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Fulltext Genome-wide transposon mutagenesis analysis of Burkholderia pseudomallei reveals essential genes for in vitro and in vivo survival

Wong YC, Naeem R, Abd El Ghany M, Hoh CC, Pain A, Nathan S

Front Cell Infect Microbiol, 2022;12:1062682.
PMID: 36619746 DOI: 10.3389/fcimb.2022.1062682

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.

Matched MeSH terms: Caenorhabditis elegans/microbiology
Stenotrophomonas maltophilia: pathogenesis model using Caenorhabditis elegans

Thomas R, Hamat RA, Neela V

J Med Microbiol, 2013 Nov;62(Pt 11):1777-1779.
PMID: 23988629 DOI: 10.1099/jmm.0.063230-0
Matched MeSH terms: Caenorhabditis elegans/microbiology*
Fulltext Molecular characterization of putative virulence determinants in Burkholderia pseudomallei

Puah SM, Puthucheary SD, Wang JT, Pan YJ, Chua KH

ScientificWorldJournal, 2014;2014:590803.
PMID: 25215325 DOI: 10.1155/2014/590803

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

Matched MeSH terms: Caenorhabditis elegans/microbiology
Fulltext Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen

Lee SH, Ooi SK, Mahadi NM, Tan MW, Nathan S

PLoS One, 2011;6(3):e16707.
PMID: 21408228 DOI: 10.1371/journal.pone.0016707

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Matched MeSH terms: Caenorhabditis elegans/microbiology*
Pathogenicity and phenotypic analysis of sopB, sopD and pipD virulence factors in Salmonella enterica serovar typhimurium and Salmonella enterica serovar Agona

Khoo CH, Sim JH, Salleh NA, Cheah YK

Antonie Van Leeuwenhoek, 2015 Jan;107(1):23-37.
PMID: 25312847 DOI: 10.1007/s10482-014-0300-7

Salmonella is an important food-borne pathogen causing disease in humans and animals worldwide. Salmonellosis may be caused by any one of over 2,500 serovars of Salmonella. Nonetheless, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Agona are the second most prevalent serovars isolated from humans and livestock products respectively. Limited knowledge is available about the virulence mechanisms responsible for diarrheal disease caused by them. To investigate the contribution of sopB, sopD and pipD as virulence factors in intracellular infections and the uniqueness of these bacteria becoming far more prevalent than other serovars, the infection model of Caenorhabditis elegans and phenotypic microarray were used to characterize their mutants. The strains containing the mutation in sopB, sopD and pipD genes were constructed by using latest site-specific group II intron mutagenesis approach to reveal the pathogenicity of the virulence factors. Overall, we observed that the mutations in sopB, sopD and pipD genes of both serovars did not exhibit significant decrease in virulence towards the nematode. This may indicate that these virulence effectors may not be universal virulence factors involved in conserved innate immunity. There are significant phenotypic differences amongst strains carrying sopB, sopD and pipD gene mutations via the analysis of biochemical profiles of the bacteria. Interestingly, mutant strains displayed different susceptibility to chemical stressors from several distinct pharmacological and structural classes when compared to its isogenic parental strains. These metabolic and chemosensitivity assays also revealed multiple roles of Salmonella virulence factors in nutrient metabolism and antibiotic resistance.

Matched MeSH terms: Caenorhabditis elegans/microbiology
Fulltext Burkholderia pseudomallei suppresses Caenorhabditis elegans immunity by specific degradation of a GATA transcription factor

Lee SH, Wong RR, Chin CY, Lim TY, Eng SA, Kong C, et al.

Proc Natl Acad Sci U S A, 2013 Sep 10;110(37):15067-72.
PMID: 23980181 DOI: 10.1073/pnas.1311725110

Burkholderia pseudomallei is a Gram-negative soil bacterium that infects both humans and animals. Although cell culture studies have revealed significant insights into factors contributing to virulence and host defense, the interactions between this pathogen and its intact host remain to be elucidated. To gain insights into the host defense responses to B. pseudomallei infection within an intact host, we analyzed the genome-wide transcriptome of infected Caenorhabditis elegans and identified ∼6% of the nematode genes that were significantly altered over a 12-h course of infection. An unexpected feature of the transcriptional response to B. pseudomallei was a progressive increase in the proportion of down-regulated genes, of which ELT-2 transcriptional targets were significantly enriched. ELT-2 is an intestinal GATA transcription factor with a conserved role in immune responses. We demonstrate that B. pseudomallei down-regulation of ELT-2 targets is associated with degradation of ELT-2 protein by the host ubiquitin-proteasome system. Degradation of ELT-2 requires the B. pseudomallei type III secretion system. Together, our studies using an intact host provide evidence for pathogen-mediated host immune suppression through the destruction of a host transcription factor.

Matched MeSH terms: Caenorhabditis elegans/microbiology*
Fulltext Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model

Kong C, Yehye WA, Abd Rahman N, Tan MW, Nathan S

BMC Complement Altern Med, 2014;14:4.
PMID: 24393217 DOI: 10.1186/1472-6882-14-4

The limited antibiotic options for effective control of methicillin-resistant Staphylococcus aureus infections has led to calls for new therapeutic approaches to combat this human pathogen. An alternative approach to control MRSA is through the use of anti-infective agents that selectively disrupt virulence-mediated pathways without affecting microbial cell viability or by modulating the host natural immune defenses to combat the pathogen.

Matched MeSH terms: Caenorhabditis elegans/microbiology*
Swietenia macrophylla extract promotes the ability of Caenorhabditis elegans to survive Pseudomonas aeruginosa infection

Dharmalingam K, Tan BK, Mahmud MZ, Sedek SA, Majid MI, Kuah MK, et al.

J Ethnopharmacol, 2012 Jan 31;139(2):657-63.
PMID: 22193176 DOI: 10.1016/j.jep.2011.12.016

Swietenia macrophylla or commonly known as big leaf mahogany, has been traditionally used as an antibacterial and antifungal agent.

Matched MeSH terms: Caenorhabditis elegans/microbiology
Black sea cucumber (Holothuria atra Jaeger, 1833) rescues Pseudomonas aeruginosa-infected Caenorhabditis elegans via reduction of pathogen virulence factors and enhancement of host immunity

Lee WT, Tan BK, Eng SA, Yuen GC, Chan KL, Sim YK, et al.

Food Funct, 2019 Sep 01;10(9):5759-5767.
PMID: 31453615 DOI: 10.1039/c9fo01357a

A strategy to circumvent the problem of multidrug resistant pathogens is the discovery of anti-infectives targeting bacterial virulence or host immunity. Black sea cucumber (Holothuria atra) is a tropical sea cucumber species traditionally consumed as a remedy for many ailments. There is a paucity of knowledge on the anti-infective capacity of H. atra and the underlying mechanisms involved. The objective of this study is to utilize the Caenorhabditis elegans-P. aeruginosa infection model to elucidate the anti-infective properties of H. atra. A bioactive H. atra extract and subsequently its fraction were shown to have the capability of promoting the survival of C. elegans during a customarily lethal P. aeruginosa infection. The same entities also attenuate the production of elastase, protease, pyocyanin and biofilm in P. aeruginosa. The treatment of infected transgenic lys-7::GFP worms with this H. atra fraction restores the repressed expression of the defense enzyme lys-7, indicating an improved host immunity. QTOF-LCMS analysis revealed the presence of aspidospermatidine, an indole alkaloid, and inosine in this fraction. Collectively, our findings show that H. atra possesses anti-infective properties against P. aeruginosa infection, by inhibiting pathogen virulence and, eventually, reinstating host lys-7 expression.

Matched MeSH terms: Caenorhabditis elegans/microbiology*
Fulltext Burkholderia pseudomallei kills Caenorhabditis elegans through virulence mechanisms distinct from intestinal lumen colonization

Ooi SK, Lim TY, Lee SH, Nathan S

Virulence, 2012 Oct 01;3(6):485-96.
PMID: 23076282 DOI: 10.4161/viru.21808

The nematode Caenorhabditis elegans is hypersusceptible to Burkholderia pseudomallei infection. However, the virulence mechanisms underlying rapid lethality of C. elegans upon B. pseudomallei infection remain poorly defined. To probe the host-pathogen interaction, we constructed GFP-tagged B. pseudomallei and followed bacterial accumulation within the C. elegans intestinal lumen. Contrary to slow-killing by most bacterial pathogens, B. pseudomallei caused fairly limited intestinal lumen colonization throughout the period of observation. Using grinder-defective mutant worms that allow the entry of intact bacteria also did not result in full intestinal lumen colonization. In addition, we observed a significant decline in C. elegans defecation and pharyngeal pumping rates upon B. pseudomallei infection. The decline in defecation rates ruled out the contribution of defecation to the limited B. pseudomallei colonization. We also demonstrated that the limited intestinal lumen colonization was not attributed to slowed host feeding as bacterial loads did not change significantly when feeding was stimulated by exogenous serotonin. Both these observations confirm that B. pseudomallei is a poor colonizer of the C. elegans intestine. To explore the possibility of toxin-mediated killing, we examined the transcription of the C. elegans ABC transporter gene, pgp-5, upon B. pseudomallei infection of the ppgp-5::gfp reporter strain. Expression of pgp-5 was highly induced, notably in the pharynx and intestine, compared with Escherichia coli-fed worms, suggesting that the host actively thwarted the pathogenic assaults during infection. Collectively, our findings propose that B. pseudomallei specifically and continuously secretes toxins to overcome C. elegans immune responses.

Matched MeSH terms: Caenorhabditis elegans/microbiology