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Fulltext Bioactive Compounds from the Bornean Endemic Plant Goniothalamus longistipetes

Teo SP, Bhakta S, Stapleton P, Gibbons S

Antibiotics (Basel), 2020 Dec 16;9(12).
PMID: 33339285 DOI: 10.3390/antibiotics9120913

The present study aimed to screen plants for bioactive compounds with potential antibacterial activities. In our efforts to evaluate plants from Borneo, we isolated and elucidated the structures of four natural products from the bioactive fraction of a chloroform extract of Goniothalamus longistipetes using various chromatographic and spectroscopic techniques. The bioactive compounds were identified as a known styryllactone, (+)-altholactone ((2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (1), a new styryllactone, (2S,3R,3aS,7aS)-3-hydroxy-2-phenyl-2,3,3a,7a-tetrahydrobenzo-5(4H)-5-one) (2) as well as a new alkaloid, 2,6-dimethoxyisonicotinaldehyde (3) and a new alkenyl-5-hydroxyl-phenyl benzoic acid (4). 1 and 4 showed broad-spectrum anti-bacterial activities against Gram-positive and Gram-negative bacteria as well as acid-fast model selected for this study. Compound 2 only demonstrated activities against Gram-positive bacteria whilst 3 displayed selective inhibitory activities against Gram-positive bacterial strains. Additionally, their mechanisms of anti-bacterial action were also investigated. Using Mycobacterium smegmatis as a fast-growing model of tubercle bacilli, compounds 1, 2 and 4 demonstrated inhibitory activities against whole-cell drug efflux and biofilm formation; two key intrinsic mechanisms of antibiotic resistance. Interestingly, the amphiphilic compound 4 exhibited inhibitory activity against the conjugation of plasmid pKM101 in Escherichia coli using a plate conjugation assay. Plasmid conjugation is a mechanism by which Gram-positive and Gram-negative-bacteria acquire drug resistance and virulence. These results indicated that bioactive compounds isolated from Goniothalamus longistipetes can be potential candidates as 'hits' for further optimisation.

Matched MeSH terms: Virulence
Fulltext Use of the waxworm Galleria mellonella larvae as an infection model to study Acinetobacter baumannii

Ten KE, Muzahid NH, Rahman S, Tan HS

PLoS One, 2023;18(4):e0283960.
PMID: 37018343 DOI: 10.1371/journal.pone.0283960

Galleria mellonella larvae have been increasingly used in research, including microbial infection studies. They act as suitable preliminary infection models to study host-pathogen interactions due to their advantages, such as the ability to survive at 37°C mimicking human body temperature, their immune system shares similarities with mammalian immune systems, and their short life cycle allowing large-scale studies. Here, we present a protocol for simple rearing and maintenance of G. mellonella without requiring special instruments and specialized training. This allows the continuous supply of healthy G. mellonella for research purposes. Besides, this protocol also provides detailed procedures on the (i) G. mellonella infection assays (killing assay and bacterial burden assay) for virulence studies and (ii) bacterial cell harvesting from infected larvae and RNA extraction for bacterial gene expression studies during infection. Our protocol could not only be used in the studies of A. baumannii virulence but can also be modified according to different bacterial strains.

Matched MeSH terms: Virulence
Genetic variation analysis of Vibrio cholerae using multilocus sequencing typing and multi-virulence locus sequencing typing

Teh CS, Chua KH, Thong KL

Infect Genet Evol, 2011 Jul;11(5):1121-8.
PMID: 21511055 DOI: 10.1016/j.meegid.2011.04.005

This paper describes the development and application of multilocus sequencing typing (MLST) and multi-virulence locus sequencing typing (MVLST) methods in determining the genetic variation and relatedness of 43 Vibrio cholerae strains of different serogroups isolated from various sources in Malaysia. The MLST assay used six housekeeping genes (dnaE, lap, recA, gyrB, cat and gmd), while the MVLST assay incorporated three virulence genes (ctxAB, tcpA and tcpI) and three virulence-associated genes (hlyA, toxR and rtxA). Our data showed that the dnaE and rtxA genes were the most conserved genes in V. cholerae O1 strains. Among the 12 studied genes, transitional substitutions that led to silent mutations were observed in all, except for gmd and hlyA, while non-synonymous substitutions occurred more frequently in virulence and virulence-associated genes. Five V. cholerae O1 strains were found to be the El Tor variant O1 strains because they harboured the classical ctxB gene. In addition, the classical ctxB gene was also observed in O139 V. cholerae. A total of 29 MLST types were observed, and this assay could differentiate V. cholerae within the non-O1/non-O139 serogroups. A total of 27 MVLST types were obtained. MVLST appeared to be more discriminatory than MLST because it could differentiate V. cholerae strains from two different outbreaks and could separate the toxigenic from the non-toxigenic subtypes. Although the O1 V. cholerae strains were closely related, the combined MLST and MVLST analyses differentiated the strains isolated from different localities. In conclusion, sequence-based analysis in this study provided a better understanding of mutation points and the type of mutations in V. cholerae. The MVLST assay is useful to characterise O1 V. cholerae strains, while combined analysis may improve the discriminatory power and is suitable for the local epidemiological study of V. cholerae.

Matched MeSH terms: Virulence Factors/metabolism*
Fulltext Multiple-locus variable-number tandem repeat analysis of Vibrio cholerae in comparison with pulsed field gel electrophoresis and virulotyping

Teh CS, Chua KH, Thong KL

J Biomed Biotechnol, 2010;2010:817190.
PMID: 20671932 DOI: 10.1155/2010/817190

Molecular analysis of Malaysian Vibrio cholerae was carried out using a multiple-locus variable-number tandem repeat analysis (MLVA) assay based on 7 loci of V. cholerae. The discriminatory ability of the assay was compared with pulsed-field gel electrophoresis (PFGE) using 43 Malaysian V. cholerae isolated from various sources. In addition, the virulotypes of the strains were determined. Based on MLVA, 38 allelic profiles were obtained (F = 0.63) while PFGE generated 35 pulsotypes (F = 0.71). Simpson's index of diversity for different VNTR loci ranged from 0.59 to 0.92. The combined loci increased the discriminatory index to 0.99 which was comparable with PFGE (D = 0.99). Most of the environmental non-O1/non-O139 strains harbored rtxA, rstR, toxR, and hlyA only, and the virulotype of this serogroup was significantly different (P < .01) from clinical/environmental O1 and environmental O139 strains. In conclusion, the MLVA assay developed in this study was a useful genotyping tool with comparable discriminatory power with PFGE. In addition, the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation.

Matched MeSH terms: Virulence/genetics
Fulltext Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive enterovirus A71 virulence in mice

Tee HK, Tan CW, Yogarajah T, Lee MHP, Chai HJ, Hanapi NA, et al.

PLoS Pathog, 2019 11;15(11):e1007863.
PMID: 31730673 DOI: 10.1371/journal.ppat.1007863

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.

Matched MeSH terms: Virulence; Virulence Factors/genetics; Virulence Factors/metabolism*; Virulence Factors/chemistry
In vitro demonstration of the hemolytic, cytotoxic activities and induction of apoptosis in Orientia tsutsugamushi infected L929 mouse fibroblast cells

Tay ST, Rohani MY, Ho TM, Devi S

Southeast Asian J. Trop. Med. Public Health, 2003 Jun;34(2):352-6.
PMID: 12971561

Using cultured mouse fibroblast L929 cells, this study demonstrated the hemolytic and cytotoxic activities and induction of apoptosis in cells infected with Orientia tsutsugamushi. Low levels of hemolytic activity were detected using heavily infected cells. No hemolysin or cytotoxin were detected in the infected culture fluid regardless of the pathogenicity of the O. tsutsugamushi strains in mice. Using propidium iodide uptake assay, acridine orange/ethidium bromide staining and terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labeling assay, apoptosis was observed in L929 cells infected with Karp and Gilliam strains.

Matched MeSH terms: Virulence Factors
Fulltext Phylogeny and putative virulence gene analysis of Bartonella bovis

Tay ST, Kho KL, Lye SF, Ngeow YF

J Vet Med Sci, 2018 Apr 18;80(4):653-661.
PMID: 29311425 DOI: 10.1292/jvms.17-0448

Bartonella bovis is a small Gram-negative bacterium recognized as an etiological agent for bacteremia and endocarditis in cattle. As few reports are available on the taxonomic position of B. bovis and its mechanism of virulence, this study aims to resolve the phylogeny of B. bovis and investigate putative virulence genes based on whole genome sequence analysis. Genome-wide comparisons based on single nucleotide polymorphisms (SNP) and orthologous genes were performed in this study for phylogenetic inference of 27 Bartonella species. Rapid Annotation using Subsystem Technology (RAST) analysis was used for annotation of putative virulence genes. The phylogenetic tree generated from the genome-wide comparison of orthologous genes exhibited a topology almost similar to that of the tree generated from SNP-based comparison, indicating a high concordance in the nucleotide and amino acid sequences of Bartonella spp. The analyses show consistent grouping of B. bovis in a cluster related to ruminant-associated species, including Bartonella australis, Bartonella melophagi and Bartonella schoenbuchensis. RAST analysis revealed genes encoding flagellar components, in corroboration with the observation of flagella-like structure of BbUM strain under negative straining. Genes associated with virulence, disease and defence, prophages, membrane transport, iron acquisition, motility and chemotaxis are annotated in B. bovis genome. The flagellin (flaA) gene of B. bovis is closely related to Bartonella bacilliformis and Bartonella clarridgeiae but distinct from other Gram-negative bacteria. The absence of type IV secretion systems, the bona fide pathogenicity factors of bartonellae, in B. bovis suggests that it may have a different mechanism of pathogenicity.

Matched MeSH terms: Virulence; Virulence Factors/genetics*
Detection of haemolytic activity of campylobacters by agarose haemolysis and microplate assay

Tay ST, Devi S, Puthucheary SD, Kautner IM

J Med Microbiol, 1995 Mar;42(3):175-80.
PMID: 7884798

There are several methods for the detection of haemolytic activity in campylobacters. However, we found the haemolytic effect of campylobacters on conventional blood agar plates to be variable, inconsistent and difficult to interpret. Blood agarose plates showed campylobacter haemolytic activity more clearly. The incubation conditions (temperature and gaseous) appear to be important for the expression of this activity. Ninety four percent of the Campylobacter isolates examined were found to be haemolytic by the microplate assay with minimal haemolytic units that ranged from 1 to 64. Haemolytic activity was detected only from live bacterial cultures and not from any of the 50 bacterial culture supernates, which suggests that campylobacters may possess a cell-associated haemolysin. The identification of such haemolytic activity in a large number of campylobacters (94%) suggests its potential role as a virulence factor in campylobacter gastroenteritis.

Matched MeSH terms: Virulence
Fulltext Significant replication time-points of avian influenza a virus strain H5N1 in Madin-Darby Canine Kidney cells

Tan, Toong Seng, Yap, Wei Boon, Sharifah Syed Hassan

Jurnal Sains Kesihatan Malaysia, 2016;14(1):17-21.
MyJurnal

The occasional influenza pandemics and the seasonal influenza epidemics have destroyed millions of lives since
the last century. It is therefore necessary to understand the virus replication patterns as this provides essential
information on the virus infectivity, pathogenicity and spread patterns. This study aimed to investigate the replication
of avian influenza A virus H5N1 (A/Chicken/Malaysia/5858/2004) in MDCK cells. In this study, the TCID50 (50% tissue
culture infectious dose) of AIV H5N1 was first determined. The MDCK cells were then infected with AIV H5N1 at TCID50
for 0-48 h. The CPE (cytopathic effect) was observed and cell death was determined hourly. The virus-infected cells
and media were subsequently collected for gene analysis. The results showed that the TCID50 of AIV H5N1 was 10-9
dilution. The CPE percentage showed a strong and positive correlation with the infection period (r = 1.0, n = 9, p <
0.01). The amount of a highly conserved influenza viral gene, M2 gene amplified from infected media (r = 0.471, n =
9, p= > 0.05) and infected cell (r = 0.73, n = 9, p < 0.05) were also positively correlated with the infection period. In
conclusion, although CPE started to be observed in the early time points of infection, however, the M2 gene was only
amplified from the infected media and cells after 48 h and 24 h, respectively. This signifies that AIV H5N1 used in this
study is pathogenic and it is able to cause severe cytopathology to host cells even at low virus load.

Matched MeSH terms: Virulence
Oxidative stress resistance and fitness-compensatory response in vancomycin-intermediate Staphylococcus aureus (VISA)

Tan XE, Neoh HM, Cui L, Hiramatsu K, Jamal R

Can J Microbiol, 2019 Aug;65(8):623-628.
PMID: 31063703 DOI: 10.1139/cjm-2019-0048

In this study, vancomycin-intermediate Staphylococcus aureus (VISA) cells carrying vraS and (or) graR mutations were shown to be more resistant to oxidative stress. Caenorhabditis elegans infected with these strains in turn demonstrated lower survival. Altered regulation in oxidative stress response and virulence appear to be physiological adaptations associated with the VISA phenotype in the Mu50 lineage.

Matched MeSH terms: Virulence/drug effects
Fulltext Evolutionary study of Yersinia genomes deciphers emergence of human pathogenic species

Tan SY, Tan IK, Tan MF, Dutta A, Choo SW

Sci Rep, 2016 10 31;6:36116.
PMID: 27796355 DOI: 10.1038/srep36116

On record, there are 17 species in the Yersinia genus, of which three are known to be pathogenic to human. While the chromosomal and pYV (or pCD1) plasmid-borne virulence genes as well as pathogenesis of these three species are well studied, their genomic evolution is poorly understood. Our study aims to predict the key evolutionary events that led to the emergence of pathogenic Yersinia species by analyzing gene gain-and-loss, virulence genes, and "Clustered regularly-interspaced short palindromic repeats". Our results suggest that the most recent ancestor shared by the human pathogenic Yersinia was most probably an environmental species that had adapted to the human body. This might have led to ecological specialization that diverged Yersinia into ecotypes and distinct lineages based on differential gene gain-and-loss in different niches. Our data also suggest that Y. pseudotuberculosis group might be the donor of the ail virulence gene to Y. enterocolitica. Hence, we postulate that evolution of human pathogenic Yersinia might not be totally in parallel, but instead, there were lateral gene transfer events. Furthermore, the presence of virulence genes seems to be important for the positive selection of virulence plasmid. Our studies provide better insights into the evolutionary biology of these bacteria.

Matched MeSH terms: Virulence/genetics
Fulltext YersiniaBase: a genomic resource and analysis platform for comparative analysis of Yersinia

Tan SY, Dutta A, Jakubovics NS, Ang MY, Siow CC, Mutha NV, et al.

BMC Bioinformatics, 2015;16:9.
PMID: 25591325 DOI: 10.1186/s12859-014-0422-y

Yersinia is a Gram-negative bacteria that includes serious pathogens such as the Yersinia pestis, which causes plague, Yersinia pseudotuberculosis, Yersinia enterocolitica. The remaining species are generally considered non-pathogenic to humans, although there is evidence that at least some of these species can cause occasional infections using distinct mechanisms from the more pathogenic species. With the advances in sequencing technologies, many genomes of Yersinia have been sequenced. However, there is currently no specialized platform to hold the rapidly-growing Yersinia genomic data and to provide analysis tools particularly for comparative analyses, which are required to provide improved insights into their biology, evolution and pathogenicity.

Matched MeSH terms: Virulence/genetics*
Fulltext Occurrence of virulent multidrug-resistant Enterococcus faecalis and Enterococcus faecium in the pigs, farmers and farm environments in Malaysia

Tan SC, Chong CW, Teh CSJ, Ooi PT, Thong KL

PeerJ, 2018;6:e5353.
PMID: 30123701 DOI: 10.7717/peerj.5353

Background: Enterococcus faecalis and Enterococcus faecium are ubiquitous opportunistic pathogens found in the guts of humans and farmed animals. This study aimed to determine the occurrence, antimicrobial resistance, virulence, biofilm-forming ability and genotypes of E. faecalis and E. faecium from swine farms. Correlations between the genotypes, virulotypes, antibiotic resistance, and the environmental factors such as locality of farms and farm hygiene practice were explored.
Methods: E. faecalis and E. faecium strains were isolated from the oral, rectal and fecal samples of 140 pigs; nasal, urine and fecal samples of 34 farmers working in the farms and 42 environmental samples collected from seven swine farms located in Peninsular Malaysia. Antibiotic susceptibility test was performed using the disk diffusion method, and the antibiotic resistance and virulence genes were detected by Polymerase Chain Reaction. Repetitive Extragenic Palindromic-Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis were performed to determine the clonality of the strains. Crosstab/Chi-square test and DistLM statistical analyses methods were used to determine the correlations between the genotypes, virulence factors, antibiotic resistance, and the environmental factors.
Results: A total of 211 E. faecalis and 42 E. faecium were recovered from 140 pigs, 34 farmers and 42 environmental samples collected from seven swine farms in Peninsular Malaysia. Ninety-eight percent of the strains were multidrug-resistant (resistant to chloramphenicol, tetracycline, ciprofloxacin and erythromycin). Fifty-two percent of the strains formed biofilms. Virulence genes efa, asaI, gelE, esp, cyl and ace genes were detected. Virulence genes efa and asaI were most prevalent in E. faecalis (90%) and E. faecium (43%), respectively. Cluster analyses based on REP-PCR and PFGE showed the strains were genetically diverse. Overall, the strains isolated from pigs and farmers were distinct, except for three highly similar strains found in pigs and farmers. The strains were regional- and host-specific.
Discussion: This study revealed alarming high frequencies of multidrug-resistant enterococci in pigs and swine farmers. The presence of resistance and virulence genes and the ability to form biofilm further enhance the persistence and pathogenicity of the strains. Although the overall clonality of the strains were regionals and host-specific, strains with high similarity were found in different hosts. This study reiterates a need of a more stringent regulation to ensure the proper use of antibiotics in swine husbandry to reduce the wide spread of multidrug-resistant strains.

Matched MeSH terms: Virulence; Virulence Factors
Fulltext Comprehensive genome analysis of a pangolin-associated Paraburkholderia fungorum provides new insights into its secretion systems and virulence

Tan KY, Dutta A, Tan TK, Hari R, Othman RY, Choo SW

PeerJ, 2020;8:e9733.
PMID: 32953261 DOI: 10.7717/peerj.9733

Background: Paraburkholderia fungorum (P. fungorum) is a Gram-negative environmental species that has been commonly used as a beneficial microorganism in agriculture as an agent for biocontrol and bioremediation. Its use in agriculture is controversial as many people believe that it could harm human health; however, there is no clear evidence to support.
Methodology: The pangolin P. fungorum (pangolin Pf) genome has a genomic size of approximately 7.7 Mbps with N50 of 69,666 bps. Our study showed that pangolin Pf is a Paraburkholderia fungorum supported by evidence from the core genome SNP-based phylogenetic analysis and the ANI analysis. Functional analysis has shown that the presence of a considerably large number of genes related to stress response, virulence, disease, and defence. Interestingly, we identified different types of secretion systems in the genome of pangolin Pf, which are highly specialized and responsible for a bacterium's response to its environment and in physiological processes such as survival, adhesion, and adaptation. The pangolin Pf also shared some common virulence genes with the known pathogenic member of the Burkholderiales. These genes play important roles in adhesion, motility, and invasion.
Conclusion: This study may provide better insights into the functions, secretion systems and virulence of this pangolin-associated bacterial strain. The addition of this genome sequence is also important for future comparative analysis and functional work of P. fungorum.

Matched MeSH terms: Virulence
Fulltext Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri

Tan KH, How KY, Tan JY, Yin WF, Chan KG

Front Microbiol, 2017;8:72.
PMID: 28197135 DOI: 10.3389/fmicb.2017.00072

The process of intercellular communication among bacteria, termed quorum sensing (QS), is mediated by small diffusible molecules known as the autoinducers. QS allows the population to react to the change of cell density in unison, in processes such as biofilm formation, plasmid conjugation, virulence, motility and root nodulation. In Gram-negative proteobacteria, N-acyl homoserine lactone (AHL) is the common "language" to coordinate gene expression. This signaling molecule is usually synthesized by LuxI-type proteins. We have previously discovered that a rare bacterium, Cedecea neteri, exhibits AHL-type QS activity. With information generated from genome sequencing, we have identified the luxIR gene pair responsible for AHL-type QS and named it cneIR. In this study, we have cloned and expressed the 636 bp luxI homolog in an Escherichia coli host for further characterization. Our findings show that E. coli harboring cneI produced the same AHL profile as the wild type C. neteri, with the synthesis of AHL known as N-butyryl-homoserine lactone. This 25 kDa LuxI homolog shares high similarity with other AHL synthases from closely related species. This work is the first documentation of molecular cloning and characterization of luxI homolog from C. neteri.

Matched MeSH terms: Virulence
Fulltext Comparative genomic analysis of Mycobacterium iranicum UM_TJL against representative mycobacterial species suggests its environmental origin

Tan JL, Ngeow YF, Wee WY, Wong GJ, Ng HF, Choo SW

Sci Rep, 2014;4:7169.
PMID: 25417557 DOI: 10.1038/srep07169

Mycobacterium iranicum is a newly reported mycobacterial species. We present the first comparative study of M. iranicum UM_TJL and other mycobacteria. We found M. iranicum to have a close genetic association with environmental mycobacteria infrequently associated with human infections. Nonetheless, UM_TJL is also equipped with many virulence genes (some of which appear to be the consequence of transduction-related gene transfer) that have been identified in established human pathogens. Taken all together, our data suggest that M. iranicum is an environmental bacterium adapted for pathogenicity in the human host. This comparative study provides important clues and forms the basis for future functional studies on this mycobacterium.

Matched MeSH terms: Virulence Factors/genetics
Role of Helicobacter pylori virulence factor and genotypes in non-ulcer dyspepsia

Tan HJ, Rizal AM, Rosmadi MY, Goh KL

J Gastroenterol Hepatol, 2006 Jan;21(1 Pt 1):110-5.
PMID: 16706821

The role of Helicobacter pylori (HP) in non-ulcer dyspepsia is debatable. Eradicating HP will help a small group of non-ulcer dyspeptic patients. However, it is unclear which subgroup of patients will benefit from eradication therapy. The aim of the present study was to compare the cagA and cagE status, as well as vacA genotypes, of HP in non-ulcer dyspeptic patients who responded successfully to eradication therapy compared with those patients who did not.

Matched MeSH terms: Virulence; Virulence Factors/genetics*
Distribution of Helicobacter pylori cagA, cagE and vacA in different ethnic groups in Kuala Lumpur, Malaysia

Tan HJ, Rizal AM, Rosmadi MY, Goh KL

J Gastroenterol Hepatol, 2005 Apr;20(4):589-94.
PMID: 15836708

There is a geographic variation in Helicobacter pylori (HP) genotypes and virulence factors. Cytotoxin associated genes A (cagA) and E (cagE), and certain vacuolating cytotoxin (vacA) genotypes are associated with peptic ulcer disease (PUD). There is also a different prevalence of PUD among different ethnic groups in Malaysia. The present study compared the distribution of vacA alleles and cagA and cagE status in three ethnic groups residing in Kuala Lumpur, Malaysia, and their association with clinical outcome.

Matched MeSH terms: Virulence/genetics
Fulltext Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

Tan CW, Tee HK, Lee MH, Sam IC, Chan YF

PLoS One, 2016;11(9):e0162771.
PMID: 27617744 DOI: 10.1371/journal.pone.0162771

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3' ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71.

Matched MeSH terms: Virulence
Fulltext Interaction between Metarhizium anisopliae and Its Host, the Subterranean Termite Coptotermes curvignathus during the Infection Process

Syazwan SA, Lee SY, Sajap AS, Lau WH, Omar D, Mohamed R

Biology (Basel), 2021 Mar 25;10(4).
PMID: 33806225 DOI: 10.3390/biology10040263

Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host's cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host's integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites' body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host's body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.

Matched MeSH terms: Virulence

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