins in polyacrylamide in a multiwell format.

Processing multiple protein samples from polyacrylamide at significant sensitivity represents a major chokepoint for raising the success rate in high-volume protein identification projects. A multiwell filterplate method for processing proteins in polyacrylamide was optimized for sensitivity using a protein standard. The results demonstrate this process to be a reliable and reproducible method over a range of gel loadings and suitable for the identification of proteins near the threshold of silver stain. This high-throughput manual method requires a minimum of specialized equipment, and can be performed disconnected from a proteomics infrastructure for the preparation of mass spectrometry-ready samples.

Gene expression profiling of the response of Streptococcus pneumoniae to penicillin.

OBJECTIVES: The aim of this study was to identify changes in the gene expression profile of Streptococcus pneumoniae in response to a subinhibitory concentration of penicillin in an effort to better understand mechanisms by which this organism copes with this stress. METHODS: S. pneumoniae serotype 2 strain D39 was grown for 1 h in the presence or absence of penicillin at a concentration equivalent to half the MIC (0.03 mg/L). RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time RT-PCR. RESULTS: A total of 386 genes were found to be responsive to penicillin. Up-regulated genes included those of the ciaR-ciaH operon, luxS, genes encoding cell envelope proteins and genes of the pst locus. Down-regulated genes included genes involved in competence, genes encoding capsular polysaccharide biosynthesis proteins, genes involved in fatty acid chain elongation and genes of the polyamine transporter operon. CONCLUSIONS: Altered expression of these genes reflects a protective response to perturbation of the bacterial cell wall by penicillin. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism and provide insight into novel mechanisms of penicillin resistance.

Proteomic analysis of experimentally induced azole resistance in Candida glabrata.

OBJECTIVES: The aim of the present study was to identify changes in the proteome of a laboratory-derived azole-resistant strain of Candida glabrata compared with its susceptible parent strain in an effort to identify proteins that are differentially expressed in association with azole resistance. METHODS: Soluble and membrane protein fractions were isolated from mutant strain F15 (fluconazole MIC>128 mg/L) and parent strain 66032 (fluconazole MIC=16 mg/L) grown to mid-log phase. Soluble proteins were resolved by both two-dimensional (2D) and one-dimensional (1D) polyacrylamide gel electrophoresis (GE) whereas membrane proteins were resolved by 1D GE. Spots or bands representing differentially expressed proteins were identified by matrix-assisted desorption ionization-time of flight mass spectroscopy (MALDI-TOF MS) and peptide mass fingerprinting. RESULTS: A total of 22 proteins were found to be more abundantly represented, and 3 proteins were found to be less abundantly represented, in strain F15 compared with strain 66032. These included up-regulation of the ATP-binding cassette transporter Cdr1p, the ergosterol biosynthesis enzyme Erg11p, proteins involved in glycolysis and glycerol metabolism, and proteins involved in the response to oxidative stress and cadmium exposure. CONCLUSIONS: In addition to transcriptional regulation of Cdr1p, this study identified the differential expression of several proteins that may contribute to azole resistance and suggests the possibility for a post-transcriptional mechanism for increased expression of Erg11p.

Heterogeneous nuclear ribonucleoprotein P2 is an autoantibody target in mice deficient for Mer, Axl, and Tyro3 receptor tyrosine kinases.

Deficiencies in clearance of apoptotic cells predispose to the development of autoimmune disease. This is evident in mice lacking the receptor tyrosine kinases Tyro3, Axl, and Mer. Deficient mice exhibit an increased abundance of apoptotic cells in tissues and manifest diverse autoimmune conditions. To test these mice for the presence of autoantibodies to apoptotic cells, we generated spontaneous splenic B cell hybridomas and used a novel microscopy screen to detect Ab binding to apoptotic Jurkat cells. From hybridomas secreting IgG Abs reactive with apoptotic cells, we selected one that recreated the major serum specificity for apoptotic cells. The Ab LHC7.15 bound to an Ag that is differentially distributed between the nucleus and the cytoplasm in live and apoptotic cells. In late apoptotic cells, the Ag coalesces into aggregates that bleb from the cell surface. Immunopurification of the Ag, followed by mass spectrometry, identified a protein of 69 kDa whose partial sequence matched heterogeneous nuclear ribonucleoprotein P2. This multifunctional protein binds DNA, RNA, and several known ribonucleoprotein autoantigens. Our observations indicate that a ribonucleoprotein complex, formed and translocated to the cell surface in apoptosis, represents a potent stimulus for breaking tolerance and inducing systemic autoimmunity in mice with defective clearance of cell remnants.

A mass spectrometric determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins.

Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.

Application of proteomic analysis to the study of azole antifungal resistance in Candida albicans.

The sequencing of the Candida albicans genome and recent refinements in protein resolution and identification techniques have greatly enhanced the application of proteomics for the study of this fungal pathogen. Proteome analysis includes the separation and isolation of proteins by two-dimensional polyacrylamide gel electrophoresis and subsequent protein identification by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This technique has been used for the study of the proteomes of Candida species in the context of virulence, drug response, and resistance. We describe here the application of this approach to the study of azole antifungal resistance in C. albicans.

A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.

Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.

Proteomic analysis of azole resistance in Candida albicans clinical isolates.

Changes in protein expression within a matched set of Candida albicans isolates representing the acquisition of azole resistance were examined by two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting. Proteins differentially expressed in association with azole resistance included Grp2p, Ifd1p, Ifd4p, Ifd5p, and Erg10p, a protein involved in the ergosterol biosynthesis pathway.

Vascular oxygen sensing: detection of novel candidates by proteomics and organ culture.

We have shown that the specific inhibition of hypoxia-induced relaxation by organ culture in porcine coronary arteries can be mimicked by treatment of control vessels with the protein synthesis inhibitor, cycloheximide. We hypothesize that organ culture of vascular smooth muscle results in the decreased expression of proteins that are critical for vascular oxygen sensing. Using two-dimensional gel electrophoresis and mass spectroscopy, we identified such candidate proteins. The expressions of the smooth muscle-specific protein, SM22, and tropomyosin are decreased after 24 h in organ culture. These results were confirmed by Western blot analysis. Other smooth muscle proteins (actin and calponin) exhibited little change. We also demonstrate a 50% downregulation in the small G protein, Rho, a potent modulator of Ca(2+)-independent force. These results indicate that organ culture preferentially inhibits the expression of certain smooth muscle proteins. This change in protein expression after organ culture correlates with the specific inhibition of hypoxic vasorelaxation. These results provide novel target pathways for investigation that are potentially important for vascular oxygen sensing.

The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass specrometry study.

The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable cross-linking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.

Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets.

Recent evidence indicates that Pseudomonas aeruginosa residing as biofilms in airway mucus of cystic fibrosis (CF) patients is undergoing anaerobic metabolism, a form of growth requiring gene products that are not utilized during aerobic growth. The outer membrane protein, OprF, and the rhl quorum sensing circuit are two previously unrecognized cellular factors that are required for optimal anaerobic biofilm viability. Without OprF, bacteria grow extremely poorly because they lack nitrite reductase activity while lacking rhlR or rhlI forces bacteria to undergo metabolic suicide by overproduction of nitric oxide. Furthermore, anaerobic growth favors maintenance of the mucoid, alginate-overproducing phenotype. Thus, with increasing age of CF patients, mucoid populations predominate, indicating that anaerobic bacteria reside in the inspissated airway mucus. Because many frontline antibiotics used in the treatment of CF airway disease are either ineffective or show reduced efficacy during anaerobic conditions, we propose development of new drugs to combat anaerobic metabolism by P. aeruginosa for more effective treatment of chronic CF lung infections.

Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis.

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.