Chi-Won Choi

Acinetobacter baumannii outer membrane protein a modulates the biogenesis of outer membrane vesicles

Acinetobacter baumannii secretes outer membrane vesicles (OMVs) during both in vitro and in vivo growth, but the biogenesis mechanism by which A. baumannii produces OMVs remains undefined. Outer membrane protein A of A. baumannii (AbOmpA) is a major protein in the outer membrane and the C-terminus of AbOmpA interacts with diaminopimelate of peptidoglycan. This study investigated the role of AbOmpA in the biogenesis of A. baumannii OMVs. Quantitative and qualitative approaches were used to analyze OMV biogenesis in A. baumannii ATCC 19606T and an isogenic ΔAbOmpA mutant. OMV production was significantly increased in the ΔAbOmpA mutant compared to wild-type bacteria as demonstrated by quantitation of proteins and lipopolysaccharides (LPS) packaged in OMVs. LPS profiles prepared from OMVs from wild-type bacteria and the ΔAbOmpA mutant had identical patterns, but proteomic analysis showed different protein constituents in OMVs from wild-type bacteria compared to the ΔAbOmpA mutant. In conclusion, AbOmpA influences OMV biogenesis by controlling OMV production and protein composition.

Proteomic analysis of outer membrane proteins from Acinetobacter baumannii DU202 in tetracycline stress condition

Acinetobacter baumannii readily developed antimicrobial resistance to clinically available antibiotics. A. baumannii DU202 is a multi-drug resistant strain, and is highly resistant to tetracycline (MIC>1,024 μ/ml). The surface proteome of A. baumannii DU202 in response to the sub-minimal inhibitory concentration (subMIC) of tetracycline was analyzed by 2-DE/MS-MS and 1-DE/LC/MS-MS to understand the pathways that form barriers for tetracycline. Membrane expression of major outer membrane proteins (Omps) was significantly decreased in response to the subMIC of tetracycline. These Omps with sizes of 38, 32, 28, and 21 kDa were identified as OmpA38, OmpA32, CarO, and OmpW, respectively. However, transcription level of these Omps was not significantly changed. 1-DE/LC/MS-MS analysis of secreted proteins showed that OmpA38, CarO, OmpW, and other Omps were increasingly secreted at tetracycline condition. This result suggests that A. baumannii actively regulates the membrane expression and the secretion of Omps to overcome antibiotic stress condition.

Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.

Transcription factor σB plays an important role in the production of extracellular membrane-derived vesicles in Listeria monocytogenes.

Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs) that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs) in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σB is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σB affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σB such as transporters (OpuCA and OpuCC), stress response (Kat), metabolism (LacD), translation (InfC), and cell division protein (FtsZ). Gene Ontology (GO) enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance) and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB) was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σB plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.

Analysis of Streptococcus pneumoniae secreted antigens by immuno-proteomic approach

Streptococcus pneumoniae is an important human pathogen that causes a variety of diseases in both adults and children, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Despite their clinical importance, to date, there have been few proteomic studies of these strains for screening of virulence factors or diagnostic markers. In the present study, secreted proteins (secretome) of Streptococcus pneumoniae strains were enriched using ammonium sulfate precipitation and identified by the shotgun proteomic method using 1-dimensional electrophoresis liquid chromatography-mass spectrometry/mass spectrometry analysis. Characterization of the identified proteins revealed that 17.8% (42) of the secreted proteins possessed signal peptides. Twenty-one secreted proteins belonged to the extracellular group, and 4 secreted proteins belonged to the cell wall group. Well-known virulence factors (PrtA, PspC, PsaA, PbpA, PhtD, AmiA, ZmpB, Eno, and Ply) were included in the secreted protein fraction. Western blotting using antiserum against secreted protein mixtures showed that Gsp-781, Sphtra, NagA, PhtD, ZmpB, and Eno were strongly immunogenic. Our data suggest that the immuno-proteomic approach is a useful method for high-throughput identification of secreted proteins and screening of candidate vaccine antigens or diagnostic markers. Gsp-781 is introduced as a novel secreted antigen of Streptococcus pneumoniae.

Proteomic characterization of the outer membrane vesicle of Pseudomonas putida KT2440.

Outer membrane vesicles (OMVs) are produced by various pathogenic Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. In this study, we isolated OMVs from a representative soil bacterium, Pseudomonas putida KT2440, which has a biodegradative activity toward various aromatic compounds. Proteomic analysis identified the outer membrane proteins (OMPs) OprC, OprD, OprE, OprF, OprH, OprG, and OprW as major components of the OMV of P. putida KT2440. The production of OMVs was dependent on the nutrient availability in the culture media, and the up- or down-regulation of specific OMPs was observed according to the culture conditions. In particular, porins (e.g., benzoate-specific porin, BenF-like porin) and enzymes (e.g., catechol 1,2-dioxygenase, benzoate dioxygenase) for benzoate degradation were uniquely found in OMVs prepared from P. putida KT2440 that were cultured in media containing benzoate as the energy source. OMVs of P. putida KT2440 showed low pathological activity toward cultured cells that originated from human lung cells, which suggests their potential as adjuvants or OMV vaccine carriers. Our results suggest that the protein composition of the OMVs of P. putida KT2440 reflects the characteristics of the total proteome of P. putida KT2440.

Proteomic Characterization of Influenza H5N1 virus-like particles and their protective immunogenicity

Recombinant virus-like particles (VLPs) have been shown to induce protective immunity. Despite their potential significance as promising vaccine candidates, the protein composition of VLPs produced in insect cells has not been well characterized. Here we report a proteomic analysis of influenza VLPs containing hemagglutinin (HA) and matrix M1 proteins from a human isolate of avian influenza H5N1 virus (H5 VLPs) produced in insect cells using the recombinant baculovirus expression system. Comprehensive proteomic analysis of purified H5 VLPs identified viral proteins and 37 additional host-derived proteins, many of which are known to be present in other enveloped viruses. Proteins involved in different cellular structures and functions were found to be present in H5 VLPs including those from the cytoskeleton, translation, chaperone, and metabolism. Immunization with purified H5 VLPs induced protective immunity, which was comparable to the inactivated whole virus containing all viral components. Unpurified H5 VLPs containing excess amounts of noninfluenza soluble proteins also conferred 100% protection against lethal challenge although lower immune responses were induced. These results provide important implications consistent with the idea that VLP production in insect cells may involve similar cellular machinery as other RNA enveloped viruses during synthesis, assembly, trafficking, and budding processes.

Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1

Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LC-MS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1.

A global proteome study of Mycobacterium gilvum PYR-GCK grown on pyrene and glucose reveals the activation of glyoxylate, shikimate and gluconeogenetic pathways through the central carbon metabolism highway

Various hydrocarbons have been released into the environment as a result of industrialization. An effective way of removing these materials without further environmental contamination is microbial bioremediation. Mycobacterium gilvum PYR-GCK, a bacteria isolated from a PAH polluted estuary, was studied using comparative shotgun proteomics to gain insight on its molecular activity while using pyrene and glucose as sole carbon and energy sources. Based on annotated genomic information, a confirmation analysis was first performed to confirm its pyrene degradation activity, using gas chromatography–mass spectrometry technology. One dimensional gel electrophoresis and liquid chromatography–mass spectrometry technologies employed in the proteomics analysis revealed the expression of pyrene degrading gene products along with upregulated expression of proteins functioning in the glyoxylate and shikimate pathways, in the pyrene-induced cells. The study also revealed the pathway of pyrene degraded intermediates, via partial gluconeogenesis, into the pentose phosphate pathway to produce precursors for nucleotides and amino acids biosynthesis.