Multilayered proteomics reveals that JAM-A promotes breast cancer progression via regulation of amino acid transporter LAT1.

Recent studies have shown that transmembrane-type tight junction proteins are upregulated in various cancers compared with their levels in normal tissues and are involved in cancer progression, suggesting that they are potential therapeutic targets. Here, we demonstrated the expression profile and a novel role of junctional adhesion molecule-A (JAM-A) in breast cancer. Immunohistochemistry of surgical specimens showed that JAM-A was highly expressed from carcinoma in situ lesions, as in other adenocarcinomas, with higher expression in invasive carcinomas. High expression of JAM-A contributed to malignant aspects such as lymph node metastasis and lymphatic involvement positivity. In breast cancer cells, JAM-A expression status affects malignant potentials including proliferation and migration. Multilayered proteomics revealed that JAM-A interacts with the amino acid transporter LAT1 in breast cancer cells. JAM-A regulates the expression of LAT1 and interacts with it on the whole cell membrane, leading to enhanced amino acid uptake to promote tumor growth. Double high expression of JAM-A and LAT1 predicts poor prognosis in patients with breast cancer. Of note, an antibody against an extracellular domain of JAM-A suppressed the proliferation of breast cancer cells. Our findings indicate the possibility of JAM-A-targeted therapy ideally combined with LAT1-targeted therapy as a new therapeutic strategy against breast cancer.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.

BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

Invasive pulmonary aspergillosis with candidiasis: usefulness of molecular and ultrastructural morphological analysis on FFPE tissue for invasive fungal infections.

Invasive pulmonary aspergillosis (IPA) is one of the most frequent forms of invasive fungal infections (IFI); however, it is often difficult to identify the pathogenic fungal species and to select appropriate treatments for patients with IFI including IPA. Here, we describe the detailed pathophysiology of an autopsy case of severe respiratory failure due to IPA with candidiasis. The patient developed severe respiratory failure after influenza infection and died, and the autopsy revealed a mixed disease of IPA with candidiasis. In this study, in addition to the routine pathological examination, we further examined formalin-fixed paraffin-embedded (FFPE) tissues by scanning electron microscopy (SEM) and partial genomic DNA sequencing. Although optical microscopy alone was insufficient to identify the pathogenic organisms, SEM clearly depicted the characteristic morphology of Aspergillus sp. and Candida sp. as closely overlapping in a nested fashion, providing evidence of mixed infection of both fungal species in a focal site. The technique using FFPE tissue in combination with ultrastructural observation by SEM, elemental analysis by SEM-EDX, and DNA sequencing is promising for analyzing the pathophysiology of IFI.

Aberrant expression of junctional adhesion molecule-A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155.

Recent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule-A (JAM-A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM-A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM-A significantly suppressed cell proliferation and colony-forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM-A reduced cell proliferation ability and that loss of JAM-A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM-A and formed a physical interaction with JAM-A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155-positive cases expressed a high level of JAM-A, and patients with the expression pattern of PVR/CD155 positive/JAM-A high had significantly shorter periods of relapse-free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM-A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM-A is therefore a potential therapeutic target for this malignancy.

Sitafloxacin has a potent activity for eradication of extended spectrum ß-lactamase-producing fluoroquinolone-resistant Escherichia coli forming intracellular bacterial communities in uroepithelial cells.

INTRODUCTION: Eradication of asymptomatic bacteriuria (ASB) before urological procedures is important to reduce the risk for infectious complications after surgery. However, the appropriate regimen for antimicrobial treatment has not been fully determined. We experienced continuous (over 10 months) isolation of extended spectrum ß-lactamase (ESBL)-producing fluoroquinolone-resistant Escherichia coli from urine of an asymptomatic patient. The four isolates obtained (SMESC1 to 4) were international high-risk clones of O25b:H4-ST131-H30R, and originated from one strain, as revealed by the whole genome sequences. Although the patient received meropenem (MEPM) and fosfomycin (FOM), to which the strains were susceptible before the urological procedures, they could not be eradicated. METHODS: To explore the reason for the continuous isolation even after MEPM and FOM administration, antimicrobial killing of adherent and/or intracellular bacterial communities (IBC) formed by coculture of the E. coli cells and T24 bladder epithelial cells were examined. RESULTS: FOM and levofloxacin did not decrease viable E. coli cells compared with gentamicin. MEPM partly decreased them, and sitafloxacin (STFX) decreased them most potently. These observations indicate that E. coli can survive in the urinary tract under antimicrobial administration, and some antimicrobials such as FOM and MEPM cannot eradicate E. coli in uroepithelial cells. Adhesion on urinary epithelial cells and/or IBC formation might result in continuous isolation from the urinary tract and recurrence of ASB and urinary tract infections. CONCLUSIONS: The present study suggests that STFX is a promising optional agent for the eradication of ESBL-producing fluoroquinolone-resistant E. coli in the urinary tract before urological procedures.

A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease.

Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid ß (Aß) peptides. Yet the role of autophagy in Aß production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aß clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/Becn1 in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. Becn1F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aß oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by Becn1 gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar Becn1-dependent protective effects on Aß removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of Becn1-mediated autophagy hyperactivation in the prevention of AD.

Comparison of measurements of anti-PLA2R antibodies in Japanese patients with membranous nephropathy using in-house and commercial ELISA.

BACKGROUND: The prevalence of antibodies against M-type anti-phospholipase A2 receptor (PLA2R) was reported to be ~ 70-80% in early studies on idiopathic membranous nephropathy (iMN) cohorts from Western countries, China, and Korea, and ~ 50% in recent studies on two Japanese iMN cohorts. METHODS: We developed an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of anti-PLA2R antibodies, and examined sera from 217 patients with iMN, 22 patients with secondary MN (sMN), and 50 healthy individuals. All patients and healthy individuals were Japanese. The relationships between levels of anti-PLA2R antibodies and clinical parameters were analyzed. Serum samples were also tested using a standardized commercial ELISA (Euroimmun, Germany). RESULTS: In our ELISA, OD values greater than the mean + 3 standard deviation of healthy subjects were considered to be positive for anti-PLA2R antibodies. Of the patients with iMN, 33.6% (73/217) were positive, but all sMN patients were negative. Our ELISA and the Euroimmun ELISA had a high concordance (93.5%). The proportion of patients with nephrotic syndrome was significantly higher in anti-PLA2R antibody-positive patients than in antibody-negative patients (65.8 vs. 37.5%, P < 0.001). Levels of anti-PLA2R antibodies were significantly correlated with levels of urinary protein and serum albumin (P = 0.004 and P < 0.001, respectively). CONCLUSIONS: The prevalence of anti-PLA2R antibodies in our Japanese iMN cohort was lower than that in the previous studies from other countries and other Japanese institutes. The low prevalence of antibodies may be related with the characteristics of enrolled patients with mild proteinuria and undetectable antibody levels.

Evaluation of consistency in quantification of gene copy number by real-time reverse transcription quantitative polymerase chain reaction and virus titer by plaque-forming assay for human respiratory syncytial virus.

The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

Tigecycline Nonsusceptibility Occurs Exclusively in Fluoroquinolone-Resistant Escherichia coli Clinical Isolates, Including the Major Multidrug-Resistant Lineages O25b:H4-ST131-H30R and O1-ST648.

Tigecycline (TGC) is a last-line drug for multidrug-resistant Enterobacteriaceae We investigated the mechanism(s) underlying TGC nonsusceptibility (TGC resistant/intermediate) in Escherichia coli clinical isolates. The MIC of TGC was determined for 277 fluoroquinolone-susceptible isolates (ciprofloxacin [CIP] MIC, <0.125 mg/liter) and 194 fluoroquinolone-resistant isolates (CIP MIC, >2 mg/liter). The MIC50 and MIC90 for TGC in fluoroquinolone-resistant isolates were 2-fold higher than those in fluoroquinolone-susceptible isolates (MIC50, 0.5 mg/liter versus 0.25 mg/liter; MIC90, 1 mg/liter versus 0.5 mg/liter, respectively). Two fluoroquinolone-resistant isolates (O25b:H4-ST131-H30R and O125:H37-ST48) were TGC resistant (MICs of 4 and 16 mg/liter, respectively), and four other isolates of O25b:H4-ST131-H30R and an isolate of O1-ST648 showed an intermediate interpretation (MIC, 2 mg/liter). No TGC-resistant/intermediate strains were found among the fluoroquinolone-susceptible isolates. The TGC-resistant/intermediate isolates expressed higher levels of acrA and acrB and had lower intracellular TGC concentrations than susceptible isolates, and they possessed mutations in acrR and/or marR The MICs of acrAB-deficient mutants were markedly lower (0.25 mg/liter) than those of the parental strain. After continuous stepwise exposure to CIP in vitro, six of eight TGC-susceptible isolates had reduced TGC susceptibility. Two of them acquired TGC resistance (TGC MIC, 4 mg/liter) and exhibited expression of acrA and acrB and mutations in acrR and/or marR In conclusion, a population of fluoroquinolone-resistant E. coli isolates, including major extraintestinal pathogenic lineages O25b:H4-ST131-H30R and O1-ST648, showed reduced susceptibility to TGC due to overexpression of the efflux pump AcrAB-TolC, leading to decreased intracellular concentrations of the antibiotics that may be associated with the development of fluoroquinolone resistance.

Mitochondrial proteins NIP-SNAP-1 and -2 are a target for the immunomodulatory activity of clarithromycin, which involves NF-κB-mediated cytokine production.

Macrolide antibiotics have immunomodulatory activities, including suppression of cytokine production, cell adhesion molecule expression, and mucin production. These immunomodulatory activities improve the symptoms of respiratory diseases associated with chronic inflammation. However, the underlying molecular mechanism(s) is not well understood yet. To address this, we prepared clarithromycin (CAM)-conjugated Sepharose and examined bound cellular proteins by proteome analysis. We identified mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25-like protein homolog (NIP-SNAP)-1 and -2 and very long-chain acyl-CoA dehydrogenase (VLCAD) as CAM-binding proteins. Production of proinflammatory cytokines (IL-8 and IL-6) induced by lipopolysaccharides (LPSs) and Pam3-CSK4 in human epithelial cell lines BEAS-2B and T24 were suppressed by knockdown of NIP-SNAP-1 or -2, and partly by knockdown of VLCAD. Also, knockdown of NIP-SNAP-1 or -2 in various cell lines suppressed LPS-induced expression of IL-8 and IL-6 mRNA and NF-κB activity. Thus, CAM suppresses NF-κB-mediated proinflammatory cytokine production by interacting with mitochondrial proteins, NIP-SNAP-1 and -2.

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3.

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-ß and -λ production. Furthermore, IFN-ß promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

ATPase activity and ATP-dependent conformational change in the co-chaperone HSP70/HSP90-organizing protein (HOP).

Co-chaperones help to maintain cellular homeostasis by modulating the activities of molecular chaperones involved in protein quality control. The HSP70/HSP90-organizing protein (HOP) is a co-chaperone that cooperates with HSP70 and HSP90 in catalysis of protein folding and maturation in the cytosol. We show here that HOP has ATP-binding activity comparable to that of HSP70/HSP90, and that HOP slowly hydrolyzes ATP. Analysis of deletion mutants revealed that the ATPase domain of HOP is in the N-terminal TPR1-DP1-TPR2A segment. In addition, HOP changes its conformation in the presence of ATP. These results indicate that HOP is a unique co-chaperone that undergoes an ATP-dependent conformational change.

Intrafamilial, Preferentially Mother-to-Child and Intraspousal, Helicobacter pylori Infection in Japan Determined by Mutilocus Sequence Typing and Random Amplified Polymorphic DNA Fingerprinting.

BACKGROUND: The infection route of Helicobacter pylori has been recognized to be mainly intrafamilial, preferentially mother-to-child, especially in developed countries. To determine the transmission route, we examined whether multilocus sequence typing (MLST) was useful for analysis of intrafamilial infection. The possibility of intraspousal infection was also evaluated. MATERIALS AND METHODS: Clonal relationships between strains derived from 35 index Japanese pediatric patients, and their family members were analyzed by two genetic typing procedures, MLST and random amplified polymorphic DNA (RAPD) fingerprinting. RESULTS: Mostly coincident results were obtained by MLST and RAPD. By MLST, the allele of loci in the isolates mostly matched between the index child and both the father and mother for 9 (25.7%) of the 35 patients, between the index child and the mother for 25 (60.0%) of the 35 patients. CONCLUSIONS: MLST is useful for analyzing the infection route of H. pylori as a highly reproducible method. Intrafamilial, especially mother-to-children and sibling, infection is the dominant transmission route. Intraspousal infection is also thought to occur in about a quarter in the Japanese families.

Aminoglycosides suppress the protein folding activity of the molecular chaperone HSC70: implication of a structure-activity relationship.

BACKGROUND: The mechanism of aminoglycoside (AG)-induced nephrotoxicity has not been fully elucidated. We previously reported that gentamicin suppresses the functions of HSC70, a heat shock cognate protein, which is a cytosolic molecular chaperone in mammalian cells. However, the relationship between HSC70 dysfunction and nephrotoxicity has not been clarified yet. METHODS: The effects of 11 AGs, including gentamicin, on the function, protein refolding and substrate binding of HSC70 were examined in vitro. The protein refolding activity of HSC70 was examined by enzymatic activity restoration of the denatured firefly luciferase. Substrate binding activity of HSC70 was examined by the binding of fluorescent-dye-labeled reduced carboxymethlylated α-lactalbumin determined by HSC70/substrate complex formation by native polyacrylamide gel electrophoresis. RESULTS: Strong inhibitory activity of AGs on the protein refolding activity of HSC70 was observed for arbekacin, gentamicin, neomycin and sisomicin, but not spectinomycin, streptomycin, ribostamycin and paromomycin, and inhibition was weak for tobramycin, amikacin and kanamycin. Neomycin strongly suppressed the formation of the HSC70/substrate complex and had the lowest concentration that resulted in 50% inhibition (IC50; in the order of 10(-4) M). Arbekacin, gentamicin, sisomicin and tobramycin had IC50 values in the order of 10(-3) M. Amikacin, ribostamycin and paromomycin had no effective potencies. CONCLUSIONS: The inhibition efficacies of AGs for protein refolding of HSC70 were as follows: neomycin >> gentamicin, arbekacin, and sisomicin > tobramycin, amikacin and kanamycin > spectinomycin, streptomycin, ribostamycin and paromomycin. Amino groups and/or hydroxyl groups located at 2', 3', 4', and 6' of amino sugar residue II, as well as the number of amino sugar rings were closely associated with the inhibition efficacies of AGs. Inhibition efficacies of AGs towards the function of HSC70 correlated well with the nephrotoxicity of AGs deduced from the in vivo studies previously reported. Amino groups/hydroxyl groups and the number of amino sugar rings of AGs were shown to be important for inhibition efficacies. The results strongly suggest that the AG-induced dysfunction of HSC70 is involved in the nephrotoxicity.

The clarithromycin-binding proteins NIPSNAP1 and 2 regulate cytokine production through mitochondrial quality control.

The mechanism underlying the anti-inflammatory effect of macrolide antibiotics, such as clarithromycin (CAM), remains to be clarified. The CAM-binding proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25 (SNAP25)-like protein homolog (NIPSNAP) 1 and 2 are involved in the immune response and mitochondrial homeostasis. However, the axis between CAM-NIPSNAP-mitochondria and Toll-like receptor (TLR) and their molecular mechanisms remain unknown. In this study, we sought to elucidate the relationship between mitochondrial homeostasis mediated by NIPSNAP1 and 2 and the immunomodulatory effect of CAM. NIPSNAP1 or 2 knockdown (KD) by RNA interference impaired TLR4-mediated interleukin-8 (IL-8) production. Similar impairment was observed upon treatment with mitochondrial function inhibitors. However, IL-8 secretion was not impaired in NIPSNAP1 and 2 individual knockout (KO) and double KO (DKO) cells. Moreover, the oxygen consumption rate (OCR) in mitochondria measured using a flex analyzer was significantly reduced in NIPSNAP1 or 2 KD cells, but not in DKO cells. CAM also dose-dependently reduced the OCR. These results indicate that CAM suppresses the IL-8 production via the mitochondrial quality control regulated by temporary functional inhibition of NIPSNAP1 and 2. Our findings provide new insight into the mechanisms underlying cytokine production, including the TLR-mitochondria axis, and the immunomodulatory effects of macrolides.

Affinity of ß-Lactam Antibiotics for Neisseria gonorrhoeae Penicillin-Binding Protein 2 Having Wild, Cefixime-Reduced-Susceptible, and Cephalosporin (Ceftriaxone)-Resistant penA Alleles.

Multidrug-resistant Neisseria gonorrhoeae is a serious concern worldwide. Resistance to ß-lactam antibiotics occurs through mutations in penicillin-binding proteins (PBPs), acquisition of ß-lactamases, and alteration of antibiotic penetration. Mosaic structures of penA, which encodes PBP2, play a major role in resistance to ß-lactams, especially cephalosporins. Ceftriaxone (CRO) is recognized as the only satisfiable antibiotic for the treatment of gonococcal infections; however, CRO-resistant isolates have emerged in the community. Here, we examined the affinity of ß-lactam antibiotics for recombinant PBP2 in a competition assay using fluorescence-labeled penicillin. We found no or little difference in the affinities of penicillins and meropenem (MEM) for PBP2 from cefixime (CFM)-reduced-susceptible strain and cephalosporin-resistant strain. However, the affinity of cephalosporins, including CRO, for PBP2 from the cephalosporin-resistant strain was markedly lower than that for PBP2 from the CFM-reduced-susceptible-resistant strain. Notably, piperacillin (PIP) showed almost the same affinity for PBP2 from penicillin-susceptible, CFM-reduced-susceptible, and cephalosporin (including CRO)-resistant strains. Thus, PIP/tazobactam and MEM are candidate antibiotics for the treatment of CRO-resistant/multidrug-resistant N. gonorrhoeae.

Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Imiquimod suppresses propagation of herpes simplex virus 1 by upregulation of cystatin A via the adenosine receptor A1 pathway.

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A(1) and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.

Positive relationship between a polymorphism in Helicobacter pylori neutrophil-activating protein a gene and iron-deficiency anemia.

BACKGROUND: Numerous studies have suggested a link between iron-deficiency anemia (IDA) and Helicobacter pylori infection. Previously, we found that strains isolated from IDA patients showed higher levels of Fe ion uptake and Fe-ion-dependent rapid proliferation than those of strains derived from patients without IDA. MATERIALS AND METHODS: Twenty-four H. pylori strains from IDA patients (IDA strains) and 25 strains from patients who had H. pylori gastritis without anemia (non-IDA strains) were examined. Their nucleotide sequences of napA, fur, and feoB, which contribute to Fe ion uptake, were determined. RESULTS: Numerous polymorphisms of the three genes were found in both strains. Frequency of neutrophil-activating protein A (NapA), which encoded by napA, with threonine at amino acid residue No. 70 (Thr70-type NapA) was significantly higher in IDA strains than in non-IDA strains. Strains with Thr70-type NapA showed significantly higher levels of Fe(3+) and Fe(2+) uptake than did strains with other types, Ser70-type of NapA, which is found in standard strains. Other significantly different occurrences of polymorphisms between IDA and non-IDA groups were not observed in these genes. CONCLUSION: The results suggest that H. pylori strains with Thr70-type NapA have enhanced Fe ion uptake ability and are associated with the pathogenesis of IDA.