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Infection by enterotoxigenic Escherichia coli (ETEC) causes severe watery diarrhea and dehydration in humans. Heat-labile enterotoxin (LT) is a major virulence factor produced by ETEC. LT is one of AB 5 -type toxins, such as Shiga toxin (Stx) and cholera toxin (Ctx), and the B-subunit pentamer is responsible for high affinity binding to the LT-receptor, ganglioside GM1, through multivalent interaction. In this report, we found that Glu51 of the B-subunit plays an essential role in receptor binding compared with other amino acids, such as Glu11, Arg13, and Lys91, all of which were previously shown to be involved in the binding. By targeting Glu51, we identified four tetravalent peptides that specifically bind to the B-subunit pentamer with high affinity by screening tetravalent random-peptide libraries, which were tailored to bind to the B-subunit through multivalent interaction. One of these peptides, GGR-tet, efficiently inhibited the cell-elongation phenotype and the elevation of cellular cAMP levels, both induced by LT. Furthermore, GGR-tet markedly inhibited LT-induced fluid accumulation in the mouse ileum. Thus, GGR-tet represents a novel therapeutic agent against ETEC infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cholera toxin (Ctx) is a major virulence factor produced by Vibrio cholerae that can cause gastrointestinal diseases, including severe watery diarrhea and dehydration, in humans. Ctx binds to target cells through multivalent interactions between its B-subunit pentamer and the receptor ganglioside GM1 present on the cell surface. Here, we identified a series of tetravalent peptides that specifically bind to the receptor-binding region of the B-subunit pentamer using affinity-based screening of multivalent random-peptide libraries. These tetravalent peptides efficiently inhibited not only the cell-elongation phenotype but also the elevated cAMP levels, both of which are induced by Ctx treatment in CHO cells or a human colon carcinoma cell line (Caco-2 cells), respectively. Importantly, one of these peptides, NRR-tet, which was highly efficient in these two activities, markedly inhibited fluid accumulation in the mouse ileum caused by the direct injection of Ctx. In consistent, NRR-tet reduced the extensive Ctx-induced damage of the intestinal villi. After NRR-tet bound to Ctx, the complex was incorporated into the cultured epithelial cells and accumulated in the recycling endosome, affecting the retrograde transport of Ctx from the endosome to the Golgi, which is an essential process for Ctx to exert its toxicity in cells. Thus, NRR-tet may be a novel type of therapeutic agent against cholera, which induces the aberrant transport of Ctx in the intestinal epithelial cells, detoxifying the toxin., Competing Interests: Declaration of competing interest We declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to circulate in humans since its emergence in 2019. While infection in humans continues, numerous spillover events to at least 32 animal species, including companion and zoo animals, have been reported. Since dogs and cats are highly susceptible to SARS-CoV-2 and have direct contact with their owners and other household members, it is important to know the prevalence of SARS-CoV-2 in dogs and cats. Here, we established an ELISA to detect serum antibodies against the receptor-binding domain and the ectodomain of the SARS-CoV-2 spike and nucleocapsid proteins. Using this ELISA, we assessed seroprevalence in 488 dog serum samples and 355 cat serum samples that were collected during the early pandemic period (between May and June of 2020) and 312 dog serum samples and 251 cat serum samples that were collected during the mid-pandemic period (between October 2021 and January 2022). We found that two dog serum samples (0.41%) collected in 2020, one cat serum sample (0.28%) collected in 2020, and four cat serum samples (1.6%) collected in 2021 were positive for antibodies against SARS-CoV-2. No dog serum samples collected in 2021 were positive for these antibodies. We conclude that the seroprevalence of SARS-CoV-2 antibodies in dogs and cats in Japan is low, suggesting that these animals are not a major SARS-CoV-2 reservoir., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors.)

We previously reported that Vibrio cholerae in a viable but non-culturable (VBNC) state can be converted to a culturable state by treatment with catalase. This finding enabled us to develop an assay system to observe the time course of the conversion from VBNC to culturable in V. cholerae. VBNC cells began to convert to culturable cells as early as 2 h after catalase supplementation. Gene expression in VBNC cells during catalase treatment was analyzed using RNA microarray. Many ribosomal DNA genes were stimulated 6 h post catalase exposure, suggesting that the conversion-driving signal started prior to 6 h. Focusing on the period prior to cell proliferation, we found that 16 genes might be involved in the conversion mechanism in V. cholerae, and they showed enhanced expression at 2 h and 4 h after catalase addition. These upregulated genes included phage shock proteins (pspA, B, and C), alternative sigma factor (rpoE) and its negative regulator (rseA), cobW C terminal domain-containing protein, damage-inducible helicase (dinG), cholerae toxin secretion protein epsM, HTH-type transcription regulator (iscR), mechanosensitive ion channel family protein, anthranilate synthase component I, fructose-specific IIBC component, molybdenum import ATP-binding protein (modC), LysE family translocator, putative organic hydroperoxide resistance protein, and a hypothetical protein. This study identified genes involved in the catalase-induced conversion of V. cholerae VBNC cells to a culturable state and provided valuable insights into the mechanisms involved in the conversion process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Japan has reported a relatively small number of COVID-19 cases. Because not all infected persons receive diagnostic tests for COVID-19, the reported number must be lower than the actual number of infections. We assessed SARS-CoV-2 seroprevalence by analyzing >60,000 samples collected in Japan (Tokyo Metropolitan Area and Hokkaido Prefecture) during February 2020-March 2022. The results showed that ≈3.8% of the population had become seropositive by January 2021. The seroprevalence increased with the administration of vaccinations; however, among the elderly, seroprevalence was not as high as the vaccination rate. Among children, who were not eligible for vaccination, infection was spread during the epidemic waves caused by the SARS-CoV-2 Delta and Omicron variants. Nevertheless, seroprevalence for unvaccinated children <5 years of age was as low as 10% as of March 2022. Our study underscores the low incidence of SARS-CoV-2 infection in Japan and the effects of vaccination on immunity at the population level.

Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter ( P Stx1 ), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1 . In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe 2+ -responsive transcription factor. The intracellular Fe 2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe 2+ availability drove the formation of less Fe 2+ -Fur, less DNA binding to the P Stx1 region, and an increase in Stx1 production.