Discovery and analysis of Paraburkholderia fungorum genome associated with Manis javanica / Tan Ka Yun

Tan , Ka Yun (2022) Discovery and analysis of Paraburkholderia fungorum genome associated with Manis javanica / Tan Ka Yun. PhD thesis, Universiti Malaya.

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Abstract

The Paraburkholderia fungorum, a Gram-negative environmental species has been commonly used in agriculture, as an agent of biodegradation and bioremediation. However, there are debates on whether these bacterial species could affect human health as there are cases where P. fungorum have been identified in clinical specimens, but no evidence to show that they can cause disease in humans nor in other mammals. We previously sequenced and analysed the genomic DNA from brain tissue of a pregnant Malayan pangolin (“UM3”), which is a placental mammal and endangered species. Interestingly, sequencing data revealed a considerable amount of foreign bacterial DNA that had high similarity with the sequence of Paraburkholderia fungorum (hereinafter referred to as “Pf”), indicating that the brain tissue might be infected by Pf. The pangolin associated Pf sequences were further analysed and characterized using molecular and bioinformatics approaches. To examine and confirm the presence of this bacterial species in pangolin, DNA extracted from different organ specimens of the pangolin were screened by performing PCR using in-house designed primers targeting specific genomic regions of the Pf and a generic 16S primer set. Clear positive PCR bands were found in samples from the pangolin cerebrum, cerebellum, blood, lung, and fetal gastrocnemius muscle. To confirm whether the presence of Pf in pangolin UM3 was an isolated case, the blood samples of seven individual pangolins (seized in two independent anti-animal trafficking operations) were screened. Of the seven pangolins, DNA from four of the pangolins were PCR positive, suggesting that the presence of this bacterial species in pangolin is not an isolated case. Histological staining of sections of the positive specimen showed the presence of Gram-negative rod-shaped bacteria in the pangolin brain, which could likely be Pf. To further study the taxonomic position and potential gene functions of the pangolin associated Pf, the genome sequence was assembled using CLC Assembly Cell with a reference genome of P. fungorum ATCC-BAA-463, which yielded a genome size of 7.7Mbps with 86% of genome completeness. The sequence was further confirmed to be Pf as supported by evidence from phylogenetic analyses generated using selected marker genes and the core-genome SNPs, and ANI analysis. Genome annotation revealed genes related to stress response that may play a role in bacterial adaptation to various stress environments such as oxidative stress, periplasmic stress, heat shock, cold shock, and detoxification; genes annotated related to virulence disease and a defense subsystem, that may help the bacteria to survive in harsh environments and that may also play a role in its pathogenic potential. Urease (Urea ABC transporter urtBCDE) and toxin-antitoxin system (higAB and ygiUT) that are associated with emerging roles to withstand different stress environments and reduce production of virulence factors as well as biofilm formation were also predicted. Interestingly, genome annotation also revealed several secretion systems that are also found in infectious Burkholderial species such as T6SS, a virulence factor associated with potential pathogenic bacterial-host interactions and is associated with tolerance to the innate immune response in Gram-negative bacteria. This study provides the first evidence on the possibility that Pf can infect a non-human placental mammal and specifically a pangolin. The likelihood that Pf has pathogenic capabilities suggests that caution should be taken for its use and application in agriculture or bioremediation.

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