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Outer membrane proteome analysis of Indian strain of Pasteurella multocida serotype B:2 by MALDI-TOF/MS analysis.

ABSTRACT: Identification of outer membrane proteins (OMPs) is important to understand the bacteria structure and function, host-pathogen interaction, development of novel vaccine candidates, and diagnostic antigens. But till now the key antigens of P. multocida B:2 isolate causing haemorrhagic septicaemia (HS) in animals are not clearly defined. In this study, P52 strain of P. multocida serotype B:2 was grown in vitro under iron-rich and iron-limited condition. The OMPs were extracted by sarkosyl method followed by SDS-PAGE and the proteins were identified by MALDI-TOF/MS analysis. In total, 22 proteins were identified, of which 7 were observed exclusively under iron-limited condition. Most of the high molecular weight proteins (TbpA, HgbA, HgbB, HasR, IroA, and HemR) identified in this study were involved in iron acquisition. Some hypothetical proteins (HP-KCU-10206, HP and AAUPMB 08244, HP AAUPMB 21592, HP AAUPMB 19766, AAUPMB 11295) were observed for the first time in this study which could be unique to serotype B:2. Further functional in vivo study of the proteins identified are required to explore the utility of these proteins in developing diagnostics and vaccine against HS.

SUBMITTER: Prasannavadhana A

PROVIDER: S-EPMC4283227 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Outer membrane proteome analysis of Indian strain of Pasteurella multocida serotype B:2 by MALDI-TOF/MS analysis.

Prasannavadhana A A Kumar Santosh S Thomas Prasad P Sarangi Laxmi Narayan LN Gupta Santosh Kumar SK Priyadarshini Adyasha A Nagaleekar Viswas Konasagara VK Singh Vijendra Pal VP

TheScientificWorldJournal 20141222

Identification of outer membrane proteins (OMPs) is important to understand the bacteria structure and function, host-pathogen interaction, development of novel vaccine candidates, and diagnostic antigens. But till now the key antigens of P. multocida B:2 isolate causing haemorrhagic septicaemia (HS) in animals are not clearly defined. In this study, P52 strain of P. multocida serotype B:2 was grown in vitro under iron-rich and iron-limited condition. The OMPs were extracted by sarkosyl method f ...[more]

PMID: 25587569

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Project description:BackgroundOuter membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome.ResultsIn the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane β-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane β-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes.ConclusionsThe bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria.

Project description:Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

Project description:MALDI-TOF mass spectrometry (MS) on whole cells enables the detection of different cell types and cell activation. Here, we wondered whether this approach would be useful to investigate the host response in sepsis.Peripheral blood mononuclear cells (PBMCs) from patients with severe sepsis and healthy donors were analyzed with MALDI-TOF MS. PBMCs from healthy donors were also stimulated with lipopolysaccharide, peptidoglycan, CpG oligonucleotides, polyinosinic polycytidylic acid, and with heat-inactivated bacteria. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software and matching scores between each spectrum from patients and averaged spectra from the database were calculated.We show that the MALDI-TOF MS signature of PBMCs from septic patients was specific, compared with healthy controls. As the fingerprints observed in patients may be related to PBMC activation, PBMCs from healthy controls were stimulated with cytokines, soluble Pathogen-Associated Molecular Patterns (PAMPs) and heat-killed bacteria, and we created a database of reference spectra. The MALDI-TOF MS profiles of PBMCs from septic patients were then compared with the database. No differences were found between patients with documented infection (n?=?6) and those without bacteriological documentation (n?=?6). The spectra of PBMCs from septic patients matched with those of interferon-?- and interleukin-10-stimulated PBMCs, confirming that sepsis is characterized by both inflammatory and immunoregulatory features. Interestingly, the spectra of PBMCs from septic patients without documented infection matched with the reference spectrum of PBMCs stimulated by CpG oligonucleotides, suggesting a bacterial etiology in these patients.Despite the limits of this preliminary study, these results indicate that the monitoring of functional status of PBMCs in peripheral blood by whole cell MALDI-TOF MS could provide unique opportunities to assess disease progression or resolution in clinical settings.

Dataset Information

Outer membrane proteome analysis of Indian strain of Pasteurella multocida serotype B:2 by MALDI-TOF/MS analysis.

Publications

Outer membrane proteome analysis of Indian strain of Pasteurella multocida serotype B:2 by MALDI-TOF/MS analysis.

Similar Datasets

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