Biology subjects

Inaoka, D.

Publications and source records attributed to Inaoka, D..

4 recordsLinked to original sources

bioRxiv2026

Characterisation of novel Campylobacter jejuni Type VI secretion system (T6SS) effectors and exploration of the roles of the C. jejuni T6SS in bacterial antagonism and human host cell interaction

Campylobacter jejuni is a leading global cause of acute foodborne gastroenteritis however, C. jejuni lacks some of the classic virulence determinants associated with other common enteric bacterial pathogens. In recent years an increasing number of C. jejuni isolates have been identified to encode Type Six Secretion System (T6SS), an apparatus utilised by Gram-negative bacteria to secrete toxic bacterial effectors into neighbouring cells. Despite the prevalence of the T6SS and previous investigations, the roles of the C. jejuni T6SS are still not well characterised especially when compared to our knowledge of other clinically relevant T6SS-positive bacterial species. Additionally, as of yet, no C. jejuni T6SS cargo effectors have been characterised. In this study, we show the C. jejuni 488 strain T6SS displays contact-dependent antagonistic behaviour towards T6SS-negative C. jejuni, Campylobacter coli, Escherichia coli and Enterococcus faecium strains suggesting the presence of the T6SS contributes to the competitive capacity of this C. jejuni T6SS-positive strain. Moreover, this antagonistic activity is linked to the functionality of CJ488_0980 and CJ488_0982, two novel putative Tox-REase-7 domain-containing effectors, which were identified through bioinformatical analysis of the C. jejuni 488 strain genome. Additionally, our investigations propose the C. jejuni 488 T6SS contributes to interaction, invasion and intracellular survival in human intestinal epithelial cells (IEC). Collectively, these initial findings are the first examples of in vitro investigation of putative cargo effectors in Campylobacter spp. and provide valuable insights into the roles of C. jejuni T6SS effectors in bacterial competition and pathogenesis. This study highlights the importance of T6SS as an emerging virulence determinant in Campylobacter spp. warranting further investigation.

microbiology↗

bioRxiv2024

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei operates in the gluconeogenic direction in cellulo under in vivo-like conditions

In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis glucose 6- phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We showed here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, (i) fructose-1,6- bisphosphatase (FBPase), the canonical enzyme performing this reaction, (ii) sedoheptulose- 1,7-bisphosphatase (SBPase) and (iii) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyse the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas a 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we showed for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is probably due to the supramolecular organisation of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channelling.

microbiology↗

bioRxiv2021

The trypanosome UDP-glucose pyrophosphorylase is imported by piggybacking into glycosomes where unconventional sugar nucleotide synthesis takes place

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways usually present in the cytosol of other eukaryotes, such as glycolysis and biosynthesis of sugar nucleotides. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localised in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisomal targeting signal (PTS1 and PTS2). The questions we addressed here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotide functional and (ii) how the PTS-free UGP is imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3-10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knock-down UGP mutant cell line (RNAiUGP) was rescued by expressing a recoded UGP in the organelle (RNAiUGP/EXPrUGP-GPDH). Our conclusion was supported by targeted metabolomic analyses (IC-HRMS) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. ImportanceUnusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localisation of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here we showed that the cytosolic and glycosomal pathways are functional. Like in all other eukaryotes, the cytosolic pathways feed glycosylation reactions, however the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK), which are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

microbiology↗

bioRxiv2019

PITX1 protein interacts with ZCCHC10 to regulate hTERT mRNA transcription

Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, resulting in continuous cell division that is a crucial factor for malignant transformation. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is a new functional unit that suppresses hTERT transcription, and plays a crucial role in cancer development through regulation of telomerase activity.

cancer biology↗