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Fulltext Hijacking of the Host's Immune Surveillance Radars by Burkholderia pseudomallei

Mariappan V, Vellasamy KM, Barathan M, Girija ASS, Shankar EM, Vadivelu J

Front Immunol, 2021;12:718719.
PMID: 34456925 DOI: 10.3389/fimmu.2021.718719

Burkholderia pseudomallei (B. pseudomallei) causes melioidosis, a potentially fatal disease for which no licensed vaccine is available thus far. The host-pathogen interactions in B. pseudomallei infection largely remain the tip of the iceberg. The pathological manifestations are protean ranging from acute to chronic involving one or more visceral organs leading to septic shock, especially in individuals with underlying conditions similar to COVID-19. Pathogenesis is attributed to the intracellular ability of the bacterium to 'step into' the host cell's cytoplasm from the endocytotic vacuole, where it appears to polymerize actin filaments to spread across cells in the closer vicinity. B. pseudomallei effectively evades the host's surveillance armory to remain latent for prolonged duration also causing relapses despite antimicrobial therapy. Therefore, eradication of intracellular B. pseudomallei is highly dependent on robust cellular immune responses. However, it remains ambiguous why certain individuals in endemic areas experience asymptomatic seroconversion, whereas others succumb to sepsis-associated sequelae. Here, we propose key insights on how the host's surveillance radars get commandeered by B. pseudomallei.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Identification of immunoreactive secretory proteins from the stationary phase culture of Burkholderia pseudomallei

Vellasamy KM, Mariappan V, Hashim OH, Vadivelu J

Electrophoresis, 2011 Jan;32(2):310-20.
PMID: 21254130 DOI: 10.1002/elps.201000355

Bacterial secreted proteins are known to be involved in virulence and may mediate important host-pathogen interactions. In this study, when the stationary phase culture supernatant of Burkholderia pseudomallei was subjected to 2-DE, 113 protein spots were detected. Fifty-four of the secreted proteins, which included metabolic enzymes, transcription/translation regulators, potential virulence factors, chaperones, transport regulators, and hypothetical proteins, were identified using MS and database search. Twelve of these proteins were apparently reactive to antisera of mice that were immunised with B. pseudomallei secreted proteins. These proteins might be excellent candidates to be used as diagnostic markers or putative candidate vaccines against B. pseudomallei infections.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Burkholderia thailandensis whole cell antigen cross-reacts with B. pseudomallei antibodies from patients with melioidosis in an immunofluorescent assay

Puthucheary SD, Anuar AS, Tee TS

Southeast Asian J. Trop. Med. Public Health, 2010 Mar;41(2):395-400.
PMID: 20578523

An immunofluorescent assay (IFAT) using whole cell antigen derived from Burkholderia thailandensis used for detection of total antibodies to Burkholderia pseudomallei, was found to compare favorably with a previous published report on a B. pseudomallei IFAT assay. At a 1:20 cut-off titer, the assay had high sensitivity (98.9%) and satisfactory specificity (92.3%), when tested against sera from 94 patients suspected of melioidosis. Sera from 12 patients with culture proven melioidosis gave absolute concordance with the 2 test antigens. No sera from 50 blood donors had a titer of > or =20. Cross-reactivity with patients' sera positive for Chlamydia, Mycoplasma, Legionella and typhoid was not observed, except for 3 sera from typhus patients and one from a patient with leptospirosis. The major advantage of this assay is that the cultivation and preparation of B. thailandensis as antigen can be carried out in any laboratory with basic microbiological set-up. The serodiagnosis of melioidosis can be made safe for medical laboratory personnel, particularly in B. pseudomallei endemic regions.

Matched MeSH terms: Burkholderia pseudomallei/immunology
Fulltext Evaluation of recombinant antigens for diagnosis of melioidosis

Allwood EM, Logue CA, Hafner GJ, Ketheesan N, Norton RE, Peak IR, et al.

FEMS Immunol. Med. Microbiol., 2008 Oct;54(1):144-53.
PMID: 18657105 DOI: 10.1111/j.1574-695X.2008.00464.x

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 h after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days. This delayed diagnosis is a major contributing factor to high mortality rates. Rapid diagnosis is vital for successful management of the disease. This study describes the purification and evaluation of three recombinant antigenic proteins, BPSL0972, BipD and OmpA from B. pseudomallei 08, for their potential in the serodiagnosis of melioidosis using an indirect enzyme-linked immunosorbent assay (ELISA) method. The recombinant proteins were evaluated using 74 serum samples from culture-confirmed melioidosis patients from Malaysia, Thailand and Australia. In addition, 62 nonmelioidosis controls consisting of serum samples from clinically suspected melioidosis patients (n=20) and from healthy blood donors from an endemic region (n=18) and a nonendemic region (n=24) were included. The indirect ELISAs using BipD and BPSL0972 as antigens demonstrated poor to moderate sensitivities (42% and 51%, respectively) but good specificity (both 100%). In contrast, the indirect ELISA using OmpA as an antigen achieved 95% sensitivity and 98% specificity. These results highlight the potential for OmpA to be used in the serodiagnosis of melioidosis in an endemic area.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin

Su YC, Lim KP, Nathan S

J. Biochem. Mol. Biol., 2003 Sep 30;36(5):493-8.
PMID: 14536033

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
An immunohistochemical method for the diagnosis of melioidosis

Wong KT, Vadivelu J, Puthucheary SD, Tan KL

Pathology, 1996 May;28(2):188-91.
PMID: 8743829

In order to assess the usefulness of immunohistochemistry in the diagnosis of melioidosis, an infection by Burkholderia pseudomallei, polyclonal antibodies were applied to tissues from known cases of melioidosis and to other infected tissues. Formalin-fixed, paraffin-embedded tissues were stained by a modified immunoperoxidase technique. In autopsy tissues with inflammatory lesions of melioidosis, the cytoplasm of phagocytes and intact bacilli, both intra- and extracellular, were stained very strongly positive. Relatively more focal positive staining was observed in some but not all surgical biopsies from proven cases of melioidosis. In granulomas staining was mainly found in the central necrotic areas, with little staining of peripheral phagocytes. All control materials stained negative. Immunohistochemistry appears to be a useful diagnostic tool in melioidosis.

Matched MeSH terms: Burkholderia pseudomallei/immunology
Distribution of immunoglobulin classes and IgG subclasses against a culture filtrate antigen of Burkholderia pseudomallei in melioidosis patients

Chenthamarakshan V, Kumutha MV, Vadivelu J, Puthucheary SD

J Med Microbiol, 2001 Jan;50(1):55-61.
PMID: 11192506 DOI: 10.1099/0022-1317-50-1-55

The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups. The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups. However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7. The IgA response was intermediate with GMTIs ranging from 119.2 to 170. The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections. As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts. None of the sera from the control groups had any significant titres of antibodies.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Fulltext Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis

Puah SM, Puthucheary SD, Chua KH

Int J Med Sci, 2013;10(5):539-47.
PMID: 23532805 DOI: 10.7150/ijms.5516

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Fulltext Multiple-antigen ELISA for melioidosis--a novel approach to the improved serodiagnosis of melioidosis

Hara Y, Chin CY, Mohamed R, Puthucheary SD, Nathan S

BMC Infect Dis, 2013;13:165.
PMID: 23556548 DOI: 10.1186/1471-2334-13-165

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of disease are diverse, ranging from chronic infection to acute septicaemia. The current gold standard of diagnosis involves bacterial culture and identification which is time consuming and often too late for early medical intervention. Hence, rapid diagnosis of melioidosis is crucial for the successful management of melioidosis.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Immunization with the recombinant Burkholderia pseudomallei outer membrane protein Omp85 induces protective immunity in mice

Su YC, Wan KL, Mohamed R, Nathan S

Vaccine, 2010 Jul 12;28(31):5005-11.
PMID: 20546831 DOI: 10.1016/j.vaccine.2010.05.022

Burkholderia pseudomallei is resistant to a wide range of antibiotics, leading to relapse and recrudescence of melioidosis after cessation of antibiotic therapy. More effective immunotherapies are needed for better management of melioidosis. We evaluated the prophylactic potential of the immunogenic outer membrane protein Omp85 as a vaccine against murine melioidosis. Immunization of BALB/c mice with recombinant Omp85 (rOmp85) triggered a Th2-type immune response. Up to 70% of the immunized animals were protected against infectious challenge of B. pseudomallei with reduced bacterial load in extrapulmonary organs. Mouse anti-rOmp85 promoted complement-mediated killing and opsonophagocytosis of B. pseudomallei by human polymorphonuclear cells. In conclusion, we demonstrated that B. pseudomallei Omp85 is potentially able to induce protective immunity against melioidosis.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Fulltext Immunogenic Burkholderia pseudomallei outer membrane proteins as potential candidate vaccine targets

Hara Y, Mohamed R, Nathan S

PLoS One, 2009 Aug 05;4(8):e6496.
PMID: 19654871 DOI: 10.1371/journal.pone.0006496

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium's surface and secreted proteins are currently being evaluated as vaccine candidates.
METHODOLOGY/PRINCIPAL FINDINGS: With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1x10(6) colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.
CONCLUSIONS/SIGNIFICANCE: We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics

Chan SW, Nathan S

FEMS Immunol. Med. Microbiol., 2005 Jan 1;43(1):37-44.
PMID: 15607634

Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.

Matched MeSH terms: Burkholderia pseudomallei/immunology
Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei

Chenthamarakshan V, Vadivelu J, Puthucheary SD

Diagn Microbiol Infect Dis, 2001 Jan;39(1):1-7.
PMID: 11173184

IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Diagnostic and prognostic value of an immunofluorescent assay for melioidosis

Vadivelu J, Puthucheary SD

Am J Trop Med Hyg, 2000 Feb;62(2):297-300.
PMID: 10813488

Melioidosis caused by Burkholderia pseudomallei is endemic in southeast Asia. The clinical manifestations range from wound infections to acute septicemia. In some cases, recurrence can also occur following complete recovery. Case fatality rates are high and a major factor is the delay in the culture and identification of the bacterium. An immunofluorescent assay (IFAT) using whole-cell antigen for the detection of total antibodies to B. pseudomallei was tested with 650 sera. Using a cut-off value of 1:80, 66 sera from culture-confirmed cases were positive with titers > or = 320. In another 523 sera from patients in which no other etiology could be found, 149 (23.4%) were positive. To monitor disease activity, persistence of antibody levels was investigated on 61 serial sera samples collected from 14 other confirmed cases on follow-up visits while on oral maintenance therapy. The IFAT demonstrated a reduction in titers in cases of localized infections, suggesting that either the infection was being resolved or arrested while septicemic patients maintained high IFAT titers on follow-up, suggesting the possibility of continuous sequestration of antigen from an intracellular source.

Matched MeSH terms: Burkholderia pseudomallei/immunology
Fulltext Indirect hemagglutination antibodies against Burkholderia pseudomallei in normal blood donors and suspected cases of melioidosis in Malaysia

Norazah A, Rohani MY, Chang PT, Kamel AG

Southeast Asian J. Trop. Med. Public Health, 1996 Jun;27(2):263-6.
PMID: 9279987

Interpretation of the indirect hemagglutination test (IHA) for melioidosis in endemic areas is difficult because of the presence of antibodies in apparently healthy individuals. Fifty-three out of 200 healthy blood donors in Malaysia showed positive antibody titers (> or = 1 : 40) against Burkholderia pseudomallei. Seven percent had an IHA titer of 1 : 40, 11% had an IHA titer of 1 : 80 while 8.5% had a titer > or = 1 : 160. Out of 258 sera sent for melioidosis serology, 7% of the patients had an IHA titer of 1 : 40, 9% had an IHA titer of 1 : 80 while 20% had an IHA titer of > or = 1 : 160. If a titer of > or = 1 : 80 is taken as cut off point for positivity, 29% of the patients had positive melioidosis serology. Increasing the positivity threshold may jeopardize the sensitivity of the test. A more specific and sensitive test is needed.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Fulltext Immunogenic recombinant Burkholderia pseudomallei MprA serine protease elicits protective immunity in mice

Chin CY, Tan SC, Nathan S

Front Cell Infect Microbiol, 2012;2:85.
PMID: 22919676 DOI: 10.3389/fcimb.2012.00085

Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG(1), proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis.

Matched MeSH terms: Burkholderia pseudomallei/immunology
A genome level survey of Burkholderia pseudomallei immunome expressed during human infection

Su YC, Wan KL, Mohamed R, Nathan S

Microbes Infect., 2008 Oct;10(12-13):1335-45.
PMID: 18761419 DOI: 10.1016/j.micinf.2008.07.034

Burkholderia pseudomallei is the etiological agent of melioidosis, a severe infectious disease of humans and animals. The role of the bacterium's proteins expressed in vivo during human melioidosis continues to remain an enigma. This study's aim was to identify B. pseudomallei target proteins that elicit the humoral immune response in infected humans. A small insert genomic expression library was constructed and immunoscreened to identify peptides that reacted exclusively with melioidosis patients' sera. Sero-positive clones expressing immunogenic peptides were sequenced and annotated, and shown to represent 109 proteins involved in bacterial cell envelope biogenesis, cell motility and secretion, transcription, amino acid, ion and protein metabolism, energy production, DNA repair and unknown hypothetical proteins. Western blot analysis of three randomly selected full-length immunogenic polypeptides with patients' sera verified the findings of the immunome screening. The patients' humoral immune response to the 109 proteins suggests the induction or significant upregulation of these proteins in vivo during human infection and thus may play a role in the pathogenesis of B. pseudomallei. Identification of B. pseudomallei immunogens has shed new light on the elucidation of the bacterium's pathogenesis mechanism and disease severity. These immunogens can be further evaluated as prophylactic and serodiagnostic candidates as well as drug targets.

Matched MeSH terms: Burkholderia pseudomallei/immunology
Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

Chan SW, Ong GI, Nathan S

J. Biochem. Mol. Biol., 2004 Sep 30;37(5):556-64.
PMID: 15479619

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin

Nathan S, Li H, Mohamed R, Embi N

J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):45-53.
PMID: 12186782

We have used the phagemid pComb3H to construct recombinant phages displaying the single chain variable fragment (ScFv) towards exotoxin of Burkholderia pseudomallei. Variable heavy and light chain fragments were amplified from the hybridoma 6E6A8F3B line, with a wide spectrum of primers specific to mouse antibody genes. Through overlapping extension polymerase chain reaction, the heavy and light chain fragments were linked to form the ScFv which was subsequently cloned into the phage display vector and transformed into ER2537 cells to yield a complexity of 10(8) clones. The transformants were screened by four rounds of biopanning against the exotoxin and resulted in selective enrichment of exotoxin-binding antibodies by 301 fold. The phage pool from the final round of selection displayed antibodies of high-affinity to the exotoxin as demonstrated by ELISA. Several clones were selected randomly from this pool and analysed by restriction enzyme digestion, fingerprinting and sequencing. Restriction analysis confirmed that all clones carried a 700-800 bp insert whose sequences, in general, corresponded to that of mouse IgG. Fingerprinting profiles delineated the antibodies into two families with different CDR sequences.

Matched MeSH terms: Burkholderia pseudomallei/immunology*
Fulltext Regulatory role of GSK3β in the activation of NF-κB and modulation of cytokine levels in Burkholderia pseudomallei-infected PBMC isolated from streptozotocin-induced diabetic animals

Maniam P, Nurul Aiezzah Z, Mohamed R, Embi N, Hasidah MS

Trop Biomed, 2015 Mar;32(1):36-48.
PMID: 25801253

Increased susceptibility of diabetics to melioidosis, a disease caused by the Burkholderia pseudomallei bacterium is believed to be attributed to dysfunction of the innate immune system. However, the underlying mechanism of the innate susceptibility is not well-understood. Glycogen synthase kinase-3β (GSK3β) plays an important role in the innate inflammatory response caused by bacterial pathogens. The present study was conducted to investigate the effects of GSK3β inhibition by LiCl on levels of pro- and anti-inflammatory cytokines; and the activity of transcription factor NF-κB in B. pseudomallei-infected peripheral blood mononuclear cells (PBMC) derived from diabetic-induced and normal Sprague Dawley rats. In addition, the effects of LiCl on intracellular bacterial counts were also investigated. Infection of PBMC from diabetic and normal rats with B. pseudomallei resulted in elevated levels of cytokines (TNF-α, IL-12 and IL-10) and phosphorylation of NF-κB in both cell types. Intracellular bacterial counts decreased with time in both cell types during infection. However bacterial clearance was less prominent in diabetic PBMC. Burkholderia pseudomallei infection also caused inactivation (Ser9 phosphorylation) of GSK3β in normal PBMC, an effect absent in infected diabetic PBMC. Inhibition of GSK3β by LiCl lowered the levels of pro-inflammatory cytokines (TNF-α and IL-12) in both normal and diabetic PBMC. Similarly, phosphorylated NF- κB (pNF-κB) levels in both cell types were decreased with LiCl treatment. Also, LiCl was able to significantly decrease the intracellular bacterial count in normal as well as diabetic PBMC. Interestingly, the levels of anti-inflammatory cytokine IL-10 in both normal and diabetic PBMC were further elevated with GSK3β inhibition. More importantly, GSK3β in infected diabetic PBMC was inactivated as in their non-diabetic counterparts upon LiCl treatment. Taken together, our results suggest that inhibition of dysregulated GSK3β in diabetic PBMC resulted in the inactivation of NF-κB and modulation of inflammatory cytokine levels. This is evidence that dysregulation of GSK3β is a contributing factor in the molecular basis of innate dysfunction and susceptibility of diabetic host to melioidosis infection.

Matched MeSH terms: Burkholderia pseudomallei/immunology*

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