Patterns of reactivity of lantibiotics subtilin and nisin with molecular targets in Bacillus cereus and Bacillus subtilis 168

ABSTRACT Title of Dissertation: PATTERNS OF REACTIVITY OF LANTIBIOTICS SUBTILIN AND NISIN WITH MOLECULAR TARGETS IN Bacillus cereus AND Bacillus subtilis 168 Srilatha Kuntumalla, Doctor of Philosophy, 2005 Dissertation Directed By: Professor J. Norman HansenDepartment of Chemistry and Biochemistry Subtilin and nisin belong to a unique class of antibiotics called lantibiotics that contain unusual dehydro and lanthionine amino acid residues. The gene-encoded antimicrobial peptides subtilin and nisin exhibit bactericidal effects against several Gram-positive bacteria and also inhibit bacterial spore outgrowth. Subtilin and nisin are structural analogs and possess similar mechanisms of antimicrobial action. Although nisin is very stable, subtilin previously isolated was highly unstable with loss of biological activity observed during storage. Subtilin isolated in this work using hydrophobic interaction chromatography was very stable, with biological activity retained for at least a few months after isolation. The possibility that specificity of subtilin and nisin towards sensitive Gram-positive bacteria is due to interaction of these lantibiotics with specific target proteins in susceptible bacteria was explored in this work. Phage display experiments performed to detect peptides interacting with subtilin identified a 12-mer peptide with a KTTLL motif found in ATP binding proteins such as ABC transporters and protein synthesis initiation factor IF-2 (~78 kDa). Binding of subtilin to specific ABC transporters in bacterial cell membrane would contribute to its specificity. Binding of subtilin to IF-2 would result in inhibition of protein synthesis suggesting an alternative mechanism of action for subtilin. Experiments performed to determine the nature of interaction of subtilin and nisin with bacterial cellular proteins detected both covalent and non-covalent interactions. The covalent interactions between bacterial proteins and subtilin or nisin were stable on boiling in SDS and analyzing by SDS-PAGE. These stable covalent adducts indicated that the electrophilic dehydro residues of subtilin and nisin were probably involved in covalent attachment with specific nucleophilic groups in bacterial protein targets. Covalent attachment of an antibiotic to its bacterial target has been previously observed with only a few antibiotics. Sites of nisin attachment to bacterial spores as visualized by electron microscopy showed nisin binds to highly localized regions on spore surfaces. Attempts to identify bacterial protein targets of subtilin and nisin using monomeric avidin and anti-FITC columns, respectively, resulted in isolation of proteins in ~70-80 kDa range. Further characterization of these proteins should help in understanding the specificity and antimicrobial mechanism of action of nisin and subtilin

In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

<p>Abstract</p> <p>Background</p> <p><it>Shigella dysenteriae </it>serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of <it>Shigella dysenteriae </it>serotype 1 (SD1) <it>in vitro </it>(derived from LB cell cultures) and <it>in vivo </it>(derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology.</p> <p>Results</p> <p>Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from <it>in vitro </it>and <it>in vivo </it>samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of <it>in silico </it>predicted SD1 membrane proteome) contributed to the most extensive survey of the <it>Shigella </it>membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under <it>in vivo </it>conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High <it>in vivo </it>abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers <it>in vivo</it>. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased <it>in vivo</it>.</p> <p>Conclusions</p> <p>Global proteomic profiling of SD1 comparing <it>in vivo vs. in vitro </it>proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under <it>in vivo </it>conditions, lending credence to their candidacy as potential vaccine targets.</p

Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families

The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LC-MS/MS proteomics results

Mass spectrometry (MS) based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS) data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007) described a modified spectral counting technique, Absolute Protein Expression (APEX), which improves on basic spectral counting methods by including a correction factor for each protein (called O(i) value) that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (O(i)). This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. Results: The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition thparameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a utility to merge multiple APEX results into a standardized format in preparation for further statistical analysis. Conclusion: The APEX Quantitative Proteomics Tool provides a simple means to quickly derive hundreds to thousands of protein abundance values from standard liquid chromatography-tandem mass spectrometry proteomics datasets. The APEX tool provides a straightforward intuitive interface design overlaying a highly customizable computational workflow to produce protein abundance values from LC-MS/MS datasets.National Institute of Allergy and Infectious Diseases (NIAID) N01-AI15447National Institutes of HealthNational Science Foundation, the Welsh and Packard FoundationsInternational Human Frontier Science ProgramCenter for Systems and Synthetic Biolog

Proteomic View of Interactions of Shiga Toxin-Producing <i>Escherichia coli</i> with the Intestinal Environment in Gnotobiotic Piglets

<div><p>Background</p><p>Shiga toxin (Stx)-producing <i>Escherichia coli</i> cause severe intestinal infections involving colonization of epithelial Peyer’s patches and formation of attachment/effacement (A/E) lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing <i>Escherichia coli</i> with the <i>in vivo</i> host environment, may elucidate novel molecular pathogenesis aspects.</p><p>Methodology/Principal Findings</p><p>Enterohemorrhagic <i>E. coli</i> strain 86–24 produces Shiga toxin-2 and belongs to the serotype O157:H7. Bacterial cells were scrapped from stationary phase cultures (the <i>in vitro</i> condition) and used to infect gnotobiotic piglets via intestinal lavage. Bacterial cells isolated from the piglets’ guts constituted the <i>in vivo</i> condition. Cell lysates were subjected to quantitative 2D gel and shotgun proteomic analyses, revealing metabolic shifts towards anaerobic energy generation, changes in carbon utilization, phosphate and ammonia starvation, and high activity of a glutamate decarboxylase acid resistance system <i>in vivo</i>. Increased abundance of pyridine nucleotide transhydrogenase (PntA and PntB) suggested <i>in vivo</i> shortage of intracellular NADPH. Abundance changes of proteins implicated in lipopolysaccharide biosynthesis (LpxC, ArnA, the predicted acyltransferase L7029) and outer membrane (OM) assembly (LptD, MlaA, MlaC) suggested bacterial cell surface modulation in response to activated host defenses. Indeed, there was evidence for interactions of innate immunity-associated proteins secreted into the intestines (GP340, REG3-γ, resistin, lithostathine, and trefoil factor 3) with the bacterial cell envelope.</p><p>Significance</p><p>Proteomic analysis afforded insights into system-wide adaptations of strain 86–24 to a hostile intestinal milieu, including responses to limited nutrients and cofactor supplies, intracellular acidification, and reactive nitrogen and oxygen species-mediated stress. Protein and lipopolysaccharide compositions of the OM were altered. Enhanced expression of type III secretion system effectors correlated with a metabolic shift back to a more aerobic milieu <i>in vivo</i>. Apparent pathogen pattern recognition molecules from piglet intestinal secretions adhered strongly to the bacterial cell surface.</p></div

Global adaptation of EHEC cells to the intestinal milieu.

<p>Fifteen biological role categories, as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066462#pone.0066462.s002" target="_blank">Dataset S1</a>, are displayed in the graph. The bar length represents the sum of APEX_i quantities of all proteins with a statistically significant abundance change (<i>in vitro</i> versus <i>in vivo</i>) assigned to a given biological role category. Blue bars represent the <i>in vitro</i> (cell culture) growth, red bars the <i>in vivo</i> (intestinal) environments.</p

Sus scrofa proteins identified from purified intestinal EHEC cells.

*<p>HB+ and HB-: two in vivo EHEC groups were separated based on the identification of hemoglobin, which also correlated with high vs. low abundances of the major type III secretion system effectors; ++, +, -: the estimated protein abundances based on spectral counts (++, >8; +, <8, - none); for significance of the spectral counts, the Mascot percolator was set at q-value <0.01 and PEP value <10⁻⁴;</p>∧<p>Abbrev.: PRR, pattern recognition receptor; N.K., not known; GI, gastrointestinal. Details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066462#pone.0066462.s004" target="_blank">Dataset S3</a>.</p