Displaying all 12 publications

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  1. Fulltext Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine)
    Mustafa AD, Kalyanasundram J, Sabidi S, Song AA, Abdullah M, Abdul Rahim R, et al.
    BMC Biotechnol, 2019 05 14;19(1):27.
    PMID: 31088425 DOI: 10.1186/s12896-019-0522-x
    BACKGROUND: The current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated.

    RESULTS: Expression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days).

    CONCLUSION: The presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

    Matched MeSH terms: Antigens, Bacterial/metabolism*
  2. Intrabacterial proton-dependent CagA transport system in Helicobacter pylori
    Wu H, Nakano T, Daikoku E, Morita C, Kohno T, Lian HH, et al.
    J Med Microbiol, 2005 Dec;54(Pt 12):1117-1125.
    PMID: 16278423 DOI: 10.1099/jmm.0.46158-0
    Helicobacter pylori CagA modifies the signalling of host cells and causes gastric diseases. Although CagA is injected into gastric epithelial cells through the type IV secretion machinery, it remains unclear how CagA is transported towards the machinery in the bacterial cytoplasm. In this study, it was determined that the proton-dependent intracytoplasmic transport system correlates with the priming of CagA secretion from H. pylori. The cytotoxicity of neutral-pH- and acidic-pH-treated H. pylori was examined in the AGS cell line. The amount of phosphorylated CagA in AGS cells incubated with acidic-pH- and neutral-pH-treated H. pylori was determined by enzyme immunoassay and Western blot. The production of CagA and adherence of the treated bacteria were examined by enzyme immunoassay and light microscopy, respectively. To clarify how CagA is transported towards the inner membrane of the treated bacteria, the localization of CagA was analysed by immunoelectron microscopy. The proportion of hummingbird cells in the AGS cell line rapidly increased following the inoculation of acidic-pH-treated H. pylori but increased more slowly with neutral-pH-treated H. pylori, and the phenomenon correlated with the amount of phosphorylated CagA in AGS cells. CagA was densely localized near the inner membrane in the acidic-pH-treated bacterial cytoplasm, but this localization was not observed in the neutral-pH-treated bacterial cytoplasm, suggesting that CagA shifts from the centre to the peripheral portion of the cytoplasm as a result of an extracellular decrease in pH. This phenomenon depended on the presence of UreI, a proton-dependent urea channel, but not on the presence of urea. The pH treatments did not enhance CagA production or the adherence of the bacterium to AGS cells. The authors propose that H. pylori possesses a proton-dependent intracytoplasmic transport system that probably accelerates priming for CagA injection.
    Matched MeSH terms: Antigens, Bacterial/metabolism*
  3. Fulltext Variant of Helicobacter pylori CagA proteins induce different magnitude of morphological changes in gastric epithelial cells
    Alfizah H, Ramelah M
    Malays J Pathol, 2012 Jun;34(1):29-34.
    PMID: 22870595 MyJurnal
    Infection with Helicobacter pylori cagA-positive strains is associated with gastroduodenal diseases. The CagA protein is injected into gastric epithelial cells and supposedly induces morphological changes termed the 'hummingbird phenotype', which is associated with scattering and increased cell motility. The molecular mechanisms leading to the CagA-dependent morphological changes are only partially known. The present study was carried out to investigate the effect of CagA variants on the magnitude of gastric epithelial cell morphological changes. Recombinant 3' terminal domains of cagA were cloned and expressed in a gastric epithelial cell line and the hummingbird phenotype was quantified by microscopy. The 3' region of the cagA gene of Malaysian H. pylori isolates showed six sub-genotypes that differed in the structural organization of the EPIYA repeat sequences. The percentage of hummingbird cells induced by CagA increased with duration of transfection. The hummingbird phenotype was observed to be more pronounced when CagA with 4 EPIYA motifs rather than 3 or 2 EPIYA motifs was produced. The activity of different CagA variants in the induction of the hummingbird phenotype in gastric epithelial cells depends at least in part on EPIYA motif variability. The difference in CagA genotypes might influence the potential of individual CagAs to cause morphological changes in host cells. Depending on the relative exposure of cells to CagA genotypes, this may contribute to the various disease outcomes caused by H. pylori infection in different individuals.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  4. Distinct cagA EPIYA motifs are associated with ethnic diversity in Malaysia and Singapore
    Schmidt HM, Goh KL, Fock KM, Hilmi I, Dhamodaran S, Forman D, et al.
    Helicobacter, 2009 Aug;14(4):256-63.
    PMID: 19674129 DOI: 10.1111/j.1523-5378.2009.00684.x
    In vitro studies have shown that the biologic activity of CagA is influenced by the number and class of EPIYA motifs present in its variable region as these motifs correspond to the CagA phosphorylation sites. It has been hypothesized that strains possessing specific combinations of these motifs may be responsible for gastric cancer development. This study investigated the prevalence of cagA and the EPIYA motifs with regard to number, class, and patterns in strains from the three major ethnic groups within the Malaysian and Singaporean populations in relation to disease development.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  5. The cag PAI is intact and functional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore
    Schmidt HM, Andres S, Nilsson C, Kovach Z, Kaakoush NO, Engstrand L, et al.
    Eur J Clin Microbiol Infect Dis, 2010 Apr;29(4):439-51.
    PMID: 20157752 DOI: 10.1007/s10096-010-0881-7
    Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  6. Fulltext Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism
    Guan HH, Yoshimura M, Chuankhayan P, Lin CC, Chen NC, Yang MC, et al.
    Sci Rep, 2015 Nov 13;5:16441.
    PMID: 26563565 DOI: 10.1038/srep16441
    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 (TYPHIDOT(TM)). 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.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  7. Fulltext Helicobacter pylori Virulence Factor Cytotoxin-Associated Gene A (CagA)-Mediated Gastric Pathogenicity
    Ansari S, Yamaoka Y
    Int J Mol Sci, 2020 Oct 08;21(19).
    PMID: 33050101 DOI: 10.3390/ijms21197430
    Helicobacter pylori causes persistent infection in the gastric epithelium of more than half of the world's population, leading to the development of severe complications such as peptic ulcer diseases, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Several virulence factors, including cytotoxin-associated gene A (CagA), which is translocated into the gastric epithelium via the type 4 secretory system (T4SS), have been indicated to play a vital role in disease development. Although infection with strains harboring the East Asian type of CagA possessing the EPIYA-A, -B, and -D sequences has been found to potentiate cell proliferation and disease pathogenicity, the exact mechanism of CagA involvement in disease severity still remains to be elucidated. Therefore, we discuss the possible role of CagA in gastric pathogenicity.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  8. Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi
    Choong YS, Lim TS, Chew AL, Aziah I, Ismail A
    J Mol Graph Model, 2011 Apr;29(6):834-42.
    PMID: 21371926 DOI: 10.1016/j.jmgm.2011.01.008
    The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  9. Fulltext Investigation on the conA binding properties of Klebsiella pneumoniae
    Anuar AS, Tay ST
    Trop Biomed, 2014 Dec;31(4):802-12.
    PMID: 25776607 MyJurnal
    Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  10. Fulltext Immunogenicity of recombinant Mycobacterium bovis bacille Calmette-Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens
    Mohamud R, Azlan M, Yero D, Alvarez N, Sarmiento ME, Acosta A, et al.
    BMC Immunol, 2013;14 Suppl 1:S5.
    PMID: 23458635 DOI: 10.1186/1471-2172-14-S1-S5
    Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  11. Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology
    Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F, et al.
    Int J Biol Macromol, 2021 Jan 31;168:289-300.
    PMID: 33310091 DOI: 10.1016/j.ijbiomac.2020.12.062
    Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
    Matched MeSH terms: Antigens, Bacterial/metabolism
  12. Antigenic proteins of Helicobacter pylori of potential diagnostic value
    Khalilpour A, Santhanam A, Wei LC, Saadatnia G, Velusamy N, Osman S, et al.
    Asian Pac J Cancer Prev, 2013;14(3):1635-42.
    PMID: 23679248
    Helicobacter pylori antigen was prepared from an isolate from a patient with a duodenal ulcer. Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcers, gastric ulcers and gastritis (n=30). As controls, three kinds of sera without detectable H. pylori IgG antibodies were used: 30 from healthy individuals without history of gastric disorders, 30 from patients who were seen in the endoscopy clinic but were H. pylori culture negative and 30 from people with other diseases. OFF-GEL electrophoresis, SDS-PAGE and Western blots of individual serum samples were used to identify protein bands with good sensitivity and specificity when probed with the above sera and HRP-conjugated anti-human IgG. Four H. pylori protein bands showed good (≥ 70%) sensitivity and high specificity (98-100%) towards anti-Helicobacter IgG antibody in culture- positive patients sera and control sera, respectively. The identities of the antigenic proteins were elucidated by mass spectrometry. The relative molecular weights and the identities of the proteins, based on MALDI TOF/ TOF, were as follows: CagI (25 kDa), urease G accessory protein (25 kDa), UreB (63 kDa) and proline/pyrroline- 5-carboxylate dehydrogenase (118 KDa). These identified proteins, singly and/or in combinations, may be useful for diagnosis of H. pylori infection in patients.
    Matched MeSH terms: Antigens, Bacterial/metabolism
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