Quantitative proteomic analysis of host—pathogen interactions: a study of Acinetobacter baumannii responses to host airways

BMC Genomics

Germán Bou²  Margarita Poza²  María Tomás²  María Lopez²  Alejandro Beceiro²  Jesús Mateos¹  Jose Antonio Méndez² 

[1] Grupo de Proteomica-PBR2-ProteoRed/ISCIII-Servicio de Reumatologia, A Coruña, Spain;Microbiology Division, INIBIC-Complejo Hospitalario Universitario de la Coruña, A Coruña, Spain

关键词: Virulence;    Ex vivo;    Host-pathogen interaction;    Acinetobacter baumannii;    Proteome;   

Others  :  1208957
DOI  :  10.1186/s12864-015-1608-z

 received in 2014-12-02, accepted in 2015-05-01,  发布年份 2015

【 摘 要 】

Background

Acinetobacter baumannii is a major health problem. The most common infection caused by A. baumannii is hospital acquired pneumonia, and the associated mortality rate is approximately 50 %. Neither in vivo nor ex vivo expression profiling has been performed at the proteomic or transcriptomic level for pneumonia caused by A. baumannii. In this study, we characterized the proteome of A. baumannii under conditions that simulate those found in the airways, to gain some insight into how A. baumannii adapts to the host and to improve knowledge about the pathogenesis and virulence of this bacterium. A clinical strain of A. baumannii was grown under different conditions: in the presence of bronchoalveolar lavage fluid from infected rats, of RAW 264.7 cells to simulate conditions in the respiratory tract and in control conditions. We used iTRAQ labelling and LC-MALDI-TOF/TOF to investigate how A. baumannii responds on exposure to macrophages/BALF.

Results

179 proteins showed differential expression. In both models, proteins involved in the following processes were over-expressed: (i) pathogenesis and virulence (OmpA, YjjK); (ii) cell wall/membrane/envelope biogenesis (MurC); (iii) energy production and conversion (acetyl-CoA hydrolase); and (iv) translation (50S ribosomal protein L9). Proteins involved in the following were under-expressed: (i) lipid metabolism (short-chain dehydrogenase); (ii) amino acid metabolism and transport (aspartate aminotransferase); (iii) unknown function (DNA-binding protein); and (iv) inorganic ion transport and metabolism (hydroperoxidase).

Conclusions

We observed alterations in cell wall synthesis and identified 2 upregulated virulence-associated proteins with >15 peptides/protein in both ex vivo models (OmpA and YjjK), suggesting that these proteins are fundamental for pathogenesis and virulence in the airways. This study is the first comprehensive overview of the ex vivo proteome of A. baumannii and is an important step towards identification of diagnostic biomarkers, novel drug targets and potential vaccine candidates in the fight against pneumonia caused by A. baumannii.

【 授权许可】

   
2015 Méndez et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Howard A, Donoghue MO, Feeney A, Sleator RD. Acinetobacter baumannii an emerging opportunistic pathogen. Virulence. 2012; 3:243-250.
• [2]Visca P, Seifert H, Towner KJ. Acinetobacter infection—an emerging threat to human health. IUBMB Life. 2011; 63:1048-1054.
• [3]Anstey NM, Currie BJ, Hassell M, Palmer D, Dwyer B, Seifert H. Community-acquired bacteremic Acinetobacter pneumonia in tropical Australia is caused by diverse strains of Acinetobacterbaumannii with carriage in the throat in at-risk groups. J Clin Microbiol. 2002; 40:685-686.
• [4]Leung WS, Chu CM, Tsang KY, Lo FH, Lo KF, Ho PL. Fulminant community-acquired Acinetobacter baumannii pneumonia as a distinct clinical syndrome. Chest. 2006; 129:102-109.
• [5]Giamarellou H, Antoniadou AK, Kanellakopoulou K. Acinetobacterbaumannii: a universal threat to public health? Int J Antimicrob Agents. 2008; 32:106-119.
• [6]Theaker C, Azadian B, Soni N. The impact of Acinetobacter baumannii in the intensive care unit. Anaesthesia. 2003; 58:271-274.
• [7]Chen CH, Lin LC, Chang YJ, Huang CC, Liu CE, Young TG. Analysis of prognostic factors in 95 patients with Acinetobacter baumannii bacteremia. Infection. 2003; 31:331-335.
• [8]Rodríguez-Baño J, Cisneros JM, Fernández-Cuenca F, Ribera A, Vila J, Pascual A, Martínez-Martínez L, Bou G, Pachón J. Clinical features and epidemiology of Acinetobacter baumannii colonization and infection in Spanish hospitals. Infect Control Hosp Epidemiol. 2004; 25:819-24.
• [9]Gayoso CM, Mateos J, Méndez JA, Fernández-Puente P, Rumbo C, Tomás M, Martínez de Ilarduya O, Bou G. Molecular mechanisms involved in the response to desiccation stress and persistence in Acinetobacterbaumannii. J Proteome Res. 2014; 13:460-76.
• [10]Eveillard M, Kempf M, Belmonte O, Pailhoriès H, Joly-Guillou ML. Reservoirs of Acinetobacter baumannii outside the hospital and potential involvement in emerging human community-acquired infections. Int J Infect Dis. 2013; 17:e802-5.
• [11]Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol. 2008; 21:538-582.
• [12]Seifert H, Dijkshoorn L, Gerner-Smidt P, Pelzer N, Tjernberg I, Vaneechoutte M. Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods. J Clin Microbiol. 1997; 35:2819-25.
• [13]Chu YW, Leung CM, Houang ET, Ng KC, Leung CB, Leung HY, Cheng AF. Skin carriage of acinetobacters in Hong Kong. J Clin Microbiol. 1999; 35:2962-2967.
• [14]Fournier PE, Richet H. The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis. 2006; 42:692-9.
• [15]Camp C, Tatum OL. A review of Acinetobacter baumannii as a highly successful pathogen in times of war. Lab Medicine. 2010; 41:649-657.
• [16]Hanlon GW. The emergence of multidrug resistant Acinetobacter species: a major concern in the hospital setting. Lett Appl Microbiol. 2005; 41:375-8.
• [17]Morgan DJ, Liang SY, Smith CL, Johnson JK, Harris AD, Furuno JP, Thom KA, Snyder GM, Day HR, Perencevich EN. Frequent multidrug-resistant Acinetobacter baumannii contamination of gloves gowns and hands of healthcare workers. Infect Control Hosp Epidemiol. 2010; 31:716-721.
• [18]Wendt C, Dietze B, Dietz E, Ruden H. Survival of Acinetobacter baumannii on dry surfaces. J Clin Microbiol. 1997; 35:1394-1397.
• [19]Rao R, Karthika R, Singh S, Shashikala P, Kanungo R, Jayachandran S, Prashanth K. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Indian J Med Microbiol. 2008; 4:333-337.
• [20]Gales AC, Jones RN, Sader HS. Global assessment of the antimicrobial activity of polymyxin B against 54 731 clinical isolates of Gram-negative bacilli: report from the SENTRY antimicrobial surveillance programme (2001–2004). Clin Microbiol Infect. 2006; 12:315-321.
• [21]Ko KS, Suh JY, Kwon KT, Jung SI, Park KH, Kang CI, Chung DR, Peck KR, Song JH. High rates of resistance to colistin and polymyxin B in subgroups of Acinetobacter baumannii isolates from Korea. J Antimicrob Chemother. 2007; 60:1163-1167.
• [22]Barbe V, Vallenet D, Fonknechten N, Kreimeyer A, Oztas S, Labarre L, Cruveiller S, Robert C, Duprat S, Wincker P, Ornston LN, Weissenbach J, Marlière P, Cohen GN, Médigue C. Unique features revealed by the genome sequence of Acinetobacter sp ADP1 a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 2004; 32:5766-5779.
• [23]Smith MG, Gianoulis TA, Pukatzki S, Mekalanos JJ, Ornston LN, Gerstein M, Snyder M. New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes Dev. 2007; 21:601-614.
• [24]Mendez JA, Soares NC, Mateos J, Gayoso C, Rumbo C, Aranda J, Tomas M, Bou G. Extracellular proteome of a highly invasive multidrug-resistant clinical strain of Acinetobacter baumannii. J Proteome Res. 2012; 11:5678-94.
• [25]Kwon SO, Gho YS, Lee JC, Kim SI. Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate. FEMS Microbiol Lett. 2009; 297:150-6.
• [26]Tiwari V, Vashistt J, Kapil A, Moganty RR. Comparative proteomics of inner membrane fraction from carbapenem-resistant Acinetobacter baumannii with a reference strain. PLoS ONE. 2012; 7: Article ID e39451
• [27]Yun SH, Choi CW, Kwon SO, Park GW, Cho K, Kwon KH, Kim JY, Yoo JS, Lee JC, Choi JS, Kim S, Kim SI. Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii. J Proteome Res. 2011; 10:459-69.
• [28]Vashist J, Tiwari V, Kapil A, Rajeswari MR. Quantitative profiling and identification of outer membrane proteins of beta-lactam resistant strain of Acinetobacter baumannii. J Proteome Res. 2010; 9:1121-8.
• [29]Yun SH, Choi CW, Park SH, Lee JC, Leem SH, Choi JS, Kim S, Kim SI. Proteomic analysis of outer membrane proteins from Acinetobacter baumannii DU202 in tetracycline stress condition. J Microbiol. 2008; 46:720-7.
• [30]Soares NC, Cabral MP, Parreira JR, Gayoso C, Barba MJ, Bou G. 2-DE analysis indicates that Acinetobacter baumannii displays a robust and versatile metabolism. Proteome Sci. 2009; 7:37. BioMed Central Full Text
• [31]Cabral MP, Soares NC, Aranda J, Parreira JR, Rumbo C, Poza M, Valle J, Calamia V, Lasa I, Bou G. Proteomic and functional analyses reveal a unique lifestyle for Acinetobacter baumannii biofilms and a key role for histidine metabolism. J Proteome Res. 2011; 10:3399-417.
• [32]Chopra S, Ramkissoon K, Anderson DC. A systematic quantitative proteomic examination of multidrug resistance in Acinetobacter baumannii. J Proteomics. 2013; 12:17-39.
• [33]Pradet-Balade B, Boulme F, Beug H, Mullner EW, Garcia-Sanz JA. Translation control: bridging the gap between genomics and proteomics? Trends Biochem Sci. 2001; 26:225-229.
• [34]Echenique JR, Dorsey CW, Patrito LC, Petroni A, Tolmasky ME, Actis LA. Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron. Microbiology. 2001; 147:2805-2815.
• [35]Hood MI, Jacobs AC, Sayood K, Dunman PM, Skaar EP. Acinetobacter baumannii increases tolerance to antibiotics in response to monovalent cations. Antimicrob Agents Chemother. 2010; 54:1029-41.
• [36]Brossard KA, Campagnari AA. The Acinetobacter baumannii biofilm-associated protein plays a role in adherence to human epithelial cells. Infect Immun. 2012; 80:228-33.
• [37]Zimbler DL, Park TM, Arivett BA, Penwell WF, Greer SM, Woodruff TM, Tierney DL, Actis LA. Stress response and virulence functions of the Acinetobacter baumannii NfuA Fe-S scaffold protein. J Bacteriol. 2012; 194:2884-93.
• [38]Eijkelkamp BA, Hassan KA, Paulsen IT, Brown MH. Investigation of the human pathogen Acinetobacter baumannii under iron limiting conditions. BMC Genomics. 2011; 12:126. BioMed Central Full Text
• [39]Jacobs AC, Sayood K, Olmsted SB, Blanchard CE, Hinrichs S, Russell D, Dunman PM. Characterization of the Acinetobacter baumannii growth phase-dependent and serum responsive transcriptomes. FEMS Immunol Med Microbiol. 2012; 64:403-12.
• [40]Nwugo CC, Gaddy JA, Zimbler DL, Actis LA. Deciphering the iron response in Acinetobacter baumannii: a proteomics approach. J Proteomics. 2011; 74:44-58.
• [41]Zhang X, El-Hajj ZW, Newman E. Deficiency in L-Serine deaminase interferes with one-carbon metabolism and cell wall synthesis in Escherichia coli K-12. J Bacteriol. 2010; 192:5515-5525.
• [42]Emanuele JJ, Jin H, Jacobson BL, Chang CY, Einspahr HM, Villafranca JJ. Kinetic and crystallographic studies of Escherichia coli UDP-N-acety1muramate:L-alanine ligase. Protein Sci. 1996; 5:2566-2574.
• [43]Camejo A, Buchrieser C, Couvé E, Carvalho F, Reis O, Ferreira P, Sousa S, Cossart P, Cabanes D. In vivo transcriptional profiling of listeria monocytogenes and mutagenesis identify new virulence factors involved in infection. PLoS Pathog. 2009; 5:e1000449.
• [44]White TA, Kell DB. Comparative genomic assessment of novel broad-spectrum targets for antibacterial drugs. Comp Funct Genom. 2004; 5:304-327.
• [45]El Zoeiby A, Sanschagrin F, Lamoureux J, Darveau A, Levesque RC. Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase. FEMS Microbiol Lett. 2000; 183(2):281-8.
• [46]Zawadzke LE, Norcia M, Desbonnet CR, Wang H, Freeman-Cook K, Dougherty TJ. Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway. Assay Drug Dev Technol. 2008; 6:95-103.
• [47]Hameed PS, Manjrekar P, Chinnapattu M, Humnabadkar V, Shanbhag G, Kedari C, Mudugal NV, Ambady A, de Jonge BL, Sadler C, Paul B, Sriram S, Kaur P, Guptha S, Raichurkar A, Fleming P, Eyermann CJ, McKinney DC, Sambandamurthy VK, Panda M, Ravishankar S. Pyrazolopyrimidines establish MurC as a vulnerable target in Pseudomonas aeruginosa and Escherichia coli. ACS Chem Biol. 2014; 9(10):2274-82.
• [48]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215:403-410.
• [49]Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25:3389-3402.
• [50]Deng WL, Lin YC, Lin RH, Wei CF, Huang YC, Peng HL, Huang HC. Effects of galU Mutation on Pseudomonas syringae–plant interactions. Mol Plant-Microbe Interact. 2010; 23:1184-1196.
• [51]Jayakar HR, Parvathareddy J, Fitzpatrick EA, Bina XR, Bina JE, Re F, Emery FD, Miller MA. A galU mutant of francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia. BMC Microbiol. 2011; 179:1471-2180.
• [52]Jiang SS, Lin TY, Wang WB, Liu MC, Hsueh PR, Liaw SJ. Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance swarming and virulence. Antimicrob Agents Chemother. 2010; 54:2000-2009.
• [53]Mollerach M, López R, García E. Characterization of the galU gene of Streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis. J Exp Med. 1998; 188:2047-2056.
• [54]Priebe GP, Dean CR, Zaidi T, Meluleni GJ, Coleman FT, Coutinho YS, Noto MJ, Urban TA, Pier GB, Goldberg JB. The galU Gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung. Infect Immun. 2004; 72:4224-4232.
• [55]Crosa JH, Mey AR, Payne SM. Iron Transport in Bacteria. ASM Press, Washington, DC; 2004.
• [56]Skaar EP. The battle for iron between bacterial pathogens and their vertebrate hosts. PLoS Pathog. 2010; 6: Article ID e1000949
• [57]Dittrich AM, Meyer HA, Hamelmann E. The role of lipocalins in airway disease. Clin Exp Allergy. 2013; 43:503-511.
• [58]Välimäki E, Miettinen JJ, Lietzén N, Matikainen S, Nyman TA. Monosodium urate activates Src/Pyk2/PI3 Kinase and Cathepsin dependent unconventional protein secretion from human primary macrophages. Mol Cell Proteomics. 2013; 12:749-763.
• [59]Miethke M, Marahiel MA. Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev. 2007; 71:413-451.
• [60]Raffatellu M, George MD, Akiyama Y, Hornsby MJ, Nuccio SP, Paixao TA. Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype Typhimurium for growth and survival in the inflamed intestine. Cell Host Microbe. 2009; 5:476-486.
• [61]Proschak A, Lubuta P, Grün P, Löhr F, Wilharm G, Berardinis VD, Bode HB. Structure and Biosynthesis of Fimsbactins A–F, Siderophores from Acinetobacter baumannii and Acinetobacter baylyi. ChemBioChem. 2013; 14:633-638.
• [62]Wen ZT, Suntharaligham P, Cvitkovitch DG, Burne RA. Trigger factor in Streptococcus mutans is involved in stress tolerance competence development and biofilm formation. Infect Immun. 2005; 73:219-225.
• [63]Ullers RS, Schwager DAF, Georgopoulos C, Genevaux P. Trigger factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli. PNAS. 2007; 104:3101-3106.
• [64]Bigot A, Botton E, Dubail I, Charbit A. A homolog of Bacillus subtilis trigger factor in Listeria monocytogenes is involved in stress tolerance and bacterial virulence. Appl Environ Microbiol. 2006; 72:6623-6631.
• [65]Henard CA, Bourret TJ, Song M, Vázquez-Torres A. Control of redox balance by the stringent response regulatory protein promotes antioxidant defenses of Salmonella. J Biol Chem. 2010; 285:36785-36793.
• [66]Frees D, Chastanet A, Qazi S, Sørensen K, Hill P, Msadek T, Ingmer H. Clp ATPases are required for stress tolerance intracellular replication and biofilm formation in Staphylococcus aureus. Mol Microbiol. 2004; 54:1445-1462.
• [67]Eijkelkamp BA, Stroeher UH, Hassan KA, Papadimitrious MS, Paulsen IT, Brown MH. Adherence and motility characteristics of clinical Acinetobacter baumannii isolates. FEMS Microbiol Lett. 2011; 323:44-51.
• [68]Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, Hyun SH, Kim SA, Lee SK, Lee JC. Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cell Microbiol. 2005; 7:1127-1138.
• [69]Gaddy JA, Tomaras AP, Actis LA. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect Immun. 2009; 77:3150-3160.
• [70]Law RJ, Hamlin JN, Sivro A, McCorrister SJ, Cardama GA, Cardona ST. A functional phenylacetic acid catabolic pathway is required for full pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans host model. J Bacteriol. 2008; 190:7209-7218.
• [71]Wu JW, Chen XL. Extracellular metalloproteases from bacteria. Appl Microbiol Biotechnol. 2011; 92:253-262.
• [72]Moro A, Ruiz-Cabello F, Fernandez-Cano A, Stock RP, González A. Secretion by trypanosoma cruzi of a peptidyl-prolpyl cis-trans isomerase involved in cell infection. EMBO J. 1995; 14:2483-2490.
• [73]Moulder JW. Comparative biology of intracellular parasitism. Microbiol Rev. 1985; 49:298-337.
• [74]Hermans PWM, Adrian PV, Albert C, Estevao S, Hoogenboezem T, Luijendijk IHT, Kamphausen T, Hammerschmidt S. The Streptococcal lipoprotein rotamase A (SlrA) is a functional peptidyl-prolyl isomerase involved in pneumococcal colonization. J Biol Chem. 2006; 281:968-976.
• [75]Xia Q, Wang T, Taub F, Park Y, Capestany CA, Lamont RJ, Hackett M. Quantitative proteomics of intracellular Porphyromonas gingivalis. Proteomics. 2007; 7:4323-4337.
• [76]Dieppedale J, Sobral D, Dupuis M, Dubail I, Klimentova J, Stulik J, Postic G, Frapy E, Meibom KL, Barel M. Charbit: a identification of a putative chaperone involved in stress resistance and virulence in Francisella tularensis. Infect Immun. 2011; 79:1428-1439.
• [77]Umland TC, Schultz LW, MacDonald U, Beanan JM, Olson R, Russo TA. In vivo-validated essential genes identified in acinetobacter baumanniiby using human ascites overlap poorly with essential genes detected on laboratory media. mBio. 2012; 3:e00113-12.
• [78]Merino M, Alvarez-Fraga L, Gómez MJ, Aransay AM, Lavín JL, Chaves F, Bou G, Poza M. Complete genome sequence of the multiresistant Acinetobacter baumannii strain AbH12O-A2, isolated during a large outbreak in Spain. Genome Announc. 2014; 2(6):e01182-14.
• [79]Fernández-Puente P, Mateos J, Fernández-Costa C, Oreiro N, Fernández-López C, Ruiz-Romero C, Blanco FJ. Identification of a panel of novel serum osteoarthritis biomarkers. J Proteome Res. 2011; 10:5095-5101.
• [80]Wu J, Kobayashi M, Sousa EA, Liu W, Cai J, Goldman SJ, Dorner AJ, Projan SJ, Kavuru MS, Yongchang Qiu Y, Thomassen MJ. Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge. Mol Cell Proteomics. 2005; 4:1251-64.
• [81]Crowther JE, Kutala VK, Kuppusamy P, Ferguson JS, Beharka AA, Zweier JL, McCormack FX, Schlesinger LS. Pulmonary surfactant protein a inhibits macrophage reactive oxygen intermediate production in response to stimuli by reducing NADPH oxidase activity. J Immunol. 2004; 172:6866-74.
• [82]Polati R, Castagna A, Bossi A, Campostrini N, Zaninotto F, Timperio AM, Zolla L, Olivieri O, Corrocher R, Girelli D. High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics. Proteome Sci. 2009; 7:4. BioMed Central Full Text