Takaaki Shimohata

Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024

Ketone bodies serve several functions in the intestinal epithelium, such as stem cell maintenance, cell proliferation and differentiation, and cancer growth. Nevertheless, there is limited understanding of the mechanisms governing the regulation of intestinal ketone body concentration. In this study, we elucidated the factors responsible for ketone body production and excretion using shRNA-mediated or pharmacological inhibition of specific genes or functions in the intestinal cells. We revealed that a fasting-mimicked culture medium, which excluded glucose, pyruvate, and glutamine, augmented ketone body production and excretion in the Caco2 and HT29 colorectal cells. This effect was attenuated by glucose or glutamine supplementation. On the other hand, the inhibition of the mammalian target of rapamycin complex1 (mTORC1) recovered a fraction of the excreted ketone bodies. In addition, the pharmacological or shbeclin1-mediated inhibition of autophagy suppressed ketone body excretion. The knockdown of basigin, a transmembrane protein responsible for targeting monocarboxylate transporters (MCTs), such as MCT1 and MCT4, suppressed lactic acid and pyruvic acid excretion but increased ketone body excretion. Finally, we found that MCT7 (SLC16a6) knockdown suppressed ketone body excretion. Our findings indicate that the mTORC1–autophagy axis and MCT7 are potential targets to regulate ketone body excretion from the intestinal epithelium.

Campylobacter jejuni is a leading cause of food-borne disease worldwide. The pathogenicity of C. jejuni is closely associated with the internalization process in host epithelial cells, which is related to a host immune response. Autophagy indicates a key role in the innate immune system of the host to exclude invasive pathogens. Most bacteria are captured by autophagosomes and degraded by autophagosome-lysosome fusion in host cells. However, several pathogens, such as Salmonella and Shigella, avoid and/or escape autophagic degradation to establish infection. But autophagy involvement as a host immune response to C. jejuni infection has not been clarified. This study revealed autophagy association in C. jejuni infection. During infection, C. jejuni activated the Rho family small GTPase Rac1 signaling pathway, which modulates actin remodeling and promotes the internalization of this pathogen. In this study, we found the LC3 contribution to C. jejuni invasion signaling via the Rac1 signaling pathway. Interestingly, during C. jejuni invasion, LC3 was recruited to bacterial entry site depending on Rac1 GTPase activation just at the early step of the infection. C. jejuni infection induced LC3-II conversion, and autophagy induction facilitated C. jejuni internalization. Also, autophagy inhibition attenuated C. jejuni invasion step. Moreover, Rac1 recruited LC3 to the cellular membrane, activating the invasion of C. jejuni. Altogether, our findings provide insights into the new function of LC3 in bacterial invasion. We found the interaction between the Rho family small GTPase, Rac1, and autophagy-associated protein, LC3.

Ultraviolet (UV) irradiation is used to disinfect water and food and can be classified as UVA (detected at wavelengths 320–400 nm), UVB (280–320 nm), and UVC (Vibrio parahaemolyticus and selected six mutants with high sensitivity to UVA irradiation. Genes associated with this phenotype include F-type H⁺-transporting ATPases (atp), as well as those involved in general secretion (gsp), and ubiquinone and terpenoid-quinone biosynthesis (ubi). Gene complementation resulted in decreased sensitivity to UVA-LED. The atp mutants had lower intracellular adenosine triphosphate (ATP) concentrations than the wild-type treatment, with 20 mM L-serine resulting in elevated ATP concentrations and decreased sensitivity to UVA-LED. The gsp mutants exhibited high levels of extracellular protein transport and the ubi mutants exhibited significantly different intracellular concentrations of ubiquinone-8. Taken together, our results suggest that the protein products of the atp, gsp, and ubi genes may regulate sensitivity to UVA irradiation.

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock. This study aimed to determine the ideal irradiation by ultraviolet-light emitting diodes (UV-LEDs) for IAV disinfection. We irradiated the IAV H1N1 subtype with 4.8 mJ/cm² UV using eight UV-LEDs [peak wavelengths (WL) = 365, 310, 300, 290, 280, 270, and 260 nm)] or a mercury low pressure (LP)-UV lamp (Peak WL = 254 nm). Inactivation was evaluated by the infection ratio of Madin–Darby canine kidney (MDCK) cells or chicken embryonated eggs. Irradiation by the 260 nm UV-LED showed the highest inactivation among all treatments. Because the irradiation-induced inactivation effects strongly correlated with damage to viral RNA, we calculated the correlation coefficient (R_AE) between the irradiant spectrum and absorption of viral RNA. The R_AE scores strongly correlated with the inactivation by the UV-LEDs and LP-UV lamp. To increase the R_AE score, we combined three different peak WL UV-LEDs (hybrid UV-LED). The hybrid UV-LED (R_AE = 86.3) significantly inactivated both H1N1 and H6N2 subtypes to a greater extent than 260 nm (R_AE = 68.6) or 270 nm (R_AE = 42.2) UV-LEDs. The R_AE score is an important factor for increasing the virucidal effects of UV-LED irradiation.

The metabolic response of host cells upon infection is pathogen specific, and infection-induced host metabolic reprogramming may have beneficial effects on the proliferation of pathogens. V. parahaemolyticus contains a range of virulence factors to manipulate host signaling pathways and metabolic processes. In this study, we identified that the T3SS1 VopQ effector rewrites host metabolism in conjunction with the inflammation and cell death processes. Understanding how VopQ reprograms host cell metabolism during the infection could help us to identify novel therapeutic strategies to enhance the survival of host cells during V. parahaemolyticus infection.

Microbes in the human gut play a role in the production of bioactive compounds, including some vitamins. Although several studies attempted to identify definitive markers for certain vitamin deficiencies, the role of gut microbiota in these deficiencies is unclear. To investigate the role of gut microbiota in deficiencies of four vitamins, B₂, B₆, folate, and B₁₂, we conducted a comprehensive analysis of metabolites in mice treated and untreated with antibiotics. We identified glycolate (GA) as a novel marker of vitamin B₂ (VB2) deficiency, and show that gut microbiota sense dietary VB2 deficiency and accumulate GA in response. The plasma GA concentration responded to reduced VB2 supply from both the gut microbiota and the diet. These results suggest that GA is a novel marker that can be used to assess whether or not the net supply of VB2 from dietary sources and gut microbiota is sufficient. We also found that gut microbiota can provide short-term compensation for host VB2 deficiency when dietary VB2 is withheld.

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