Overexpression, Purification and Validation of Antigenic Salmonella enterica Serovar Typhi Proteins Identified from LC-MS/MS

Chin, Chai Fung; Teh, Boon Aun; Anthony, Amy Amilda; Ismail, Asma; Ong, Eugene Boon Beng; Lim, Theam Soon

doi:10.1007/s12010-014-1173-y

Cited by 4 publications

(3 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…ANTIGENpro has been successfully used to identify antigenic proteins in many pathogens including Salmonella, Leishmania, and Corynebacterium spp. 29,30 . In addition, in vitro transcription and translation (IVTT) is a useful high-throughput approach to screen a variety of proteins for functional studies, since this system can rapidly produce soluble protein in native conformation and overcome difficulties often encountered with protein expression in cell-based systems 31,32 .…”

Section: Introductionmentioning

confidence: 99%

Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

et al. 2020

View full text Add to dashboard Cite

Immunomolecular characterization of Ehrlichia chaffeensis (E. ch.) and E. canis (E. ca.) has defined protein orthologs, including tandem repeat proteins (TRPs) that have immunodominant linear antibody epitopes. In this study, we combined bioinformatic analysis and cell-free protein expression to identify undiscovered immunoreactive E. ch. and E. ca. hypothetical proteins. Antigenicity of the E. ch. and E. ca. ORFeomes (n = 1105 and n = 925, respectively) was analyzed by the sequence-based prediction model ANTIGENpro, and we identified ~250 ORFs in each respective ORFeome as highly antigenic. The hypothetical proteins (E. ch. n = 93 and E. ca. n = 98) present in the top 250 antigenic ORFs were further investigated in this study. By ELISA, 46 E. ch. and 30 E. ca. IVTT-expressed hypothetical proteins reacted with antibodies in sera from naturally E. ch.-infected patients or E. ca.-infected dogs. Moreover, 15 E. ch. and 16 E. ca. proteins consistently reacted with a panel of sera from patients or dogs, including many that revealed the immunoreactivity of “gold standard” TRPs. Antibody epitopes in most (>70%) of these proteins exhibited partial or complete conformation-dependence. The majority (23/31; 74%) of the major immunoreactive proteins identified were small (≤250 aa), and 20/31 (65%) were predicted to be secreted effectors. Unlike the strong linear antibody epitopes previously identified in TRP and OMP orthologs, there were contrasting differences in the E. ch. and E. ca. antigenic repertoires, epitopes and ortholog immunoreactivity. This study reveals numerous previously undefined immunodominant and subdominant antigens, and illustrates the breadth, complexity, and diversity of immunoreactive proteins/epitopes in Ehrlichia.

show abstract

Section: Introductionmentioning

confidence: 99%

Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

et al. 2020

View full text Add to dashboard Cite

show abstract

“…2b ), indicating that the L2 loop might have the specificity to play an important role in antibody recognition and be the immuno-dominant site in ST50. In addition, according to ELISA experiments in previous work 27 , denatured ST50 possesses reactivity against sera of typhoid patients, indicating that the discontinuous amino acid sequences of ST50 are less crucial for antibody binding, whereas the linear amino acid sequence of ST50 might serve as an epitope for paratope binding. These analyses suggest that the linear amino acid sequence of L2 in ST50 might be a primary candidate for the development of an optimized diagnostic antigen or a new drug target for typhoid fever.…”

Section: Discussionmentioning

confidence: 84%

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

Guan

Yoshimura

Chuankhayan

et al. 2015

Sci Rep

Self Cite

View full text Add to dashboard Cite

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOTTM). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

show abstract

“…ANTIGENPro provides an accuracy of up to 82%, which surpasses other antigenicity predictors (Magnan et al, 2010). In addition, antigenic proteins in other pathogens were detected by this program (Chin et al, 2014;Magnan et al, 2010). ANTIGENpro server scores 0.816380 for Pal, 0.900650 for LptD, 0.779916 for Wza, and 0.804137 for TbpA.…”

Section: Protein Antigenicity Prediction and Comparisonmentioning

confidence: 99%

Characterisation of the Putative Antigenic Genes of the Outer Membrane Proteins of Pasteurella multocida B:2 Strain PMTB2.1 through in silico Analysis

Hashimi¹,

Joyce²,

Nasir³

et al. 2023

JTAS

View full text Add to dashboard Cite

Outer membrane proteins (OMPs), usually found in Gram-negative bacteria, have long been shown to elicit immune responses in infected hosts. This tendency of OMPs to generate immune reactions makes them ideal candidates for vaccine development against pathogenic bacteria. Pasteurella multocida is a Gram-negative pathogen responsible for the economically significant veterinary disease, hemorrhagic septicemia (HS). HS is an endemic and highly fatal disease affecting buffaloes and cattle. In Malaysia, outbreaks of this disease cost about half a million USD each year. Thus, despite current treatment and prevention measures, HS is a prevalent issue that needs to be overcome. Pasteurella multocida subsp. multocida PMTB2.1, a Malaysian strain of the pathogen, has recently had its entire genome sequenced after being isolated from HS outbreaks in the region. Antigenic OMPs from this strain have since been identified and published for further characterisation. LptD, Wza, and TbpA are integral membrane proteins, while Pal is a peripheral membrane protein that has not been characterised in-depth. This study, therefore, aims to analyse these OMPs through in silico methods. First, protein homology modelling was performed using SWISS-MODEL, whereafter, the structures generated were validated using the SWISS-MODEL structure assessment page, PROCHECK, ERRAT, and PROSA programs. The Pal, Wza, and TbpA structures were good models, while the LptD structure was found to be a near-good model based on the validation performed. Analyses using BCPREDS, NetMHCpan4.1, and NetBoLAIIpan1.0 revealed that these four OMPs could potentially elicit humoral and cellular immune responses.

show abstract

Overexpression, Purification and Validation of Antigenic Salmonella enterica Serovar Typhi Proteins Identified from LC-MS/MS

Cited by 4 publications

References 22 publications

Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

Ehrlichia chaffeensis and E. canis hypothetical protein immunoanalysis reveals small secreted immunodominant proteins and conformation-dependent antibody epitopes

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

Characterisation of the Putative Antigenic Genes of the Outer Membrane Proteins of Pasteurella multocida B:2 Strain PMTB2.1 through in silico Analysis

Contact Info

Product

Resources

About