基于iTRAQ技术对植物乳杆菌FS5-5的耐盐特性分析

【目的】对大酱中耐盐性较好的植物乳杆菌进行蛋白质组学研究,为植物乳杆菌盐胁迫应激机制的研究提供实验数据。【方法】本项研究以筛选自东北传统农家大酱的耐盐性较好的植物乳杆菌FS5-5为研究对象,绘制了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的生长曲线,并利用i TRAQ技术研究了其在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下的蛋白质表达情况。【结果】植物乳杆菌FS5-5在0%、6.0%、7.0%和8.0%(W/V)Na Cl浓度下到达对数生长期中期的时间点分别为5、10、12和12 h;以差异倍数在1.2倍以上且P0.05为筛选条件对6.0%、7.0%和8.0%(W/V)Na Cl浓度下与0%进行差异蛋白质的筛选,共筛选出1271个差异蛋白质。这些差异蛋白质主要参与糖代谢、氨基酸代谢、脂肪酸代谢、核苷酸代谢、应激反应、转运、PTS系统和核糖体代谢等。【结论】植物乳杆菌在高盐浓度下生长与能量合成蛋白质、应激蛋白质以及相容性溶质转运蛋白质的表达上调有密切关系。     

Proteomic analysis of cytoplasmic and surface proteins from yeast cells,hyphae, and biofilms of Candida albicans

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non‐covalently attached cell‐surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (> 1.5‐fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi‐organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.     

Proteomic analysis of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> in biofilm and planktonic growth mode

Recently, multidrug-resistant clinical isolates of Acinetobacter baumannii have been found to have a high capacity to form biofilm. It is well known that bacterial cells within biofilms are highly resistant to antibiotics, UV light, acid exposure, dehydration, and phagocytosis in comparison to their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which proteins are up-regulated in A. baumannii biofilm cells, we performed a proteomic analysis. A clinical isolate of A. baumannii 1656-2, which was characterized to have a high biofilm forming ability, was cultivated under biofilm and planktonic conditions. Outer membrane enriched A. baumannii 1656-2 proteins were separated by two-dimensional (2-D) gel electrophoresis and the differentially expressed proteins were identified by MALDI-TOF mass spectrometry. The proteins up-regulated or expressed only in biofilm cells of A. baumannii are categorized as follows: (i) proteins processing environmental information such as the outer membrane receptor protein involved in mostly Fe transport, a sensor histidine kinase/response regulator, and diguanylate cyclase (PAS-GGEDF-EAL domain); (ii) proteins involved in metabolism such as NAD-linked malate dehydrogenase, nucleoside-diphosphate sugar epimerase, putative GalE, ProFAR isomerase, and N-acetylmuramoyl-l-alanine amidase; (iii) bacterial antibiotic resistance related proteins; and (iv) proteins related to gene repair such as exodeoxyribonuclease III and GidA. This proteomic analysis provides a fundamental platform for further studies to reveal the role of biofilm in the persistence and tolerance of A. baumannii.     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

Elevated c-di-GMP levels promote biofilm formation and biodesulfurization capacity of Rhodococcus erythropolis

Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels.     

Exoproteome of Staphylococcus aureus reveals putative determinants of nasal carriage

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage.     

Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose‐grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate‐grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose‐grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate‐grown cells. We identified mating and pheromone‐regulated proteins that were exclusive to lactate‐grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa‐specific and other niche‐specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.     

Two‐dimensional gel‐based proteomic of the caries causative bacterium Streptococcus mutans UA159 and insight into the inhibitory effect of carolacton

Streptococcus mutans is considered to be the most cariogenic organism. Carolacton, isolated from the myxobacterium Sorangium cellulosum, shows the ability to disturb S. mutans biofilm viability that makes it a potential anti‐biofilm drug. However, the molecular mechanism of carolacton remains to be elucidated. In order to use proteomics to characterize the effect of carolacton, we constructed a 2DE‐based proteome reference map of the cytoplasmic and extracellular proteins for S. mutans in the present study. In total, 239 protein spots representing 192 different cytoplasmic proteins were identified by MALDI‐TOF MS and PMF. This represents the highest number of identified proteins so far for S. mutans UA159 in the pI range of 4–7 and would benefit further research on the physiology and pathogenicity of this strain. Based on the constructed reference map, the inhibitory effects of carolacton on S. mutans biofilm and planktonic‐growing cells were investigated. The results of the comparative proteome analysis indicate that carolacton exerts its inhibitory effects by disturbing the peptidoglycan biosynthesis and degradation and thereby causes damages to the integrity of the cell envelope, leading ultimately to cell death.     

iTRAQ-based proteomic analysis of responses of Lactobacillus plantarum FS5-5 to salt tolerance

Lactobacillus plantarum FS5-5 (L. plantarum FS5-5) is a salt-tolerant probiotic strain, which had been isolated from northeast Chinese traditionally fermented Dajiang. We analyzed the underlying molecular mechanisms of L. plantarum FS5-5 after salt stress by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics and bioinformatics analysis. L. plantarum FS5-5 was treated with low (1.5, 3.0, 4.0, and 5.0% (w/v) NaCl) and high (6.0, 7.0, and 8.0% (w/v) NaCl) salt stress. Differentially expression proteins (DEPs) of all groups were measured by quantitative proteomic approach of iTRAQ with LC–MS/MS. Furthermore, DEPs were identified by Mascot and GO, and bioinformatics analysis was conducted by KEGG. Thirty DEPs (P < 0.05) between low salt stress and control condition (0% (w/v) NaCl) were mapped and classified into nine functional groups; 122 DEPs (P < 0.05) between high salt stress and control condition were mapped and classified into 15 functional groups. In all groups, most proteins were involved in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and ATP-binding cassette (ABC) transporter. We found that six proteins (metS, GshAB, GshR3, PepN, GshR4, and serA) involved in amino acid metabolism, three proteins (I526_2330, Gpd, and Gnd) involved in carbohydrate metabolism, and one protein (N876_0118940) involved in peptidoglycan hydrolysis were upregulated after salt stress. Conclusively, optimal L. plantarum FS5-5 growth was dependent on the collective action of different regulatory systems, with each system playing an important role in adapting to salt stress. There may be some relationship between the upregulated proteins of L. plantarum FS5-5 and salt stress.

     

Application of 16O/18O reverse proteolytic labeling to determine the effect of biofilm culture on the cell envelope proteome of Porphyromonas gingivalis W50

Porphyromonas gingivalis is an oral pathogen linked to chronic periodontitis. The bacterium exists as part of a polymicrobial biofilm accreted onto the tooth surface. An understanding of the changes to the proteome especially of the cell envelope of biofilm cells compared with planktonic cells could provide valuable insight into the molecular processes of biofilm formation. To establish which proteins changed in abundance between the planktonic and biofilm growth states, the cell envelope fractions of two biological replicates of P. gingivalis cultivated in a chemostat were analysed. Proteins were separated by 1-D SDS-PAGE, in-gel digested with trypsin in the presence of H216O or H218O and identified and quantified by LC-MALDI TOF/TOF-MS. Using a reverse labeling strategy we identified and quantified the changes in abundance of 81 P. gingivalis cell envelope proteins. No form of bias between the labels was observed. Twenty four proteins increased in abundance and 18 decreased in abundance in the biofilm state. A group of cell-surface located C-Terminal Domain family proteins including RgpA, HagA, CPG70 and PG99 increased in abundance in the biofilm cells. Other proteins that exhibited significant changes in abundance included transport related proteins (HmuY and IhtB), metabolic enzymes (FrdAB) and immunogenic proteins.     

Novel surface display system for heterogonous proteins on Lactobacillus plantarum

Aims: To establish a novel cell surface display system that would enable the display of target proteins on Lactobacillus plantarum. Methods and Results: Blast P analysis of the amino acids sequence data revealed that the N‐terminus of the putative muropeptidase MurO from L. plantarum contained two putative lysin motif (LysM) repeat regions, implying that the MurO was involved in bacterial cell wall binding. To investigate the potential of MurO for surface display, green fluorescent protein (GFP) was fused to MurO at its C‐terminus and the resulting fusion protein was expressed in Escherichia coli. After being mixed with L. plantarum cells in vitro, GFP was successfully displayed on the surfaces of L. plantarum cells. Increases in the fluorescence intensities of chemically pretreated L. plantarum cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for MurO. SDS sensitivity assay showed that the GFP fluorescence intensity was reduced after being treated with SDS. To demonstrate the applicability of the MurO‐mediated surface display system, β‐galactosidase from Bifidobacterium bifidium, in place of GFP, was functionally displayed on the surface of L. plantarum cells via MurO. Conclusions: The MurO was a novel anchor protein for constructing a surface display system for L. plantarum. Significance and Impact of Study: The success in surface display of GFP and β‐galactosidase opened up the feasibility of employing the cell wall anchor of MurO for surface display in L. plantarum.     

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a well‐known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S‐layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria–host interactions. The exo‐ and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty‐two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well‐known and putative moonlighting proteins. The exoproteome contained nine classically‐secreted (containing a signal sequence) and ten nonclassically‐secreted proteins, while the surface proteome contained four classically‐secreted and eight nonclassically secreted proteins. Identification of exo‐ and surface proteomes contributes describing potential protein‐mediated probiotic–host interactions.     

Proteome profile of a pandemic Vibrio parahaemolyticus SC192 strain in the planktonic and biofilm condition

Vibrio parahaemolyticus is one of the leading causative agents of foodborne diseases in humans. In this study, the proteome profiles of the pandemic strain V. parahaemolyticus SC192 belonging to the O3:K6 serovar during the planktonic and biofilm stages were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry. This non-gel-based multidimensional protein identification technology approach identified 45.5% of the proteome in the reference genome V. parahaemolyticus RIMD 2210633. This is the largest proteome coverage obtained so far in V. parahaemolyticus and provides evidence for expression of 27% of the hypothetical proteins. Comparison of the planktonic and biofilm proteomes based on their cluster of orthologous groups, gene ontologies and KEGG pathways provides basic information on biofilm specific functions and pathways. To the authors’ knowledge, this is the first study to generate a global proteome profile of the pandemic strain of V. parahaemolyticus and the method reported here could be used to rapidly obtain a snapshot of the proteome of any microorganism at a given condition.     

A physiological comparative study of acid tolerance of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZDY 2013 and <Emphasis Type="Italic">L. plantarum</Emphasis> ATCC 8014 at membrane and cytoplasm levels

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.     

Steric microstructure of mixed-species biofilm formed by interaction between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae

ABSTRACT

The mixed-species biofilm of Lactobacillus plantarum ML11-11 (LAB) and yeast had a double-layered structure with the ground layer composed of LAB cells, and the upper layer composed of coaggregates of LAB and yeast cells. The ability of LAB to adhere to both, the solid surface and the yeast cells, enabled the formation and maintenance of the biofilm as an ecosystem for LAB and yeast.     

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H₂O₂) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.