高屋 明子

The virulence of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), depends on the expression of toxins and virulence factors controlled by the quorum-sensing (QS) system, encoded on the virulence accessory gene regulator (agr) locus. The aim of this study was to identify a phytochemical that inhibits Agr-QS function and to elucidate its mechanism. We screened 577 compounds and identified physalin H, physalin B, and isophysalin B—–phytochemicals belonging to physalins found in plants of the Solanaceae family—–as novel Agr-QS modulators. Biological analyses and in vitro protein–DNA binding assays suggested that these physalins suppress gene expression related to the Agr-QS system by inhibiting binding of the key response regulator AgrA to the agr promoters, reducing the function of hemolytic toxins downstream of these genes in MRSA. Furthermore, although physalin F suppressed gene expression in the Agr-QS system, its anti-hemolytic activity was lower than that of physalins H, B, and isophysalin B. Conversely, five physalins isolated from the same plant with the ability to suppress Agr-QS did not reduce bacterial Agr-QS activity but inhibited AgrA binding to DNA in vitro. A docking simulation revealed that physalin interacts with the DNA-binding site of AgrA in three docking states. The carbonyl oxygens at C-1 and C-18 of physalins, which can suppress Agr-QS, were directed to residues N201 and R198 of AgrA, respectively, whereas these carbonyl oxygens of physalins, without Agr-QS suppression activity, were oriented in different directions. Next, 100-ns molecular dynamics simulations revealed that the hydrogen bond formed between the carbonyl oxygen at C-15 of physalins and L186 of AgrA functions as an anchor, sustaining the interaction between the carbonyl oxygen at C-1 of physalins and N201 of AgrA. Thus, these results suggest that physalin H, physalin B, and isophysalin B inhibit the interaction of AgrA with the agr promoters by binding to the DNA-binding site of AgrA, suppressing the Agr-QS function of S. aureus. Physalins that suppress the Agr-QS function are proposed as potential lead compounds in the anti-virulence strategy for MRSA infections.

The total synthesis of dragmacidins G and H was achieved for the first time by employing nucleophilic aromatic substitution and site-selective cross-coupling reactions using appropriately functionalized pyrazines as substrates. The evaluation of antibacterial activities of dragmacidin G, dragmacidin H, and synthetic analogues against Staphylococcus aureus and the efflux pump-deficient Salmonella Typhimurium revealed that the presence of a Br group on the indole ring adjacent to the sulfide unit was important for increasing antibacterial activities.

In this study, we developed a comb-shaped microfluidic device that can efficiently trap and culture a single cell (bacterium). Conventional culture devices have difficulty in trapping a single bacterium and often use a centrifuge to push the bacterium into the channel. The device developed in this study can store bacteria in almost all growth channels using the flowing fluid. In addition, chemical replacement can be performed in a few seconds, making this device suitable for culture experiments with resistant bacteria. The storage efficiency of microbeads that mimic bacteria was significantly improved from 0.2% to 84%. We used simulations to investigate the pressure loss in the growth channel. The pressure in the growth channel of the conventional device was more than 1400 PaG, whereas that of the new device was less than 400 PaG. Our microfluidic device was easily fabricated by a soft microelectromechanical systems method. The device was highly versatile and can be applied to various bacteria, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus.

Fractionation of the leaf extract from Murraya exotica led to the successful isolation of 12 compounds (1-12) with TRAIL-resistance-overcoming activity. Xanthinosin (1), 11α, 13-dihydroxanthinin (2), 11β, 13-dihydroxanthinosin (3), 4α, 11α, 13-trihydroxanthuminol (4), desacetylxanthanol (5), and lasidiol p-methoxybenzoate (6) were sesquiterpenes isolated from this plant for the first time, and 3 was isolated from natural sources for the first time. Among them, compounds 1 and 5 showed strong TRAIL-resistance-overcoming activity, but their mechanisms have already been revealed. Furthermore, dihydroxanthinin (2), 1, 5-dicaffeoylquinic acid (7), and (-) loliolide (8), which belong to different phytochemical groups, were investigated for their effects on increasing apoptosis induction to overcome TRAIL resistance using Western blot analysis. The results demonstrated that 2, 7, and 8 promoted TRAIL-induced apoptosis by increasing the expression of several proapoptotic markers, including cleaved caspases −3 and −8, and suppressing anti-apoptotic protein Bcl-2.

Results support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

<p>Metallo-β-lactamases (MBLs) are significant threats to humans because they deteriorate many kinds of β-lactam antibiotics and are key enzymes responsible for multi-drug resistance of bacterial pathogens. As a result of in vitro screening, two compounds were identified as potent inhibitors of two kinds of MBLs: imipenemase (IMP-1) and New Delhi metallo-β-lactamase (NDM-1). The binding structure of one of the identified compounds was clarified by an X-ray crystal analysis in complex with IMP-1, in which two possible binding poses were observed. Molecular dynamics (MD) simulations were performed by building two calculation models from the respective binding poses. The compound was stably bound to the catalytic site during the simulation in one pose. The binding model between NDM-1 and the compound was constructed for MD simulation. Calculation results for NDM-1 were similar to those of IMP-1. The simulation suggested that the binding of the identified inhibitory compound was also durable in the catalytic site of NDM-1. The compound will be a sound basis for the development of the inhibitors for MBLs.</p>

Atopic dermatitis (AD) is commonly associated with colonization by <italic>Staphylococcus aureus</italic> in the affected skin. To understand the role of <italic>S. aureus</italic> in the development of AD, we performed whole-genome sequencing of <italic>S. aureus</italic> strains isolated from the cheek skin of 268 Japanese infants 1 and 6 months after birth. About 45% of infants were colonized with <italic>S. aureus</italic> at 1 month regardless of AD outcome. In contrast, skin colonization by <italic>S. aureus</italic> at 6 months of age increased the risk of developing AD. Acquisition of dysfunctional mutations in the <italic>S. aureus</italic> Agr quorum-sensing (QS) system was primarily observed in strains from 6-month-old infants who did not develop AD. Expression of a functional Agr system in <italic>S. aureus</italic> was required for epidermal colonization and the induction of AD-like inflammation in mice. Thus, retention of functional <italic>S. aureus agr</italic> virulence during infancy is associated with pathogen skin colonization and the development of AD.

Serum IgG, which is mainly generated from IgG-secreting plasma cells in the bone marrow (BM), protects our body against various pathogens. We show here that the protein SiiE of Salmonella is both required and sufficient to prevent an efficient humoral immune memory against the pathogen by selectively reducing the number of IgG-secreting plasma cells in the BM. Attenuated SiiE-deficient Salmonella induces high and lasting titers of specific and protective Salmonella-specific IgG and qualifies as an efficient vaccine against Salmonella A SiiE-derived peptide with homology to laminin β1 is sufficient to ablate IgG-secreting plasma cells from the BM, identifying laminin β1 as a component of niches for IgG-secreting plasma cells in the BM, and furthermore, qualifies it as a unique therapeutic option to selectively ablate IgG-secreting plasma cells in autoimmune diseases and multiple myeloma.

The bacterial type III secretion system (T3SS) delivers virulence proteins, called effectors, into eukaryotic cells. T3SS comprises a transmembrane secretion apparatus and a complex network of specialized chaperones that target protein substrates to this secretion apparatus. However, the regulation of secretion switching from early (needle and inner rod) to middle (tip/filament and translocators) substrates is incompletely understood. Here, we investigated chaperone-mediated secretion switching from early to middle substrates in the T3SS encoded by Salmonella pathogenicity island 2 (SPI2), essential for systemic infection. Our findings revealed that the protein encoded by ssaH regulates the secretion of an inner rod and early substrate, SsaI. Structural modeling revealed that SsaH is structurally similar to class III chaperones, known to associate with proteins in various pathogenic bacteria. The SPI2 protein SsaE was identified as a class V chaperone homolog and partner of SsaH. A pulldown analysis disclosed that SsaH and SsaE form a heterodimer, which interacted with another early substrate, the needle protein SsaG. Moreover, SsaE also helped stabilize SsaH and a middle substrate, SseB. We also found that SsaE regulates cellular SsaH levels to translocate the early substrates SsaG and SsaI and then promotes the translocation of SseB by stabilizing it. In summary, our results indicate that the class III chaperone SsaH facilitates SsaI secretion, and a heterodimer of SsaH and the type V chaperone SsaE then switches secretion to SsaG. This is the first report of a chaperone system that regulates both early and middle substrates during substrate switching for T3SS assembly.

In primary infection with Salmonella, it has been reported-without consideration of Salmonella's functions-that humoral immunity plays no role in the clearance of bacteria. In fact, Salmonella targets and suppresses several aspects of humoral immunity, including B cell lymphopoiesis, B cell activation, and IgG production. In particular, the suppression of IgG-secreting plasma cell maintenance allows the persistence of Salmonella in tissues. Therefore, the critical role(s) of humoral immunity in the response to Salmonella infection, especially at the late phase, should be re-investigated. The suppression of IgG plasma cell memory strongly hinders vaccine development against non-typhoidal Salmonella (NTS) because Salmonella can also reduce humoral immune memory against other bacteria and viruses, obtained from previous vaccination or infection. We propose a new vaccine against Salmonella that would not impair humoral immunity, and which could also be used as a treatment for antibody-dependent autoimmune diseases to deplete pathogenic long-lived plasma cells, by utilizing the Salmonella's own suppression mechanism of humoral immunity.

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.

The viable but non-culturable (VBNC) state is a remarkable survival mechanism in which cells exist in a physiologically inactive state. Bacteria in the VBNC state do not form colonies, and thus, are difficult to detect using colony-based methods. As a result, VBNC bacteria are potentially virulent and can cause widespread contamination during food production. In the present study, we reported a novel biomarker, the membrane vesicle protein PagC, for the detection of VBNC Salmonella. Salmonella cells were chemically induced into the VBNC state by H2O2 treatment. The bacterial cells retained their shapes but were observed to release numerous membrane vesicles, which were accompanied by a transient PagC overexpression. Immunoblotting was performed to detect PagC in pathogenic strains, including Salmonella Enteritidis and S. Typhimurium, which are harmful and known to cause food-borne gastroenteritis in humans and other animals. Therefore, our findings demonstrated the potential use of PagC as a biomarker for the detection of VBNC Salmonella in food production.

Tosufloxacin (TFLX) is a fluoroquinolone antimicrobial agent. TFLX granules for children were initially released in Japan in 2010 to treat otitis media and pneumonia caused by drug-resistant bacteria, e.g. penicillin-resistant Streptococcus pneumoniae and beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae. The evolution of bacterial resistance since TFLX approval is not known. To clarify the influence of quinolones administered to children since their approval, we examined the resistance mechanism of TFLX-resistant S. pneumoniae isolated from paediatric patients as well as patient clinical characteristics. TFLX-resistant strains (MIC ≥ 2 mg/L) were detected among clinical isolates of S. pneumoniae derived from children (≤15 years old) between 2010 and 2014. These strains were characterised based on quinolone resistance-determining regions (QRDRs), i.e. gyrA, gyrB, parC, and parE. In addition, the antimicrobial susceptibility, serotype, and multilocus sequence type of strains were determined, pulsed-field gel electrophoresis was performed, and patient clinical characteristics based on medical records were assessed for cases with underling TFLX-resistant strains. Among 1168 S. pneumoniae isolates, two TFLX-resistant strains were detected from respiratory specimens obtained from paediatric patients with frequent exposure to TFLX. Both strains had mutations in the QRDRs of gyrA and parC. One case exhibited gradual changes in the QRDR during the clinical course. This is the first study of quinolone-resistant S. pneumoniae isolated from children, including clinical data, in Japan. These data may help prevent increases in infections of quinolone-resistant S. pneumoniae in children; specifically, the results emphasise the importance of administering fluoroquinolones only in appropriate cases.

We report an 8-year-old patient with catheter-related bacteremia caused by linezolid-resistant Staphylococcus epidermidis that was isolated after the long-term, repeated use of linezolid. Three S. epidermidis strains isolated from this patient were bacteriologically analyzed. While the strain isolated prior to linezolid initiation was susceptible to linezolid, two strains after linezolid therapy displayed low-level linezolid susceptibility (MIC, 4 mg/L) and linezolid resistance (MIC, 16 mg/L). T2500A mutation in two copies and G2575T mutations in three copies of 23S rRNA were detected in the low-susceptible strain and the resistant strain, respectively. Linezolid-resistant S. epidermidis infection is rare, but may occur with the long-term administration of linezolid. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmA(II) enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmA(II), rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmA(II) in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmA(II) activity was significantly enhanced by RlmCD-mediated premethylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmA(II), thereby facilitating TEL binding to the ribosome.

病原細菌は多様なエフェクター分子や毒素分子を産生し、宿主生体内の標的臓器/組織の摩耗・機能障害を引き起こす。さらに一部の細菌エフェクター・毒素は、免疫に代表される生体防御系を鈍化あるいは暴走させ、結果としてより複雑な感染病態を作り上げていく。これら細菌性病原因子は、宿主との相互作用を介した進化圧・淘汰圧の下、標的細胞の構造的・機能的脆弱性を巧妙に利用する姿へと変容し作り上げられてきたのであろう。それゆえ、細菌毒素・エフェクターの標的となる分子やシグナル伝達系を網羅的に解明する作業は、高等真核生物が築いてきた一見精緻な生体システムが持つ予期せぬアキレス腱を暴き出す結果につながる。そこで本稿では、こうした視点に立ち、宿主生体システムに潜む疾病感受性・疾病脆弱性の分子基盤を細菌エフェクター・細菌毒素研究を通して論じた6つの最近の知見を紹介する。(著者抄録)

Objectives: Linezolid has been reported to remain active against 98% of staphylococci with resistance identified in 0.05% of Staphylococcus aureus and 1.4% of CoNS. The objective of this study was to characterize the linezolid-resistance mechanisms in the linezolid-resistant CoNS strains isolated in Japan. Methods: Staphylococcus capitis strains exhibiting linezolid MICs &gt;8 mg/L isolated from inpatients between 2012 and 2014 were screened for cfr and mutations in 23S rRNA, L3 and L4 by PCR/sequencing. Isolates were also examined for mutations in the rlmN gene. Results: S. capitis had six 23S rRNA alleles. Five S. capitis isolates displayed linezolid MICs of 8, 16 and 32 mg/L. G2576U mutations were detected in three, four or five copies of 23S rRNA in all isolates. In two isolates exhibiting the highest linezolid MIC (32 mg/L) there was a large deletion in a single copy of 23S rRNA. Repeated 10 bp sequences were found in both 16S and 23S rRNAs, suggesting deletion by recombination between the repeats. One isolate had the mutation Ala-142 -&gt; Thr in the ribosomal protein L3. All linezolid-resistant isolates also demonstrated mutations in the gene encoding RlmN methyltransferase, leading to Thr-62 -&gt; Met and Gly-148 -&gt; Ser. Conclusions: Multiple mechanisms appeared to be responsible for the elevated linezolid resistance in S. capitis isolates: a G2576U mutation in different numbers of copies of 23S rRNA, loss of a single copy of 23S rRNA and a mutation in the ribosomal protein L3, suggesting the accumulation of independent mutational events.

病原細菌は多様なエフェクター分子や毒素分子を産生し, 宿主生体内の標的臓器／組織の摩耗・機能障害を引き起こす。さらに一部の細菌エフェクター・毒素は, 免疫に代表される生体防御系を鈍化あるいは暴走させ, 結果としてより複雑な感染病態を作り上げていく。これら細菌性病原因子は, 宿主との相互作用を介した進化圧・淘汰圧の下, 標的細胞の構造的・機能的脆弱性を巧妙に利用する姿へと変容し作り上げられてきたのであろう。それゆえ, 細菌毒素・エフェクターの標的となる分子やシグナル伝達系を網羅的に解明する作業は, 高等真核生物が築いてきた一見精緻な生体システムが持つ予期せぬアキレス腱を暴き出す結果につながる。そこで本稿では, こうした視点に立ち, 宿主生体システムに潜む疾病感受性・疾病脆弱性の分子基盤を細菌エフェクター・細菌毒素研究を通して論じた6 つの最近の知見を紹介する。

We previously reported that the ClpXP ATP-dependent protease specifically recognizes and degrades the flagellar master transcriptional activator complex, FlhD(4)C(2), to negatively control flagellar biogenesis. The flagellum-related protein, FliT, is also a negative regulator of flagellar regulon by inhibiting the binding of FlhD(4)C(2) to the promoter DNA. We have found a novel pathway of FliT inhibition of FlhD(4)C(2) activity connected to ClpXP proteolysis. An in vitro degradation assay using purified proteins shows that FliT selectively increases ClpXP proteolysis of the FlhC subunit in the FlhD(4)C(2) complex. FliT behaves specifically to ClpXP-dependent proteolysis of FlhC. An in vitro interaction assay detects the ternary complex of FliT-FlhD(4)C(2)-ClpX. FliT promotes the affinity of ClpX against FlhD(4)C(2) complex, whereas FliT does not directly interact with ClpX. Thus, FliT interacts with the FlhC in FlhD(4)C(2) complex and increases the presentation of the FlhC recognition region to ClpX. The DNA-bound form of FlhD(4)C(2) complex is resistant to ClpXP proteolysis. We suggest that the role of FliT in negatively controlling the flagellar gene expression involves increasing free molecules of FlhD(4)C(2) sensitive to ClpXP proteolysis by inhibiting the binding to the promoter DNA as well as enhancing the selective proteolysis of FlhC subunit by ClpXP.

Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmA(II), which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 mu g/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 mu g/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmA(II) to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmA(II) renders S. pneumoniae TEL susceptible.

YdiV is an EAL-like protein that acts as a post-transcriptional, negative regulator of the flagellar master transcriptional activator complex, FlhD4C2, in Salmonella enterica to couple flagellar gene expression to nutrient availability. Mutants defective in ClpXP protease no longer exhibit YdiV-dependent inhibition of FlhD4C2-dependent transcription under moderate YdiV expression conditions. ClpXP protease degrades FlhD4C2, and this degradation is accelerated in the presence of YdiV. YdiV complexed with both free and DNA-bound FlhD4C2; and stripped FlhD4C2 from DNA. A L22H substitution in FlhD was isolated as insensitive to YdiV inhibition. The FlhD L22H substitution prevented the interaction of YdiV with free FlhD4C2 and the ability of YdiV to release FlhD4C2 bound to DNA. These results demonstrate that YdiV prevents FlhD4C2-dependent flagellar gene transcription and acts as a putative adaptor to target FlhD4C2 for ClpXP-dependent proteolysis. Our results suggest that YdiV is an EAL-like protein that has evolved from a dicyclic-GMP phosphodiesterase into a dual-function regulatory protein that connects flagellar gene expression to nutrient starvation.

In enterobacteria such as Escherichia coli and Salmonella species, flagellar biogenesis is strictly dependent upon the master regulator flhDC. Here, we demonstrate that in enterohaemorrhagic E. coli (EHEC), the flagellar regulon is controlled by ClpXP, a member of the ATP-dependent protease family, through two pathways: (i) post-translational control of the FlhD/FlhC master regulator and (ii) transcriptional control of the flhDC operon. Both FlhD and FlhC proteins accumulated markedly following ClpXP depletion, and their half-lives were significantly longer in the mutant cells, suggesting that ClpXP is responsible for degrading FlhD and FlhC proteins, leading to downregulation of flagellar expression. ClpXP was involved in regulating the transcription of the flhD promoter only when the cells had entered stationary phase in a culture medium that markedly induced expression of the locus of enterocyte effacement (LEE). Comparative analyses of transcription from the flhD promoter in EHEC cells with different genetic backgrounds suggested that the downregulation of flhDC expression by ClpXP is dependent on the LEE-encoded GrIR-GrIA system. We have also shown that the degradation of FlhD and FlhC by ClpXP is responsible for downregulating flagellar expression even when LEE expression is induced.

Background: Many pathogens use a type III secretion system to translocate virulence proteins (called effectors) in order to adapt to the host environment. To date, many prediction tools for effector identification have been developed. However, these tools are insufficiently accurate for producing a list of putative effectors that can be applied directly for labor-intensive experimental verification. This also suggests that important features of effectors have yet to be fully characterized. Results: In this study, we have constructed an accurate approach to predicting secreted virulence effectors from Gram-negative bacteria. This consists of a support vector machine-based discriminant analysis followed by a simple criteria-based filtering. The accuracy was assessed by estimating the average number of true positives in the top-20 ranking in the genome-wide screening. In the validation, 10 sets of 20 training and 20 testing examples were randomly selected from 40 known effectors of Salmonella enterica serovar Typhimurium LT2. On average, the SVM portion of our system predicted 9.7 true positives from 20 testing examples in the top-20 of the prediction. Removal of the N-terminal instability, codon adaptation index and ProtParam indices decreased the score to 7.6, 8.9 and 7.9, respectively. These discrimination features suggested that the following characteristics of effectors had been uncovered: unstable N-terminus, non-optimal codon usage, hydrophilic, and less aliphathic. The secondary filtering process represented by coexpression analysis and domain distribution analysis further refined the average true positive counts to 12.3. We further confirmed that our system can correctly predict known effectors of P. syringae DC3000, strongly indicating its feasibility. Conclusions: We have successfully developed an accurate prediction system for screening effectors on a genome-wide scale. We confirmed the accuracy of our system by external validation using known effectors of Salmonella and obtained the accurate list of putative effectors of the organism. The level of accuracy was sufficient to yield candidates for gene-directed experimental verification. Furthermore, new features of effectors were revealed: non-optimal codon usage and instability of the N-terminal region. From these findings, a new working hypothesis is proposed regarding mechanisms controlling the translocation of virulence effectors and determining the substrate specificity encoded in the secretion system.

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.

A total of 132 Streptococcus pneumoniae isolates collected between 2005 and 2006 in Japan were examined for susceptibility to telithromycin (TEL) and macrolide. The overall resistance to macrolide was 80%. Among the isolates, 128 strains had low-level TEL susceptibility (minimal inhibitory concentrations [MICs] 0.03-1 mu g mL-1), suggesting that pneumococci with reduced susceptibility to TEL have appeared without prior exposure to the drug, although none of the isolates were assigned as TEL-resistant (breakpoint, &gt;= 4 mu g mL-1). Eight of these isolates (MIC 0.5-1 mu g mL-1) were analyzed for macrolide resistance determinants and genetic relatedness. They all carried mefE-mel, which encodes the macrolide efflux genetic assembly, and three also harbored ermB, which encodes rRNA methylase. Allele replacement mutagenesis of the corresponding genes in the clinical isolates revealed that reduced TEL susceptibility (MIC 1 mu g mL-1) in S. pneumoniae may be caused by acquisition of the mefE-mel element only and additionally conferred by the ermB determinant.

Salmonella enterica serovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of a host, S. enterica induces Sip secretion in response to sucrose or simple salts, such as NaCl. We analyzed induction of host-independent Sip secretion by monitoring protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that addition of sucrose or simple salts, such as NaCl, to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription of hiLA, hilC, and hilD, which encode known regulators of Salmonella virulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the BarA/SirA two-component signal transduction pathway, but not the two-component sensor kinase EnvZ, directly activated hilD and hilA transcription and thus Sip secretion in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependent csrB and csrC genes. A model is proposed for the major pathway in which sucrose or salt signals to enhance virulence gene expression.

Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of Ion disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

Salmonella enterica serovar Typhimurium delivers a variety of proteins via the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system into host cells, where they elicit several physiological changes, including bacterial invasion, macrophage apoptosis, and enteropathogenesis. Once Salmonella has established a systemic infection, excess macrophage apoptosis would be detrimental to the pathogen, as it utilizes macrophages as vectors for systemic dissemination throughout the host. Therefore, SPI1 expression must be restricted to one or a few specific locations in the host. In the present study, we have demonstrated that the expression of this complex of genes is repressed by the ATP-dependent ClpXP protease, which therefore suppresses macrophage apoptosis. Depletion of ClpXP caused significant increases in the amounts of two SPI1-encoded transcriptional regulators, HilC and HilD, leading to the stimulation of hilA induction and therefore activation of SPI1 expression. Our evidence shows that ClpXP regulates cellular levels of HilC and HilD via the control of flagellar gene expression. Subsequent experiments demonstrated that the flagellum-related gene product FliZ controls HilD posttranscriptionally, and this in turn activates HilC. These findings suggest that the ClpXP protease coregulates SPI1-related virulence phenotypes and motility. ClpXP is a member of the stress protein family induced in bacteria exposed to hostile environments such as macrophages.

細菌感染において生体防御の最前線に位置するマクロファージは細菌をファゴソームに内包し、ファゴソームを成熟させることによりリソソームと融合させ、細菌を死に至らしめる。しかしながら、細胞内寄生細菌であるサルモネラはマクロファージに取り込まれた後、エフェクターと呼ばれる病原因子を宿主細胞に輸送し殺菌機構をハイジャックすることにより、食細胞内で生存、増殖し感染を成立させる。サルモネラは2つのタイプIII分泌システムにより複数のエフェクターをファゴソーム膜に局在させる。サルモネラはファゴソームの成熟過程に応じてタイプIII分泌システムを使い分けることにより、細胞内環境に適応することが明らかにされてきている。本稿では、サルモネラのエフェクターがファゴソーム膜の成熟過程を制御する分子機構について紹介する。(著者抄録)

A Salmonella organism is one of the most important food-borne zoonoses in the world. This study illustrates the application of the dot blot hybridization assay to identify Salmonella strains isolated from eggs, chicken, and beef carcasses that possess virulence determinants that have been identified as significant factors in pathogenesis of the organism over the years. All the 59 Salmonella isolates examined were found to possess spiC and invA genes essential for Salmonella spp. to enter intestinal epithelial cells. Other effector proteins were also found in the order of sipC (66.1%), invF (37.3%), and hilA (11.9%). The presence of these virulence determinants clearly demonstrates the significance of dot blot hybridization assay to differentiate Salmonella strains in the absence of molecular typing tools.

ATP-dependent Lon protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) appeared to invade successfully the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of BALB/c mice and appeared to be easily eradicated by the host after oral immunization. As detected by flow cytometry, the population of major histocompatibility complex class I (MHC-I)-expressing macrophages and dendritic cells (DCs) was increased in the PP of mice immunized with CS2022 on day 6 after immunization. Thereafter, the population of splenic surface CD69(+) T lymphocytes prepared from mice immunized with CS2022 6 weeks prior to measurement increased as a result of the administration of the extracellular vesicles of RAW264.7 macrophage-like cells derived by Salmonella challenge. In addition, the proliferation of CD8(+) and even of CD4(+)T cells isolated from mouse spleens immunized with CS2022 was enhanced after cocultivation with naive DCs in the presence of the extracellular vesicles. These findings indicate that the extracellular vesicles prepared from the Salmonella-challenged macrophages carried salmonellae antigens to bystander DCs, thereby stimulating T-cell responses. Therefore, as antigen presentation after phagocytosis should be a central process in the T-cell activation that occurs in response to Salmonella infection, an oral immunization with CS2022 sufficiently induces T cell-mediated immunity in mice.

Transposon Tn2610, found in a conjugative plasmid from an Escherichia coli isolate recovered at a hospital in Chiba, Japan, in 1975, was completely sequenced. Tn2610 is 23,883 bp long and is bracketed by two transposition modules, a Tn1721-like module and a Tn21-derived module, which correspond, respectively, to the long inverted repeats IRa and IRb previously described for this transposon. Although both tnpA genes are intact, only that in the Tn21-derived module (IRb) functions in the transposition, while that in the Tn1721-derived module (IRa) cannot recognize the 38-bp imperfect repeat at the end of the IRb element. Both tnpR and res are present in IRa, while the tnpR gene of IRb is interrupted by the insertion of an IS26 insertion element. The intervening region, between the res site of the Tn1721 module and IS26, carries multiple integron-associated resistance genes within a Tn21 backbone, including a region identical to that found in the genome of Salmonella enterica serovar Typhimurium DT104. These findings suggest that Tn2610 originated from Tn1721 and Tn21, with extensive recombination events with other elements which have resulted in a complex mosaic structure.

The DnaK chaperone binds non-specifically to many unfolded polypeptides and also binds selectively to specific substrates. Although its involvement in targeting the unfolded polypeptides to assist proper folding is well documented, less is known about its role in targeting the folded polypeptides. We demonstrate that DnaK regulates the expression of the Salmonella flagellar regulon by modulating the FlhD and FlhC proteins, which function as master regulators at the apex of a transcription hierarchy comprising three classes of genes. FlhD and FlhC form an FlhD(2)C(2) complex that activates sigma(70) promoter of class 2 genes. In Delta dnaK cells, FlhD and FlhC proteins seemed to be assembled into hetero-tetrameric FlhD(2)C(2) but the complex was not fully active in class 2 gene transcription, suggesting that the DnaK chaperone is involved in activating native FlhD(2)C(2) complex into a regulator of flagellar regulon expression. This is the first time that involvement of the DnaK chaperone machinery in activating folded oligomerized proteins has been demonstrated.