2020 Fiscal Year Research-status Report
Identification and characterization of novel virulence factors of Staphylococcus aureus USA300 using silkworm model.
| Project/Area Number |
19K16653
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| Research Institution | Hokkaido University |
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Principal Investigator |
PAUDEL ATMIKA 北海道大学, 人獣共通感染症リサーチセンター, 特任助教 (80832774)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | host-pathogen / virulence factor / silkworm infection model / Bombyx mori / pathogenesis |
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| Outline of Annual Research Achievements |
We performed exo-protein profiling of yjbI and yjbH disruption mutants. The mutants produced more exo-protein while less surface protein than the wildtype. Introduction of gene into the mutants restored the phenotypes. In consistent with this, Gene Ontology term analysis showed that cellular components were among the highly enriched genes in the mutant. Further, there was a decrease in expression of the genes involved in pathogenesis. We further found that disruptions of yjbI and yjbH reduced the mice-killing ability of S. aureus, which was restored in the gene-complemented strain. In the presence of a free radical scavenger, the silkworm-killing ability of the mutants is restored, suggesting the role of the genes in oxidative stress tolerance in vivo. In addition, RNA-sequencing analysis of all the other 7 less-virulent mutants was performed. In other mutants, expression of specific kinds of genes were affected, the detailed analysis of which is underway. The research work was then extended to another Gram-positive bacteria Bacillus cereus group. A silkworm infection model of Bacillus anthracis vaccine strain Sterne was established using florescent protein-expressing B. anthracis strain. Antibiotics administration to the infected larvae could cure them from infection. Mutants of B. anthracis with disruption in known virulence genes had reduced killing and reduced colonization in silkworms. Apart from these, a comparative genomic analysis of Enterococcus faecalis EF-2001 was performed revealing that the strain does not harbor genes known for pathogenesis.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
As planned previously, we performed the exoprotein profiling of the yjbH strain and revealed that in the absence of protease, the strains had more exoproteins while decreased surface proteins. Complementing the mutants with intact genes restored the wildtype phenotype. We further confirmed that yjbH is involved in killing ability of S. aureus both in silkworms and mice and revealed that yjbH contributes to oxidative stress tolerance in vivo as shown by increased virulence when a radical scavenger was administered to the silkworms prior to infection. A new infection model of Bacillus anthracis vaccine strain Sterne was established using silkworms as model animal. Live bacteria were required for silkworm killing. Using florescent protein-expressing B. anthracis, we showed the progression in proliferation of bacteria in silkworm hemolymph. We showed that the infection can be cured by administration of clinically used antibiotics into the infected larvae. The mutants of B. anthracis with disruption in genes of known virulence factors atxA, pagA and lef had reduced silkworm-killing ability and reduced colonization in silkworm hemolymph, suggesting that silkworms can be used as suitable animal model of B. anthracis infection.
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| Strategy for Future Research Activity |
In the next period, we plan to take further our study of understanding the virulence mechanism of Bacillus cereus group of Gram-positive bacteria using silkworms. Functions of many genes are not yet characterized which might have roles in pathogenesis. To uncover the novel genes involved in virulence of the bacteria, we plan to utilize silkworm as a model animal to assess virulence of different gene disrupted mutants. In addition, we plan to perform in vivo RNA-seq analysis in silkworms to get an insight into host and pathogen interaction during infection condition. Genes that are expressed highly will be selected and the candidate genes will then be further analyzed to find their role in virulence of the pathogen.
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| Causes of Carryover |
I was able to use existing regents and equipment for the experiments in this term. In the next fiscal year, we plan to utilize silkworm as a model animal to assess virulence of Bacillus cereus group of Gram-positive bacteria and extend this study beyond Staphylococcus aureus.
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[Journal Article] Novel chromosomal insertions of ISEcp1-blaCTX-M-15 and diverse antimicrobial resistance genes in Zambian clinical isolates of Enterobacter cloacae and Escherichia coli2021
Author(s)
Misheck Shawa, Yoshikazu Furuta, Gillan Mulenga, Maron Mubanga, Evans Mulenga, Tuvshinzaya Zorigt, Christone Kaile, Manyando Simbotwe, Atmika Paudel, Bernard Hang’ombe, Hideaki HIGASHI
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Journal Title
BMC Antimicrobial Resistance & Infection Control
Volume: accepted
Pages: in press
Peer Reviewed / Open Access / Int'l Joint Research
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