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Burkholderia pseudomallei is the causative agent of the lethal disease melioidosis. This bacterium infects animals and humans and is increasingly resistant to multiple antibiotics. Recently, genes associated with survival of the bacterium in the infected host have been identified. One of these genes, bpsl0741, is annotated as a hypothetical protein of 185 amino acids. Here, recombinant BPSL0741 (rBPSL0741) protein was expressed, purified, verified by mass spectrometry, crystallized and analyzed by X‐ray diffraction. rBPSL0741 was crystallized by vapor diffusion using a reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30% PEG 4000. The crystals diffracted to 2.1 Å resolution using an in‐house X‐ray diffractometer and belonged to an orthorhombic space group, with unit‐cell parameters a = 62.92, b = 64.57, c = 89.16 Å. The Matthews coefficient (VM) was calculated to be 2.18 Å3 Da−1, suggesting the presence of two molecules per asymmetric unit and an estimated solvent content of 43.5%. The crystal was deemed to be suitable for further structural studies, which are currently ongoing. [ABSTRACT FROM AUTHOR]

Introduction: Melioidosis is a deadly endemic disease in northern Australia and Southeast Asia, including Sabah, Malaysia, which is caused by the bacterium Burkholderia pseudomallei. It contributes to high fatality rates, mainly due to misdiagnosis leading to the wrong treatment being administered to the patients. Local epidemiology and data on clinical features could assist clinicians during diagnosis and treatment. However, these details are still scarce, particularly in Sabah. Methods: A retrospective study of 246 culture-confirmed melioidosis cases in Queen Elizabeth Hospital, Sabah, Malaysia was performed between 2016 and 2018. The epidemiological data and clinical and laboratory findings were extracted and analysed. Results: The annual incidence of culture-confirmed melioidosis cases was estimated to be 4.97 per 100,000 people. The mean age of the patients was 50±15 years. Males and members of the Kadazan-Dusun ethnic group accounted for the majority of the melioidosis cases. The odds ratio analysis indicated that bacteraemic melioidosis in this region was significantly associated with fever (76%), and patients having at least one underlying illness (43%), including diabetes mellitus (32%). Sixty-eight patients (28%) succumbed to melioidosis. Contrary to what is known regarding factors that promote bacteraemic melioidosis, neither patients with fever nor patients with at least one comorbid disease, including diabetes mellitus, were significantly associated with death from melioidosis. There was no statistically significant difference between patients without comorbidities (24, 27%) and those with at least one comorbid disease (26, 25%), including diabetes mellitus (18, 23%). The odds ratios indicate that melioidosis mortality in this region is related to patients showing respiratory organ-associated symptoms (29%), bacteraemia (30%), and septic shock (47%). Burkholderia pseudomallei isolates in this study were highly susceptible to ceftazidime (100%), imipenem (100%), and trimethoprim-sulfamethoxazole (98%). Conclusions: Information obtained from this study can be used by clinicians to recognise individuals with the highest risk of acquiring melioidosis, estimate an accurate prognosis, and provide effective treatment for melioidosis patients to reduce death from melioidosis. Author summary: Melioidosis is an infection caused by the bacteria Burkholderia pseudomallei bacterium and is most commonly found in tropical regions such as northern Australia and Southeast Asia, including Sabah, Malaysia. Death due to the disease is often associated with failure in clinical identification, prediction of death, and treatment of the disease. A local database on melioidosis could assist doctors, in particular infectious disease physicians, in overcoming these failures. Doctors in Sabah should have a high clinical suspicion of melioidosis, particularly for male patients, and particularly those of the Kadazan-Dusun ethnic group, who present with fever and have at least one underlying disease, particularly diabetes. Patients with blood infections who develop septic shock or have affected respiratory organs should be prioritised during treatment and management due to their poor prognosis. Melioidosis patients with or without an existing illness, particularly diabetes, should also be equally prioritised to prevent death. In this region, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole remain highly effective antibiotics against B. pseudomallei. This new knowledge may contribute to better melioidosis clinical recognition, prognosis, and treatment of the disease to reduce mortality rates due to melioidosis. [ABSTRACT FROM AUTHOR]

Introduction: Burkholderia pseudomallei, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie B. pseudomallei's versatility to survive within a broad range of environments are still not well defined. Methods: We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria's ability to survive and proliferate in the nematode intestinal lumen. Results: Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice. Discussion: The B. pseudomallei conditional essential proteins should provide further insights into the bacteria's niche adaptation, pathogenesis, and virulence. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters mediate bacteria uptake or export of a variety of solutes across biological membranes. Bacterial uptake of the monosaccharides is important as a source of carbohydrate building blocks that contribute to the bacteria's major structure. Burkholderia pseudomallei is the etiological agent of melioidosis and within its genome, 33 genes related to monosaccharide ABC transporters have been predicted. The presence of these transporters is believed to assist in bacterial survival and adaptation in various environments. Despite a large number of genes in the genome, most of these systems have yet to be characterized, including the bpss0140-bpss0142 operon. Here, we predicted the 3D structure of each protein encoded by bpss0140-0142 and identified the specifically associated monosaccharides. In silico analyses of the structures demonstrated that BPSS0140 is a sugar-binding protein, BPSS0141 is a transmembrane permease and BPSS0142 is an ATPase. Through protein structure modeling and protein-ligand docking, several specific monosaccharide sugars were found to interact with the BPSS0140-BPSS0142 ABC transporter. To validate the in silico prediction, knock-out mutants for each of the genes were constructed. A growth profile between wild-type and mutants in an M9 medium supplemented with glucose, fructose, ribose, and galactose as predicted from the protein-ligand docking was then performed. The growth of mutants decreased significantly compared to the wild-type bacteria when grown in M9 supplemented with fructose as the sole carbon source indicating that this transporter is potentially the main fructose transporter in B. pseudomallei. [ABSTRACT FROM AUTHOR]

Abstract Background The sRNAs of bacterial pathogens are known to be involved in various cellular roles including environmental adaptation as well as regulation of virulence and pathogenicity. It is expected that sRNAs may also have similar functions for Burkholderia pseudomallei, a soil bacterium that can adapt to diverse environmental conditions, which causes the disease melioidosis and is also able to infect a wide variety of hosts. Results By integrating several proven sRNA prediction programs into a computational pipeline, available Burkholderia spp. genomes were screened to identify sRNA gene candidates. Orthologous sRNA candidates were then identified via comparative analysis. From the total prediction, 21 candidates were found to have Rfam homologs. RT-PCR and sequencing of candidate sRNA genes of unknown functions revealed six putative sRNAs which were highly conserved in Burkholderia spp. and two that were unique to B. pseudomallei present in a normal culture conditions transcriptome. The validated sRNAs include potential cis-acting elements associated with the modulation of methionine metabolism and one B. pseudomallei-specific sRNA that is expected to bind to the Hfq protein. Conclusions The use of the pipeline developed in this study and subsequent comparative analysis have successfully aided in the discovery and shortlisting of sRNA gene candidates for validation. This integrated approach identified 29 B. pseudomallei sRNA genes - of which 21 have Rfam homologs and 8 are novel.

Abstract The 2011 International Conference on Bioinformatics (InCoB) conference, which is the annual scientific conference of the Asia-Pacific Bioinformatics Network (APBioNet), is hosted by Kuala Lumpur, Malaysia, is co-organized with the first ISCB-Asia conference of the International Society for Computational Biology (ISCB). InCoB and the sequencing of the human genome are both celebrating their tenth anniversaries and InCoB’s goalposts for the next decade, implementing standards in bioinformatics and globally distributed computational networks, will be discussed and adopted at this conference. Of the 49 manuscripts (selected from 104 submissions) accepted to BMC Genomics and BMC Bioinformatics conference supplements, 24 are featured in this issue, covering software tools, genome/proteome analysis, systems biology (networks, pathways, bioimaging) and drug discovery and design.

Colorectal cancer (CRC) ranks second among the most commonly occurring cancers in Malaysia, and unfortunately, its pathobiology remains unknown. CRC pathobiology can be understood in detail with the implementation of omics technology that is able to generate vast amounts of molecular data. The generation of omics data has introduced a new challenge for data organization. Therefore, a knowledge-based repository, namely TCGA-My, was developed to systematically store and organize CRC omics data for Malaysian patients. TCGA-My stores the genome and metabolome of Malaysian CRC patients. The genome and metabolome datasets were organized using a Python module, pandas. The variants and metabolites were first annotated with their biological information using gene ontologies (GOs) vocabulary. The TCGA-My relational database was then built using HeidiSQL PorTable 9.4.0.512, and Laravel was used to design the web interface. Currently, TCGA-My stores 1,517,841 variants, 23,695 genes, and 167,451 metabolites from the samples of 50 CRC patients. Data entries can be accessed via search and browse menus. TCGA-My aims to offer effective and systematic omics data management, allowing it to become the main resource for Malaysian CRC research, particularly in the context of biomarker identification for precision medicine. [ABSTRACT FROM AUTHOR]

Cancer immunotherapies are preferred over conventional treatments which are highly cytotoxic to normal cells. Focus has been on T cells but natural killer (NK) cells have equal potential. Concepts in cancer control and influence of sex require further investigation to improve successful mobilization of immune cells in cancer patients. Acute lymphoblastic leukemia (ALL) is a hematological malignancy mainly of B cell (B-ALL) and T cell (T-ALL) subtypes. Influence of ALL on NK cell is still unclear. Targeted next-generation sequencing was conducted on 62 activating/inhibitory receptors, ligands, effector, and exhaustion molecules on T-ALL (6 males) and normal controls (NC) (4 males and 4 females). Quantitative PCR (q-PCR) further investigated copy number variation (CNV), methylation index (MI), and mRNA expression of significant genes in T-ALL (14 males), NC (12 males and 12 females), and B-ALL samples (N = 12 males and 12 females). Bioinformatics revealed unique variants particularly rs2253849 (T>C) in KLRC1 and rs1141715 (A>G) in KLRC2 only among T-ALL (allele frequency 0.8-1.0). Gene amplification was highest in female B-ALL compared to male B-ALL (KLRC2, KLRC4, and NCR3, p < 0.05) and lowest in male T-ALL cumulating in deletion of KLRD1 and CD69. MI was higher in male ALL of both subtypes compared to normal (KIR2DL1-2 and 4 and KIR2DS2 and 4, p < 0.05) as well as to female B-ALL (KIR3DL2 and KIR2DS2, p < 0.05). mRNA expressions were low. Thus, ALL subtypes potentially regulated NK cell suppression by different mechanisms which should be considered in future immunotherapies for ALL. [ABSTRACT FROM AUTHOR]

The proficiency of Salmonella Typhi to induce cell-mediated immunity has allowed its exploitation as a live vector against the obligate intracellular protozoan Toxoplasma gondii. T. gondii vaccine research is of great medical value due to the lack of a suitable toxoplasmosis vaccine. In the present work, we integrated T. gondii antigen into a growth-dependent chromosome locus guaBA of S. Typhi CVD910 strain to form recombinant S. Typhi monovalent CVD910-SAG1 expressed T. gondii SAG1 antigen and monovalent CVD910-GRA2 expressed T. gondii GRA2 antigen. Furthermore, a low-copy stabilized recombinant plasmid encoding SAG1 antigen was transformed into CVD910-GRA2 to form bivalent CVD910-GS strain. An osmolarity-regulated promoter was also incorporated to control the gene transcription, whereas clyA export protein was included to translocate the antigen out of the cytoplasm. Both CVD910-GRA2 and CVD910-GS displayed healthy growth fitness and readily expressed the encoded T. gondii antigens. When administered in vivo, CVD910-GS successfully induced both humoral and cellular immunity in the immunized BALB/c mice, and extended mice survival against virulent T. gondii. In particular, the mice immunized with bivalent CVD910-GS presented the highest titers of IgG, percentages of CD4+ T, CD8+ T, B cells and memory T cells, and total IgG+ memory B cells as compared to the CVD910-GRA2 and control strains. The CVD910-GS group also generated mixed Th1/Th2 cytokine profile with secretions of IFN-ɣ, IL-2 and IL-10. This study demonstrated the importance of enhancing live vector fitness to sustain heterologous antigen expression for eliciting robust immune responses and providing effective protection against pathogen. [ABSTRACT FROM AUTHOR]

Abstract Background Burkholderia pseudomallei is a facultative intracellular pathogen of phagocytic and non-phagocytic cells. How the bacterium interacts with host macrophage cells is still not well understood and is critical to appreciate the strategies used by this bacterium to survive and how intracellular survival leads to disease manifestation. Results Here we report the expression profile of intracellular B. pseudomallei following infection of human macrophage-like U937 cells. During intracellular growth over the 6 h infection period, approximately 22 % of the B. pseudomallei genome showed significant transcriptional adaptation. B. pseudomallei adapted rapidly to the intracellular environment by down-regulating numerous genes involved in metabolism, cell envelope, motility, replication, amino acid and ion transport system and regulatory function pathways. Reduced expression in catabolic and housekeeping genes suggested lower energy requirement and growth arrest during macrophage infection, while expression of genes encoding anaerobic metabolism functions were up regulated. However, whilst the type VI secretion system was up regulated, expression of many known virulence factors was not significantly modulated over the 6hours of infection. Conclusions The transcriptome profile described here provides the first comprehensive view of how B. pseudomallei survives within host cells and will help identify potential virulence factors and proteins that are important for the survival and growth of B. pseudomallei within human cells.

Abstract Background At present, very little is known about how Burkholderia pseudomallei (B. pseudomallei) interacts with its host to elicit melioidosis symptoms. We established a murine acute-phase melioidosis model and used DNA microarray technology to investigate the global host/pathogen interaction. We compared the transcriptome of infected liver and spleen with uninfected tissues over an infection period of 42 hr to identify genes whose expression is altered in response to an acute infection. Results Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42 hr course of infection. Microarray analysis of the liver and spleen over this time course demonstrated that genes involved in immune response, stress response, cell cycle regulation, proteasomal degradation, cellular metabolism and signal transduction pathways were differentially regulated. Up regulation of toll-like receptor 2 (TLR2) gene expression suggested that a TLR2-mediated signalling pathway is responsible for recognition and initiation of an inflammatory response to the acute B. pseudomallei infection. Most of the highly elevated inflammatory genes are a cohort of "core host immune response" genes commonly seen in general inflammation infections. Concomitant to this initial inflammatory response, we observed an increase in transcripts associated with cell-death, caspase activation and peptidoglysis that ultimately promote tissue injury in the host. The complement system responsible for restoring host cellular homeostasis and eliminating intracellular bacteria was activated only after 24 hr post-infection. However, at this time point, diverse host nutrient metabolic and cellular pathways including glycolysis, fatty acid metabolism and tricarboxylic acid (TCA) cycle were repressed. Conclusions This detailed picture of the host transcriptional response during acute melioidosis highlights a broad range of innate immune mechanisms that are activated in the host within 24 hrs, including the core immune response commonly seen in general inflammatory infections. Nevertheless, this activation is suppressed at 42 hr post-infection and in addition, suboptimal activation and function of the downstream complement system promotes uncontrolled spread of the bacteria.

Abstract Background Cryptocaryon irritans is a parasitic ciliate that causes cryptocaryonosis (white spot disease) in marine fish. Diagnosis of cryptocaryonosis often depends on the appearance of white spots on the surface of the fish, which are usually visible only during later stages of the disease. Identifying suitable biomarkers of this parasite would aid the development of diagnostic tools and control strategies for C. irritans. The C. irritans genome is virtually unexplored; therefore, we generated and analyzed expressed sequence tags (ESTs) of the parasite to identify genes that encode for surface proteins, excretory/secretory proteins and repeat-containing proteins. Results ESTs were generated from a cDNA library of C. irritans tomonts isolated from infected Asian sea bass, Lates calcarifer. Clustering of the 5356 ESTs produced 2659 unique transcripts (UTs) containing 1989 singletons and 670 consensi. BLAST analysis showed that 74% of the UTs had significant similarity (E-value < 10-5) to sequences that are currently available in the GenBank database, with more than 15% of the significant hits showing unknown function. Forty percent of the UTs had significant similarity to ciliates from the genera Tetrahymena and Paramecium. Comparative gene family analysis with related taxa showed that many protein families are conserved among the protozoans. Based on gene ontology annotation, functional groups were successfully assigned to 790 UTs. Genes encoding excretory/secretory proteins and membrane and membrane-associated proteins were identified because these proteins often function as antigens and are good antibody targets. A total of 481 UTs were classified as encoding membrane proteins, 54 were classified as encoding for membrane-bound proteins, and 155 were found to contain excretory/secretory protein-coding sequences. Amino acid repeat-containing proteins and GPI-anchored proteins were also identified as potential candidates for the development of diagnostic and control strategies for C. irritans. Conclusions We successfully discovered and examined a large portion of the previously unexplored C. irritans transcriptome and identified potential genes for the development and validation of diagnostic and control strategies for cryptocaryonosis.

Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 μg/μL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 μg/μL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments. [ABSTRACT FROM AUTHOR]

Introduction: Metabolomic studies on various colorectal cancer (CRC) cell lines have improved our understanding of the biochemical events underlying the disease. However, the metabolic profile dynamics associated with different stages of CRC progression is still lacking. Such information can provide further insights into the pathophysiology and progression of the disease that will prove useful in identifying specific targets for drug designing and therapeutics. Thus, our study aims to characterize the metabolite profiles in the established cell lines corresponding to different stages of CRC. Methods: Metabolite profiling of normal colon cell lines (CCD 841 CoN) and CRC cell lines corresponding to different stages, i.e., SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D), was carried out using liquid chromatography-mass spectrometry (LC-MS). Mass Profiler Professional and Metaboanalyst 4.0 software were used for statistical and pathway analysis. METLIN database was used for the identification of metabolites. Results: We identified 72 differential metabolites compared between CRC cell lines of all the stages and normal colon cells. Principle component analysis and partial least squares discriminant analysis score plot were used to segregate normal and CRC cells, as well as CRC cells in different stages of the disease. Variable importance in projection score identified unique differential metabolites in CRC cells of the different stages. We identified 7 differential metabolites unique to stage A, 3 in stage B, 5 in stage C, and 5 in stage D. Conclusion: This study highlights the differential metabolite profiling in CRC cell lines corresponding to different stages. The identification of the differential metabolites in CRC cells at individual stages will lead to a better understanding of the pathophysiology of CRC development and progression and, hence, its application in treatment strategies. [ABSTRACT FROM AUTHOR]

Introduction: The serum metabolomics approach has been used to identify metabolite biomarkers that can diagnose colorectal cancer (CRC) accurately and specifically. However, the biomarkers identified differ between studies suggesting that more studies need to be performed to understand the influence of genetic and environmental factors. Therefore, this study aimed to identify biomarkers and affected metabolic pathways in Malaysian CRC patients. Methods: Serum from 50 healthy controls and 50 CRC patients were collected at UKM Medical Centre. The samples were deproteinized with acetonitrile and untargeted metabolomics profile determined using liquid chromatographyquadrupole time-of-flight mass spectrometry (LC-QTOFMS, Agilent USA). The data were analysed using Mass Profiler Professional (Agilent, USA) software. The panel of biomarkers determined were then used to identify CRC from a new set of 20 matched samples. Results: Eleven differential metabolites were identified whose levels were significantly different between CRC patients compared to normal controls. Based on the analysis of the area under the curve, 7 of these metabolites showed high sensitivity and specificity as biomarkers. The use of the 11 metabolites on a new set of samples was able to differentiate CRC from normal samples with 80% accuracy. These metabolites were hypoxanthine, acetylcarnitine, xanthine, uric acid, tyrosine, methionine, lysoPC, lysoPE, citric acid, 5-oxoproline, and pipercolic acid. The data also showed that the most perturbed pathways in CRC were purine, catecholamine, and amino acid metabolisms. Conclusion: Serum metabolomics profiling can be used to identify distinguishing biomarkers for CRC as well as to further our knowledge of its pathophysiological mechanisms. [ABSTRACT FROM AUTHOR]