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Salmonella enterica serovar Typhimurium is an important foodborne pathogen that causes diarrhea. S. Typhimurium elicits inflammatory responses and colonizes the gut lumen by outcompeting the microbiota. Although evidence is accumulating with regard to the underlying mechanism, the infectious stage has not been adequately defined. Peptidoglycan amidases are widely distributed among bacteria and play a prominent role in peptidoglycan maintenance by hydrolyzing peptidoglycans. Amidase activation is required for the regulation of at least one of two cognate activators, NlpD or EnvC (also called YibP). Recent studies established that the peptidoglycan amidase AmiC-mediated cell division specifically confers a fitness advantage on S. Typhimurium in the inflamed gut. However, it remains unknown which cognate activators are involved in the amidase activation and how the activators influence Salmonella sp. pathogenesis. Here, we characterize the role of two activators, NlpD and EnvC, in S. Typhimurium cell division and gut infection. EnvC was found to contribute to cell division of S. Typhimurium cells through the activation of AmiA and AmiC. The envC mutant exhibited impairments in gut infection, including a gut colonization defect and reduced ability to elicit inflammatory responses. Importantly, the colonization defect of the envC mutant was unrelated to the microbiota but was conferred by attenuated motility and chemotaxis of S. Typhimurium cells, which were not observed in the amiA amiC mutant. Furthermore, the envC mutant was impaired in its induction of mucosal inflammation and sustained gut colonization. Collectively, our findings provide a novel insight into the peptidoglycan amidase/cognate activator circuits and their dependent pathogenesis.

The acetylcholinesterase inhibitor, acotiamide, improves gastric motility and is clinically used to treat functional dyspepsia. The present study aimed to identify the transporters involved in the distribution of acotiamide in stomach tissue. Acotiamide uptake by the gastric cancer-derived model cell line, Hs746 T, was Na+- and pH-independent. The initial uptake velocity of acotiamide was saturable with increasing concentrations of acotiamide and was inhibited by selective serotonin reuptake inhibitors, which are potent inhibitors of the plasma membrane monoamine transporter (PMAT). The uptake of acotiamide by PMAT gene-transfected HEK293 cells was saturable, with similar Km (197.9 μM) values to those of uptake by Hs 746T cells (106 μM). Moreover, immunoreactivity of PMAT was found in the gastric smooth muscle and vascular endothelial cells. These results suggest that PMAT contributes to the distribution of acotiamide in the stomach, where it exerts its pharmacological effects.

The twin-arginine translocation (Tat) system is involved in not only a wide array of cellular processes but also pathogenesis in many bacterial pathogens; thus, this system is expected to become a novel therapeutic target to treat infections. To the best of our knowledge, involvement of the Tat system has not been reported in the gut infection caused by Citrobacter rodentium. Here, we studied the role of Tat in C. rodentium gut infection, which resembles human infection with enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). A C. rodentium Tat loss-of-function mutant displayed prolonged gut colonization, which was explained by reduced inflammatory responses and, particularly, neutrophil infiltration. Further, the Tat mutant had colonization defects upon coinfection with the wild-type strain of C. rodentium. The Tat mutant also became hypersensitive to bile acids, and an increase in fecal bile acids fostered C. rodentium clearance from the gut lumen. Finally, we show that the chain form of C. rodentium cells, induced by a Tat-dependent cell division defect, exhibits impaired resistance to bile acids. Our findings indicate that the Tat system is involved in gut colonization by C. rodentium, which is associated with neutrophil infiltration and resistance to bile acids. Interventions that target the Tat system, as well as luminal bile acids, might thus be promising therapeutic strategies to treat human EHEC and EPEC infections.

Members of the organic anion transporting polypeptide (OATP) transporter family are involved in drug absorption and disposition, and therefore drug–drug and drug–food interactions at these transporters may have both pharmacological and toxicological effects. Accordingly, a detailed understanding of the substrates and modulators, including inhibitors and stimulators, of the transporters, as well as the resulting interactions, is required. However, this is not straightforward, since OATP transporters have multiple binding sites, which exhibit different selectivity for substrates and modulators. Here, we focused on the characterization of the binding sites of OATP2B1, which contributes to the intestinal absorption and hepatic disposition of drugs. Site-directed mutagenesis studies indicated that histidine residues His579 and His618 are involved in the low- and high-affinity sites for estrone-3-sulfate (E13S) transport, respectively. Uptakes of pravastatin, rosuvastatin and sulfasalazine were decreased by mutations of His579 and His618 of OATP2B1, whereas uptakes of fexofenadine and prostaglandin E2 were not affected. Naringin, an OATP inhibitor, reduced the uptakes of pravastatin, rosuvastatin, sulfasalazine and low-concentration E13S, but not other substrates. In addition, progesterone stimulated the uptake of low-concentration E13S but did not affect or even decreased the uptakes of other substrates. These observations confirm that multiple binding sites are present on OATP2B1, resulting in differential characteristics in the recognition of substrates and modulators. Accordingly, identification of the locations of binding sites on OATP2B1 and their detailed characterization will be necessary to improve the prediction of drug–drug and drug–food interactions on OATP2B1.

Streptomyces albulus IFO14147 produces epsilon-poly-L-lysine, which exhibits antimicrobial activity. In the MIC studies with antibiotics, S. albulus IFO14147 was shown to be resistant to kanamycin and amikacin, which are aminoglycoside (AG) antibiotics. We report here the isolation of the AG-resistance gene from S. albulus IFO14147 and the substrate specificity of the gene product, AAC(6')-Isa, which catalyzes N-acetylation at the 6' position of AGs, thereby inactivating them. Kinetic studies revealed that this enzyme has remarkably wide substrate specificity. The V(max)/K(m) values determined for AGs vary by a factor of up to 6,300, a much wider range than those observed for the AAC(6')s from Enterococcus faecium [AAC(6')-Ii] and Salmonella enteritidis [AAC(6')-Iy]. In addition, AAC(6')-Isa was able to acetylate lividomycin A, which has a hydroxy group at the 6' position. Enzymatically acetylated lividomycin A was found to be highly susceptible to mild base hydrolysis, suggesting that the enzyme also catalyzes O-acetyltransfer.

Streptomyces albulus IFO14147 produces epsilon-poly-L-lysine, which exhibits antimicrobial activity. It is necessary for its molecular breeding to develop host-vector systems. We recently found a novel cryptic plasmid, pNO33, in this strain. As part of a search for a selectable marker gene for pNO33, we report here the isolation and analysis of the beta-lactamase gene of this strain, which can grow on ampicillin-containing plates. It was shown that the beta-lactamase production in S. albulus was induced by ampicillin. By introducing a genomic library of S. albulus into Escherichia coli, a 3.6-kbp fragment was identified as the region involved in ampicillin resistance. It contained three open reading frames, all of which are highly homologous to the beta-lactamase (the blaL product) and its regulatory proteins (the blaA and blaB products) of S. cacaoi. The growth phenotypes and enzyme assaying of E. coli and S. lividans showed that the blaL homologue (blaSa) encodes a beta-lactamase required for ampicillin resistance. The beta-lactamae gene can be utilized as a selectable marker in a cloning vector of S. albulus. However, the beta-lactamase activity was decreased in E. coli and repressed in S. lividans by the blaA and blaB homologues (blaASa and blaBSa). It appears as if the blaASa product is a repressor of blaSa instead of an activator as in S. cacaoi.

A high-molecular-size plasmid (37-kb), named pNO33, has been isolated from Streptomyces albulus IFO14147, a producer of e-poly- l -lysine which exhibited antimicrobial activity. The sequence analysis of a 4.6-kb fragment in pNO33 revealed four putative open reading frames, one of which exhibited a significant homology to the bldB gene product involved in morphogenesis and antibiotic production by S. coelicolor .

Streptomyces albulus IFO14147 produces epsilon-poly-L-lysine (epsilon-PL), an amino acid homopolymer antibiotic that is used as a food preservative in many countries, including Japan, South Korea, and the United States. To construct an overproducer of the industrially important epsilon-PL and to develop a deeper understanding of its biosynthetic mechanism, we developed systems for DNA delivery into the S. albulus strain based on both polyethylene glycol-mediated protoplast transformation and intergeneric conjugation from Escherichia coli. The successes of these transformations in particular were accomplished by employing the new cryptic-plasmid-based shuttle vectors constructed in this study. The genetic systems developed here should facilitate a molecular genetic approach to S. albulus.