Biology subjects

Hamamoto, H.

Publications and source records attributed to Hamamoto, H..

6 recordsLinked to original sources

bioRxiv2021

Contribution of rsaC, a small non-coding RNA, towards the pathogenicity of Staphylococcus aureus in a mouse systemic infection model

Understanding how a pathogen responds to the host stimuli and succeeds in causing disease is crucial for developing a novel treatment approach against the pathogen. Transcriptomic analysis facilitated by RNA-Seq technologies is used to examine bacterial responses at the global level. However, the ability to understand pathogen behavior inside the host tissues is hindered by much lower pathogen biomass than host tissue. Recently, we succeeded in establishing a method to enrich Staphylococcus aureus cells from infected organs. In this research, we analyzed the small non-coding RNA (sRNA) transcriptome of S. aureus inside the host and found that rsaC was among the highly expressed sRNAs. Furthermore, by gene disruption and complementation, we demonstrated that rsaC was required for full pathogenicity of S. aureus in a murine model. Besides, we found that {Delta}rsaC showed a difference in gene expression depending on the oxygen and host stress. The findings of this study suggest rsaC acts as a novel virulence factor in S. aureus and might facilitate the adaptation of staphylococci within the host. ImportanceDrug-resistant Staphylococcus aureus is among the pathogen for which new treatment options are urgently needed. However, limited understanding of S. aureus pathogenesis in the host has hindered unearthing potential strategies to treat the infections. Here, based on the in vivo transcriptomic analysis, we present the identification of a small non-coding RNA (sRNA) rsaC as a novel virulence factor of S. aureus. Furthermore, we performed transcriptomic analysis of the rsaC disrupted mutant and identified different pathways, possibly controlled by rsaC, during aerobic, anaerobic, and in vivo conditions. These findings contribute to reveal the role of sRNA rsaC and broadens our understanding of the adaptation of S. aureus to host environments.

microbiology↗

bioRxiv2021

Hybrid assembly using ultra-long reads resolves repeats and completes the genome sequence of a laboratory strain of Staphylococcus aureus subsp. aureus RN4220

Staphylococcus aureus RN4220 has been extensively used by staphylococcal researchers as an intermediate strain for genetic manipulation due to its ability to accept foreign DNA. Despite its wide use in laboratories, its complete genome is not available. In this study, we used the hybrid genome assembly approach using the minION long reads and Illumina short reads to sequence the complete genome of S. aureus RN4220. The comparative analysis of the annotated complete genome showed the presence of 39 genes fragmented in the previous assembly, many of which were located near the repeat regions. Using RNA-Seq reads, we showed that a higher number of reads could be mapped to the complete genome than the draft genome and the gene expression profile obtained using the complete genome also differs from that obtained from the draft genome. Furthermore, by comparative transcriptomic analysis, we showed the correlation between expression levels of staphyloxanthin biosynthetic genes and the production of yellow pigment. This study highlighted the importance of long reads in completing the microbial genomes, especially those possessing repetitive elements.

genomics↗

bioRxiv2021

Purification of immunostimulatory glucose homopolymer from broccoli (Brassica oleracea var. italica)

We prepared broccoli (Brassica oleracea var. italica) neutral polysaccharides (flow-through fractions of anion exchange column chromatography from hot water extracts) from different broccoli cultivars and compared their immunostimulatory effects in the silkworm muscle contraction assay. The cultivars showed a wide range of activity, with the cultivar Winter dome showing the highest specific activity (more than 100 times higher than curdlan). Furthermore, the active substance was purified by gel filtration column chromatography. The active substance showed heterogeneous molecular weights of more than 270 kDa. Sugar composition analysis of the purified fraction revealed that more than 95% of its sugar component was glucose, suggesting that the immunostimulatory neutral polysaccharide from broccoli cultivar Winter dome was a homopolymer of glucose. The purified fraction also induced TNF production in cultured mouse macrophage cells. These results suggest that the glucose homopolymer in broccoli has an immunostimulatory effect on both arthropod and mammalian immune system.

biochemistry↗

bioRxiv2020

Alteration of menaquinone isoprenoid chain length and antibiotic sensitivity by single amino acid substitution in HepT

ObjectivesStaphylococcus aureus Smith strain is a historical strain widely used for research purposes in animal infection models for testing the therapeutic activity of antimicrobial agents. We found that it displayed higher sensitivity towards lysocin E, a menaquinone (MK) targeting antibiotic, compared to other S. aureus strains. Therefore, we further explored the mechanism of this hypersensitivity. MethodsMK production was analyzed by high-performance liquid chromatography and mass spectrometric analysis. S. aureus Smith genome sequence was completed using a hybrid assembly approach, and the MK biosynthetic genes were compared with other S. aureus strains. The hepT gene was cloned and introduced into S. aureus RN4220 strain using phage mediated recombination, and lysocin E sensitivity was analyzed by the measurement of minimum inhibitory concentration and colony-forming units. ResultsWe found that Smith strain produced MKs with the length of the side chain ranging between 8 - 10, as opposed to other S. aureus strains that produce MKs 7 - 9. We revealed that Smith strain possessed the classical pathway for MK biosynthesis like the other S. aureus. HepT, a polyprenyl diphosphate synthase involved in chain elongation of isoprenoid, in Smith strain was unique with a Q25P substitution. Introduction of hepT from Smith to RN4220 led to the production of MK-10 and an increased sensitivity towards lysocin E. ConclusionsWe found that HepT was responsible for the definition of isoprenoid chain length of MKs and antibiotic sensitivity.

microbiology↗

bioRxiv2019

Uncovering Staphylococcus aureus genes with roles in pathogenicity by silkworm infection model

The regulatory network of virulence factors production by Staphylococcus aureus, an opportunistic pathogen, is incompletely understood, and the functions of many uncharacterized genes in its genome remain to be uncovered. We screened 380 function unassigned genes disrupted mutants of the community-acquired methicillin-resistant S. aureus USA300 for pathogenicity using silkworms and identified 11 strains with reduced silkworm killing ability. Nine out of the 11 strains displayed reduced virulence in the mouse model as evidenced by reduced colony-forming units in organs of the infected mice. Three of the identified gene-disrupted mutants had reduced hemolytic activity, one among the three also had reduced proteolytic activity and pigment production. These results suggest that silkworm model could identify the genes required for virulence in the mouse model. The newly identified genes involved in virulence in this study facilitates the further understanding of the pathogenicity of S. aureus.\n\nImportanceWe performed a large scale screening of mutants of Staphylococcus aureus with disruption in function unassigned genes using silkworm infection model and identified eleven genes required for full virulence in silkworm. Nine of the eleven genes were involved in virulence in mice and were previously not known to aggravate virulence of S. aureus. The results suggest that silkworm model is suitable for quantitative measurement of virulence, which is shared between silkworms and mammals.

microbiology↗

bioRxiv2019

Transcriptomic profiling of Streptococcus pyogenes M1T1 strain in a mouse model of necrotizing fasciitis

Streptococcus pyogenes is a major cause of necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection. At the host infection site, the local environment and interaction between host and bacteria affect bacterial gene-expression profiles, but the S. pyogenes gene-expression pattern in necrotizing fasciitis remains unknown. In this study, we used a mouse model of necrotizing fasciitis and performed RNA-sequencing (RNA-seq) analysis of S. pyogenes M1T1 strain 5448 by using infected hindlimbs obtained at 24, 48, and 96 h post-infection. The RNA-seq analysis identified 483 bacterial genes whose expression was consistently altered in the infected hindlimbs as compared to their expression under in vitro conditions. The consistently enriched genes during infection included 306 genes encoding molecules involved in virulence, carbohydrate utilization, amino acid metabolism, trace-metal transport and vacuolar ATPase transport system. Surprisingly, drastic upregulation of 3 genes, encoding streptolysin S precursor (sagA), cysteine protease (speB), and secreted DNase (spd), was noted in the mouse model of necrotizing fasciitis (log2 fold-change values: >6.0, >9.4, and >7.1, respectively). Conversely, the consistently downregulated genes included 177 genes, containing genes associated with oxidative-stress response and cell division. These results suggest that S. pyogenes in necrotizing fasciitis changes its metabolism, decreases cell proliferation, and upregulates the expression of major toxins. Our findings could provide critical information for developing novel treatment strategies and vaccines for necrotizing fasciitis.\n\nAuthor summaryNecrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection, principally caused by a Streptococcus pyogenes. At infection sites in hosts, bacterial pathogens are exposed to drastically changing environmental conditions and alter global gene expression patterns for survival and pathogenesis. However, there is no previous report about transcriptomic profiling of S. pyogenes in the necrotizing fasciitis. Here, we conducted comprehensive gene-expression analyses of S. pyogenes in the mouse model of necrotizing fasciitis at three distinct time points during infection. Our results indicated that S. pyogenes drastically upregulates the expression of virulence-associated genes and shifts metabolic-pathway usage during infection. The high-level expressions in particular of toxins, such as cytolysins, proteases, and nucleases, were observed at infection sites. In addition, the consistently enriched genes identified here included genes for metabolism of arginine and histidine, and carbohydrate uptake and utilization. Conversely, the genes associated with oxidative-stress response and cell division were consistently downregulated in the mouse model of necrotizing fasciitis. These data will provide useful information necessary for establishing novel treatment strategies (166 words).

microbiology↗