PurA is the main target of aurodox, a type III secretion system inhibitor.

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

Insights into the structure-activity relationship of a type III secretion system inhibitor, aurodox.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

Persistent colonization of non-lymphoid tissue-resident macrophages by Stenotrophomonas maltophilia.

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713.

Transcriptional Downregulation of a Type III Secretion System under Reducing Conditions in Bordetella pertussis.

Bordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA-) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA- medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA- medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCE The type III secretion system (T3SS) of Bordetella pertussis forms a needlelike structure that protrudes from the bacterial cell surface. B. pertussis uses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence.

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

Bordetella bronchiseptica Bcr4 antagonizes the negative regulatory function of BspR via its role in type III secretion.

Bordetella species, including B. pertussis, have a type III secretion system that is highly conserved among gram-negative pathogenic bacteria. Genes encoding the component proteins of the type III secretion system are localized at the bsc locus in the Bordetella genome. Here, the function of a hypothetical protein Bcr4 encoded at the bsc locus in the B. bronchiseptica genome was investigated. A Bcr4-deficient mutant was created and the amounts of type III secreted proteins (e.g., BopB, BopN and Bsp22) in both the supernatant fraction and whole-cell lysates of the Bcr4-deficient mutant were determined. It was found that the amounts of these proteins were significantly lower than in the wild-type strain. The amounts of type III secreted proteins in the supernatant fraction and whole-cell lysates were much greater in a Bcr4-overproducing strain than in the wild-type strain. The type III secreted protein BspR reportedly negatively regulates the type III secretion system. Here, it was observed that a Bcr4 + BspR double-knockout mutant did not secrete type III secreted proteins, whereas the amounts of these proteins in whole-cell lysates of this mutant were nearly equal to those in whole-cell lysates of the BspR-deficient mutant. Bcr4 thus appears to play an essential role in the extracellular secretion of type III secreted proteins. Our data also suggest that Bcr4 antagonizes the negative regulatory function of BspR.

Bordetella effector BopN is translocated into host cells via its N-terminal residues.

Bordetella bronchiseptica infects a wide variety of mammals, the type III secretion system (T3SS) being involved in long-term colonization by Bordetella of the trachea and lung. T3SS translocates virulence factors (commonly referred to as effectors) into host cells, leading to alterations in the host's physiological function. The Bordetella effectors BopN and BteA are known to have roles in up-regulation of IL-10 and cytotoxicity, respectively. Nevertheless, the mechanism by which BopN is translocated into host cells has not been examined in sufficient detail. Therefore, to determine the precise mechanisms of translocation of BopN into host cells, truncated derivatives of BopN were built and the derivatives' ability to translocate into host cells evaluated by adenylate cyclase-mediated translocation assay. It was found that N-terminal amino acid (aa) residues 1-200 of BopN are sufficient for its translocation into host cells. Interestingly, BopN translocation was completely blocked by deletion of the N-terminal aa residues 6-50, indicating that the N-terminal region is critical for BopN translocation. Furthermore, BopN appears to play an auxiliary role in BteA-mediated cytotoxicity. Thus, BopN can apparently translocate into host cells and may facilitate activity of BteA.

Correlation between the spread of IMP-producing bacteria and the promoter strength of blaIMP genes.

The first report of transmissible carbapenem resistance encoded by blaIMP-1 was discovered in Pseudomonas aeruginosa GN17203 in 1988, and blaIMP-1 has since been detected in other bacteria, including Enterobacterales. Currently, many variants of blaIMPs exist, and point mutations in the blaIMP promoter have been shown to alter promoter strength. For example, the promoter (Pc) of blaIMP-1, first reported in P. aeruginosa GN17203, was a weak promoter (PcW) with low-level expression intensity. This study investigates whether point mutations in the promoter region have helped to create strong promoters under antimicrobial selection pressure. Using bioinformatic approaches, we retrieved 115 blaIMPs from 14,529 genome data of Pseudomonadota and performed multiple alignment analyses. The results of promoter analysis of the 115 retrieved blaIMPs showed that most of them used the Pc located in class 1 integrons (n = 112, 97.4%). The promoter analysis by year revealed that the blaIMP population with the strong promoter, PcS, was transient. In contrast, the PcW-TG population, which had acquired a TGn-extended -10 motif in PcW and had an intermediate promoter strength, gradually spread throughout the world. An inverse correlation between Pc promoter strength and Intl1 integrase excision efficiency has been reported previously [1]. Because of this trade-off, it is unlikely that blaIMPs with strong promoters will increase rapidly, but the possibility that promoter strength will increase with the use of other integrons cannot be ruled out. Monitoring of the blaIMP genes, including promoter analysis, is necessary for global surveillance of carbapenem-resistant bacteria.

Solid-state NMR spectroscopy reveals anomer specific transport of galactose in the milk yeast Kluyveromyces lactis.

Genetic evidence indicates that only the ß-anomer of galactose is transported to Kluyveromyces lactis cells by galactose/glucose transporter Hgt1p, and that aldose-1-epimerase encoded by GAL10 is a prerequisite for growth on galactose. Minor aldose-1-epimerases other than Gal10p also exist in K. lactis. Using a mutant defective in both aldose-1-epimerases, we show by solid-state nuclear magnetic resonance spectroscopy that only ß-anomer is transported in the cell and stays without or with a slow rate of conversion to α-anomer. Signals due to intracellular ß-galactose appeared at two positions, both of which were shifted towards higher magnetic fields than that of ß-galactose in aqueous solution, suggesting that incorporated galactose binds to cellular components, probably proteins.

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica.

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.

Bcr4 Is a Chaperone for the Inner Rod Protein in the Bordetella Type III Secretion System.

Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required to stabilize some T3SS components or localize them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pulldown assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pulldown assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we show that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS. IMPORTANCE The type III secretion system (T3SS) is a needle-like complex that projects outward from bacterial cells. Bordetella bronchiseptica uses the T3SS to inject virulence proteins into host cells. Our previous study reported that a protein named Bcr4 is essential for the secretion of virulence proteins from B. bronchiseptica bacterial cells and delivery through the T3SS. Because other bacteria lack a Bcr4 homologue, the function of Bcr4 has not been elucidated. In this study, we discovered that Bcr4 interacts with BscI, a component of the T3SS machinery. We show that a B. bronchiseptica BscI-deficient strain was unable to secrete type III secreted proteins. Furthermore, in a B. bronchiseptica strain that overproduces T3SS component proteins, Bcr4 is required to maintain BscI in bacterial cells. These results suggest that Bcr4 stabilizes BscI to allow construction of the T3SS in B. bronchiseptica.

Landscape of blaNDM genes in Enterobacteriaceae.

The blaNDM-1 gene encodes a carbapenemase, New Delhi metallo-ß-lactamase (NDM-1), and the ability to produce NDM-1 is spread among Enterobacteriaceae via horizontal gene transfer of plasmids. It has been widely accepted that blaNDM-1 is regulated by a hybrid promoter (PISAba125) consisting of a -10 box from the original blaNDM-1 and a -35 box from ISAba125. However, the conservation of this promoter and the vertical transmission of blaNDM genes by chromosomal integration have not been comprehensively analyzed. We retrieved the region containing the ORF of blaNDM-1 (>95% translated protein identity) and a region 120 bp upstream of the blaNDM-1 start codon from the complete sequence data of Enterobacteriaceae plasmids (n = 10,914) and chromosomes (n = 4908) deposited in GenBank, and the 310 extracted blaNDM genes were analyzed by an in-silico approach. The results showed that most blaNDM genes (99.0%) utilized the promoter, PISAba125. Interestingly, two blaNDM-1 genes from the genus Citrobacter utilized the ISCR1-derived outward-oriented promoters POUT (PISCR1). Furthermore, the insertion of ISAba125 and ISCR1 occurred upstream of the CCATATTT sequence, which is located upstream of the -10 box. We also confirmed that most of the blaNDM genes were disseminated by horizontal gene transfer of the plasmid, but 10 cases of the blaNDM genes were integrated into the chromosome via mobile genetic elements such as IS26, IS150, ISCR1, ICE, and Tn7-like elements. Thus, plasmid-mediated transmission of the PISAba125-blaNDM genes is predominant in Enterobacteriaceae. However, the spread of blaNDM genes with new promoters and vertical dissemination via chromosomal integrations may pose additional serious clinical problems.

[Adipocytokines and assay method in metabolic syndrome].

Adipocytokines, such as adiponectin, TNF-alpha, and leptin, are cytokines secreted by visceral adipocytes, and they are associated with metabolic syndrome. Adiponectin is one of the adipocytokines, and is a protein comprised of 244 amino acids. It is known as ACRP30, GBP28, and AdipoQ. Adiponectin is secreted by adipocytes, has three different isoforms, including trimers (low-molecular weight: LMW), hexamers (middle-molecular-weight: MMW), and higher-order oligomeric (high-molecular-weight: HMW) structures, and affects the biological activity. Adiponectin is a clinically relevant parameter measured routinely in subjects at risk of type 2 diabetes and metabolic syndrome. We investigated the adiponectin levels using a number of ELISA assay kits.

[Bacterial secretion systems: their function and contribution to disease process].

Bacterial pathogens possesses certain secretion systems to maintain their homeostasis and to exert full virulence. Currently, two translocons including Sec and Tat, and 7 secretion systems are found in Gram-positive and -negative bacteria. Some virulence factors, which are referred to as effectors, are directly translocated into the host cell via an injection apparatus, i.e., the type III secretion system. Thus, characterization of secretion systems and their delivered proteins into extracellular milieus is required for understanding of strategies of bacterial pathogens.

Guadinomines, Type III secretion system inhibitors, produced by Streptomyces sp. K01-0509. I: taxonomy, fermentation, isolation and biological properties.

Enteropathogenic Escherichia coli (EPEC) expressing the Type III secretion system (TTSS) induced hemolysis of sheep blood cells. Using this assay, six structurally related compounds designated as guadinomines were isolated as inhibitors of TTSS-induced hemolysis by ion exchange column chromatography and HPLC from the culture broth of Streptomyces sp. K01-0509. Guadinomines A and B showed potent inhibition with IC50 values of 0.02 and 0.007 microg/ml, respectively, guadinomine D showed moderate activity (IC50: 8.5 microg/ml), while guadinomines C1 and C2 and guadinomic acid had no activity.

Guadinomines, Type III secretion system inhibitors, produced by Streptomyces sp. K01-0509. II: physico-chemical properties and structure elucidation.

The structures of guadinomines, new inhibitors of a bacterial Type III secretion system produced by Streptomyces sp. K01-0509, were elucidated by spectroscopic studies including various NMR experiments. Guadinomines A, B, C(1), C(2) and D consist of a carbamoylated cyclic guanidinyl moiety, an alkyl chain moiety and an L-Ala-L-Val moiety in common, while guadinomic acid is a smaller molecule consisting of a carbamoylated cyclic guanidinyl moiety and a hydroxyl hexanoate moiety.

Ultrastructural analysis of the membrane insertion of domain 3 of streptolysin O.

Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. We performed an ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS] and native SLO. SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0 degrees C. After treatment with dithiothreitol, SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. Our morphological evidence demonstrates that an increase in temperature is necessary for the occurrence of conformational changes and for the formation of double-layered rings after the insertion of domain 3 into the host cell membrane. On the basis of a model of the oligomeric structure of SLO, we propose some new details of the mechanism of hemolysis by SLO.

Correction: BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages.

[This corrects the article DOI: 10.1371/journal.pone.0148387.].

Elevated serum levels of pigment epithelium-derived factor in the metabolic syndrome.

CONTEXT: Pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis with neuronal differentiating activity, inhibits endothelial cell injury in vitro, thus suggesting the involvement of PEDF in atherosclerosis. Therefore, elucidating the relationship between serum levels of PEDF and coronary risk factors could provide a clue to understanding the pathophysiological role of PEDF in vivo. OBJECTIVE: We examined whether serum levels of PEDF were associated with risk factors for coronary artery disease. DESIGN: The study was designed as a cross-sectional study. SETTING: The study was set within the general community. PATIENTS OR OTHER PARTICIPANTS: A total of 196 general Japanese residents (age 65.7 +/- 9.3 yr; 71 males and 125 females) without clinical evidence of coronary or peripheral arterial occlusive diseases were enrolled in this study. RESULTS: PEDF showed a normal distribution, ranging from 8-24 microg/ml, with a mean of 14.6 +/- 3.2 microg/ml. Multivariate analyses revealed that uric acid (P < 0.001), waist circumference (P = 0.009), insulin (P = 0.019), and triglycerides (P = 0.028) were significant independent determinants of serum PEDF levels. Age- and uric acid-adjusted PEDF levels were significantly higher (P = 0.048 for men and P = 0.007 for women) in proportion to the accumulation of the number of the components of the metabolic syndrome. CONCLUSIONS: The present study reveals that serum levels of PEDF are strongly associated with the metabolic syndrome. Our results suggest that serum PEDF levels may be elevated as a counter-system in the metabolic syndrome.

BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages.

BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis.