Structural basis of the PE-PPE protein interaction in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has developed multiple strategies to adapt to the human host. The five type VII secretion systems, ESX-1-5, direct the export of many virulence-promoting protein effectors across the complex mycobacterial cell wall. One class of ESX substrates is the PE-PPE family of proteins, which is unique to mycobacteria and essential for infection, antigenic variation, and host-pathogen interactions. The genome of Mtb encodes 168 PE-PPE proteins. Many of them are thought to be secreted through ESX-5 secretion system and to function in pairs. However, understanding of the specific pairing of PE-PPE proteins and their structure-function relationship is limited by the challenging purification of many PE-PPE proteins, and our knowledge of the PE-PPE interactions therefore has been restricted to the PE25-PPE41 pair and its complex with the ESX-5 secretion system chaperone EspG5. Here, we report the crystal structure of a new PE-PPE pair, PE8-PPE15, in complex with EspG5. Our structure revealed that the EspG5-binding sites on PPE15 are relatively conserved among Mtb PPE proteins, suggesting that EspG5-PPE15 represents a more typical model for EspG5-PPE interactions than EspG5-PPE41. A structural comparison with the PE25-PPE41 complex disclosed conformational changes in the four-helix bundle structure and a unique binding mode in the PE8-PPE15 pair. Moreover, homology-modeling and mutagenesis studies further delineated the molecular determinants of the specific PE-PPE interactions. These findings help develop an atomic algorithm of ESX-5 substrate recognition and PE-PPE pairing.

Association of polymorphisms in the Chr18q11.2 locus with tuberculosis in Chinese population.

A GWAS study has reported that two single nucleotide polymorphisms (SNPs) were associated with predisposition to tuberculosis (TB) in African populations. These two loci represented the long-waited GWAS hits for TB susceptibility. To determine whether these two SNPs are associated with TB in Chinese population, we attempted an replication in a cohort of over one thousand Chinese TB patients and 1,280 healthy controls using melting temperature shift allele-specific genotyping analysis. We found that only SNP rs4331426 was significantly associated with TB in Chinese population (p = 0.011). However, the effect was opposite. The G allele of the SNP in Chinese population is a protective allele (OR = 0.62, 95 % CI 0.44-0.87), while it was the risk allele for African population (OR = 1.19, 95 % CI 1.12-1.26). No significance was found for SNP rs2335704. The results provided an independent support for a role in susceptibility to TB for SNP rs4331426. However, it also indicated that direct predisposition element to TB and the association effects may vary across ethnic groups.

CISH and susceptibility to infectious diseases.

BACKGROUND: The interleukin-2-mediated immune response is critical for host defense against infectious pathogens. Cytokine-inducible SRC homology 2 (SH2) domain protein (CISH), a suppressor of cytokine signaling, controls interleukin-2 signaling. METHODS: Using a case-control design, we tested for an association between CISH polymorphisms and susceptibility to major infectious diseases (bacteremia, tuberculosis, and severe malaria) in blood samples from 8402 persons in Gambia, Hong Kong, Kenya, Malawi, and Vietnam. We had previously tested 20 other immune-related genes in one or more of these sample collections. RESULTS: We observed associations between variant alleles of multiple CISH polymorphisms and increased susceptibility to each infectious disease in each of the study populations. When all five single-nucleotide polymorphisms (SNPs) (at positions -639, -292, -163, +1320, and +3415 [all relative to CISH]) within the CISH-associated locus were considered together in a multiple-SNP score, we found an association between CISH genetic variants and susceptibility to bacteremia, malaria, and tuberculosis (P=3.8x10(-11) for all comparisons), with -292 accounting for most of the association signal (P=4.58x10(-7)). Peripheral-blood mononuclear cells obtained from adult subjects carrying the -292 variant, as compared with wild-type cells, showed a muted response to the stimulation of interleukin-2 production--that is, 25 to 40% less CISH expression. CONCLUSIONS: Variants of CISH are associated with susceptibility to diseases caused by diverse infectious pathogens, suggesting that negative regulators of cytokine signaling have a role in immunity against various infectious diseases. The overall risk of one of these infectious diseases was increased by at least 18% among persons carrying the variant CISH alleles.

In vitro compatibility study of cephalosporin with intraocular irrigating solutions and intracameral medications.

BACKGROUND: To study the compatibility of cephalosporins with intraocular irrigating solutions and intracameral medications commonly used in cataract surgery. DESIGN: The was an in vitro experiment conducted in the Research Laboratory of the Department of Microbiology, the Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. SAMPLES: Three cephalosporins--cefazolin, cefuroxime and ceftazidime--were separately diluted and mixed with irrigating solutions and intracameral medications to form 192 samples and 12 control solutions. METHODS: The cephalosporins were dissolved in normal saline and further diluted to the concentration of 1 mg in 0.1 mL with normal saline, Ringer's solution, balanced salt solution and fortified balanced salt solutions. These were mixed with balanced salt solutions or fortified balanced salt solutions, with adrenaline, acetylcholine or carbachol and kept at 37°C for 2 h. The concentrations of free cephalosporins were measured with rapid high-performance liquid chromatography at baseline (0 h) and at 2 h. MAIN OUTCOME MEASURES: Free concentrations of cephalosporins at 2 h were compared with mean baseline (0 h) value. A difference of 3 standard deviations or more was considered statistically significant. RESULTS: At 2 h there was a significant drop in the cefuroxime concentration in preparations in which cefuroxime was diluted with normal saline (P < 0.01). In all preparations, the final concentrations of cephalosporins were higher than the minimal inhibitory concentrations (MIC(90)) for microbials commonly isolated from the external eye. CONCLUSION: Cefazolin, cefuroxime and ceftazidime were compatible with irrigating solutions and intracameral medications commonly used in cataract surgery.

Genetic susceptibility to Tuberculosis: Interaction between HLA-DQA1 and age of onset.

Several genome-wide association studies (GWAS) identified new single nucleotide polymorphisms (SNPs) with susceptibility to Tuberculosis (TB). However, many of them were not replicated across ethnic groups. The cause of this phenomenon of genetic heterogeneity is uncertain. Here, we attempted to replicate and evaluate the mechanism that causes genetic heterogeneity in several putative TB predisposition loci found by previous GWAS, including chromosome 18q, ASAP1, DUSP14, and HLA-DQA1. A Chinese cohort of 1200 TB patients and 1280 population controls were genotyped. The results showed that genetic predisposition to TB might operate in an age-specific manner. While no significant association was found in the whole samples, a SNP of HLA-DQA1, rs9272785, showed suggestive association within the young-onset TB subgroup (onset at 20-40 years of age, N = 396). The results provide support for the hypothesis that there are different pathogenesis mechanisms causing clinical TB disease in different age groups, and that genetics probably play a substantial role only in young-onset TB.

[Chemical constituents and anti-tuberculosis activity of root of Rhodiola kirilowii].

The chemical constituents of Rhodiola kirilowii were separated and purified by repeated column chromatography on silica gel, RP - 18, Sephadex LH -20 and semi-preparative HPLC. Each compound was characterized by spectroscopic and physical data. Twelve compounds have been purified and identified to be beta-sitosterol (1), tyrosol (2), trans-hydroxycinnamic acid (3), geranyl beta-glucopyranoside (4), neryl beta-glucopyranoside (5), hexyl beta-glucopyranoside (6), gallic acid (7), (-) -epigallocatechin gallate (8), rhodiolgin (9), isolariciresinol-9-O-beta-glucopyranoside (10), rhodiooctanoside (11), and sacranoside B (12). Among them, compounds 3, 6, 9-12 were isolated from Rhodiola kirilowii for the first time; Compounds 4 and 5 were obtained for the first time from the genus Rhodiola. The in vitro activities against Macobacterium tuberculosis (ATCC 27294) of its extracts and pure components were evaluated by testing their MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration). The 80% (a. q.) EtOH extract, EtOAc-soluble extract, compounds 7 and 8 exhibited in-vitro inhibitory and bactericidal activities against Macobacterium tuberculosis in different extent.

Effectiveness of meibomian gland massage combined with topical levofloxacin against ocular surface flora in patients before penetrating ocular surgery.

OBJECTIVE: To investigate the bacterial profile in the conjunctiva and meibomian glands in patients before penetrating ocular surgeries, and to compare the anti-bacterial efficacy of 0.5% levofloxacin and its combination with meibomian gland massage. DESIGN: Hospital-based, case-control study. PARTICIPANTS: Two hundred and twenty-six eyes from 226 patients with non-infective ocular diseases and scheduled for penetrating ocular surgeries. METHODS: Tested eyes were administered topical 0.5% levofloxacin (4 times daily) for 2 days. Among them, 91 eyes received meibomian gland massage before levofloxacin application. Samples from the conjunctival sac and meibomian glands were collected for aerobic and anaerobic cultures. MAIN OUTCOME MEASURES: Culture-positivity and bacterial strains. RESULTS: Before treatment, aerobes and anaerobes were cultured from 38.5% and 11.0% of the conjunctival samples respectively, compared with 38.5% and 8.8% in the meibomian secretions respectively. Staphylococcus epidermidis and Propionibacterium acnes were the commonest isolated aerobe and anaerobe. Two-day application of levofloxacin reduced aerobic growth to 29.6% in the conjunctiva and 19.3% in the meibomian glands. It had no effect on the anaerobes in these regions (13.3% in the conjunctiva and 10.4% in the meibomian glands). Combined levofloxacin with meibomian gland massage further reduced aerobic growth to 19.8% in the conjunctiva and 11.0% in the meibomian glands. It also drastically decreased anaerobic growth in the meibomian glands (1.1%). CONCLUSIONS: Meibomian glands carrying considerable bacteria should be considered as a potential source of contamination in ocular surgery. Meibomian gland massage shows additional anti-bacterial effects to topical levofloxacin and could be recommended as a complementary preoperative prophylaxis.

Bacteriological profile of ocular surface flora in meibomian gland dysfunction.

PURPOSE: To compare the bacteriological profile of meibomian gland secretion and conjunctival cul-de-sac in subjects with and without meibomian gland dysfunction (MGD). METHODS: This hospital-based study enrolled 201 eyes from 201 MGD patients and 84 eyes from 84 age- and gender-matched controls. Samples from the conjunctivae and meibomian gland secretion were obtained. Both aerobic and anaerobic cultures were performed. Differences in the culture-positivity and bacterial strains between two groups were compared. RESULTS: 36.9% and 10.7% of the control meibomian gland secretion recovered aerobes and anaerobes, respectively. A higher rate of aerobic (44.0%) and similar rate of anaerobic (10.7%) bacteria were isolated from the conjunctival cul-de-sac in these controls. Patients with MGD showed significantly higher positive culture rates from both the meibomian gland secretion (75.6% for aerobes and 34.3% for anaerobes) and conjunctival cul-de-sac (64.7% for aerobes and 30.8% for anaerobes). All p values were ≤ 0.001. In both groups and at either location, the predominant species isolated were Staphylococcus epidermidis (aerobes) and Propionibacterium acnes (anaerobes). Of note, the MGD patients harbor a much more complex bacterial profile than the controls. CONCLUSION: Both aerobic and anaerobic bacteria can be isolated from the ocular surface of healthy subjects. Patients with MGD demonstrate significantly higher culture positivity and more complex bacterial profiles than the controls. Bacterial related cytotoxicity and/or inflammation may contribute to the pathological process of MGD.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolated in Jiangsu Province, China, with a focus on fluoroquinolone resistance.

In this study, the phenotypic and genotypic resistance to fluoroquinolones in Neisseria gonorrhoeae isolated in Jiangsu Province, China, was analysed. In vitro susceptibility testing of eight antimicrobial agents, including ciprofloxacin and levofloxacin, against 95 clinical isolates was carried out. Detection of mutations in the gyrA and parC genes was performed by sequence analysis. The clinical isolates demonstrated 100% resistance to ciprofloxacin and 98.9% non-susceptibility to levofloxacin. All of the isolates were susceptible to cefotaxime and ceftriaxone. For cefepime, spectinomycin and tetracycline, 98.9, 94.7 and 1.1% of the isolates were susceptible, respectively. None of the isolates was susceptible to penicillin. Five types based on gyrA mutations could be categorized among 54 isolates with seven different mutation sites found on their parC gene. Analysis of sequence results showed that the gyrA mutation Asp-95-->Ala and the parC mutations Ser-87-->Arg and Ser-87-->Asn made a significant contribution to the resistance to fluoroquinolones, in addition to double mutations found in each gene. Therefore, the use of fluoroquinolones in the treatment of N. gonorrhoeae infections in Jiangsu Province is not recommended, while the use of third- and fourth-generation cephalosporins and spectinomycin is recommended.

Seasonal pattern of tuberculosis in Hong Kong.

BACKGROUND: Summer predominance of tuberculosis (TB) was reported previously in temperate regions. No consistent data were available for lower latitudes. METHOD: The monthly TB notification data in Hong Kong from 1991 to 2002 were examined for seasonal fluctuation. A seasonal model was then developed after standardization by period, sex, age, history of TB, form of disease, and bacteriological status. RESULTS: The raw monthly counts showed remarkably consistent seasonal fluctuation across different periods, sexes, and age groups. A sine model was fitted for 82 104 notifications (adjusted R(2) = 0.373, P < 0.001). A summer peak was observed with seasonal fluctuation of 18.4% (P < 0.001), which was substantially higher than that reported previously for temperate regions. The amplitudes of fluctuation were 35.0, 15.0, 19.0, and 20.2% for those aged < or =14, 15-34, 35-64, and >/=65 years, respectively (all P < 0.001). No gender difference was noted (18.2% vs 19.0%, P = 0.790). Seasonal pattern was detected among new cases (18.6%, P < 0.001), but not retreatment cases (5.2%, P = 0.333). Culture-positive cases showed greater fluctuation than culture-negative cases (29.4% vs 6.4%, P < 0.001). No significant difference was found between pulmonary and extrapulmonary cases (16.8% vs 21.6%, P = 0.356). TB cases notified in summer were more likely to be smear-positive [odds ratio (OR) 1.100, 95% confidence interval (CI) 1.045-1.158, P < 0.001] and culture-positive (OR 1.175, 95% CI 1.121-1.232, P < 0.001) than those notified in winter, even after stratification by other key variables. CONCLUSION: A consistent seasonal pattern was found, with variable amplitudes of fluctuation in different subgroups and differing disease characteristics in different seasons. These observations are suggestive of the presence of a seasonal disease-modifying factor.

Assay of pleural fluid interleukin-6, tumour necrosis factor-alpha and interferon-gamma in the diagnosis and outcome correlation of tuberculous effusion.

OBJECTIVE: To assess the usefulness of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the diagnosis and prediction of outcome of pleural tuberculosis. PATIENTS AND METHODS: Pleural fluid from 32 TB and 34 non-TB patients was sent for assay of IL-6, TNF-alpha and IFN-gamma. Clinical parameters at presentation and residual pleural scarring at completion of treatment were assessed for pleural TB cases. RESULTS: The pleural fluid Levels of IL-6, TNF-alpha and IFN-gamma in TB patients were significantly higher than those with non-TB effusions (P values of <0.001, 0.018 and <0.001, respectively by independent t-test). Utility of these cytokines for diagnosis of pleural TB was evaluated using receiver operating characteristic (ROC) curve analysis. The cut-off values for IL-6, TNF-alpha and IFN-gamma determined in this analysis were 4000, 4 and 60 pg/ml respectively, and their sensitivity and specificity were 90.6% and 76.5%, 90.6% and 79.4%, 100% and 100%, respectively. The pretreatment pleural fluid IL-6 levels had a positive correlation with the number of febrile days after treatment (Pearson correlation test: r=0.60, P=0.009). A negative correlation was found between the percentage reduction in pleural fluid cytokines after 2 weeks treatment and the extent of residual pleural scarring (IL-6: r=-0.62, P=0.041; TNF-alpha: r=-0.65, P=0.030; IFN-gamma: r=0.83, P=0.002). CONCLUSION: Pleural fluid IL-6, TNF-alpha and IFN-gamma assays are useful in the diagnosis of pleural TB. The initial IL-6 level correlates with the number of febrile days. The percentage change of cytokines after 2 weeks of treatment also helps to predict residual pleural scarring.

Preoperative Antisepsis With Povidone-Iodine 5% in Cataract Surgery.

PURPOSE: This study aimed to evaluate the efficacy of povidone-iodine (PI) 5% as the only preoperative antiseptic in cataract surgery. DESIGN: Prospective interventional case series. METHODS: Fifty patients undergoing phacoemulsification under topical anesthesia in an outpatient surgery setting in a teaching hospital participated in the study. The preoperative antiseptic regimen consisted of instillation into the conjunctival sac and periorbital scrub with PI 5%. No topical antibiotic was given. Eyelid margin swabs were obtained for bacteriological culture before and after PI antisepsis and at the conclusion of surgery. The number of bacterial species isolated was compared with the Wilcoxon test. Anterior chamber aspirates were taken immediately before and after surgery to determine the rate of contamination. RESULTS: Bacteria were isolated from the eyelid margins of 48 patients (96%; 95% exact confidence interval, 86.3%-99.5%) before surgery, from the eyelid margins of 28 patients (56%) after antisepsis with PI 5%, and from the eyelid margins of 26 patients (56%; 95% exact confidence interval, 41.3%-70.0%) at the conclusion of surgery. The reduction of positive eyelid margin culture was statistically significant (P < 0.0001). Anterior chamber aspirates were positive in 3 patients. One patient with negative growth from anterior chamber aspirates developed culture-negative postoperative endophthalmitis. CONCLUSIONS: Because 56% of eyelids still have positive culture after PI 5% antisepsis, it might not be relied on as the sole preoperative antiseptic measure. Meticulous surgical technique, together with other modalities of preoperative and intraoperative antisepsis, should be used to prevent postoperative endophthalmitis.

Effective photocatalytic disinfection of E. coli K-12 using AgBr-Ag-Bi2WO6 nanojunction system irradiated by visible light: the role of diffusing hydroxyl radicals.

Urgent development of effective and low-cost disinfecting technologies is needed to address the problems caused by an outbreak of harmful microorganisms. In this work, we report an effective photocatalytic disinfection of E. coli K-12 by using a AgBr-Ag-Bi(2)WO(6) nanojunction system as a catalyst under visible light (lambda >or= 400 nm) irradiation. The visible-light-driven (VLD) AgBr-Ag-Bi(2)WO(6) nanojunction could completely inactivate 5 x 10(7) cfu mL(-1) E. coli K-12 within 15 min, which was superior to other VLD photocatalysts such as Bi(2)WO(6) superstructure, Ag-Bi(2)WO(6) and AgBr-Ag-TiO(2) composite. Moreover, the photochemical mechanism of bactericidal action for the AgBr-Ag-Bi(2)WO(6) nanojunction was investigated by using different scavengers. It was found that the diffusing hydroxyl radicals generated both by the oxidative pathway and the reductive pathway play an important role in the photocatalytic disinfection. Moreover, direct contact between the AgBr-Ag-Bi(2)WO(6) nanojunction and bacterial cells was not necessary for the photocatalytic disinfection of E. coli K-12. Finally, the photocatalytic destruction of the bacterial cells was directly observed by TEM images and further confirmed by the determination of potassium ion (K(+)) leakage from the killed bacteria. This work provides a potential effective VLD photocatalyst to disinfect the bacterial cells, even to destruct the biofilm that can provide shelter and substratum for microorganisms and resist to disinfection.

Zn:In(OH)ySz solid solution nanoplates: synthesis, characterization, and photocatalytic mechanism.

Zn:In(OH)ySz solid solution nanoplates (Zn:In(OH)ySz-SSNs) with uniform nanoparticle size were synthesized through a simple sodium dodecyl sulfate (SDS)-assisted hydrothermal process. To achieve better photoabsorption in the visible light (VL) region and suitable redox potentials of the Zn:In(OH)ySz solid solution (Zn:In(OH)ySz-SS), the substitution of S(2-) for OH was carried out by adjusting the concentration of thiourea and SDS in the synthesis solution, while the doping of Zn2+ was realized by adjusting Zn2+ concentration. In addition, the morphology and crystallinity of Zn:In(OH)ySz-SSs were also controlled by the concentration of SDS. Using Rhodamine B (RhB) as a target pollutant the photocatalytic performance of these Zn:In(OH)ySz-SSs with different components, diameter sizes, and morphologies was investigated. Remarkably, Zn:In(OH)ySz-SSNs prepared with atomic ratio of Zn2+ and In3+ of 0.6, 45 mmol L(-1) thiourea, and 26 mmol L(-1) SDS, have the highest visible-light-driven (VLD) photocatalytic activity, exceeding 95% for the degradation of RhB after 60 min. The investigation of photocatalylic mechanism further indicates that the holes, superoxide radical (*O2(-)) and surficial hydroxyl radical (*OHs) are the major reactive species for the photocatalytic reactions. More importantly, for the first time, a simple and versatile strategy is developed to confirm the fact that direct contact between the Zn:In(OH)ySz-SS and RhB is the prerequisite for the photocatalytic degradation of RhB. Therefore, we report not only the preparation of a novel and effective VL-driven photocatalyst, but also provide mechanistic insight into semiconductor photocatalysis.

Genetic association between a chemokine gene CXCL-10 (IP-10, interferon gamma inducible protein 10) and susceptibility to tuberculosis.

BACKGROUND: Previous studies showed that activation of CXCL-10 and other chemokines were prominent in many infectious diseases. These chemokines are components of innate immune response to respiratory tract pathogens. We examined the promoter variants of CXCL-10 and their role in predisposition to tuberculosis (TB). METHODS: The promoter 1.8 kb of CXCL-10 was sequenced in 24 healthy Chinese individuals to identify genetic polymorphisms. Three tagging SNPs in CXCL-10 promoter (-1447A>G, -872G>A, -135G>A) were selected, and genotyping were performed in 240 TB patients and 176 healthy Chinese subjects. Disease associations were examined by chi(2) and Fisher exact test. RESULTS: A promoter SNP (-135G>A) with minor allele frequency of 0.1 showed a moderate association with TB both in genotype analysis (p=0.01) and allelic analysis (p=0.03); other tagging SNPs (-1447A>G, -872G>A) were not associated with TB. The odd ratio of the protective allele -135G>A was 0.51(C.I 0.29 -0.91) for homozygotes and heterozygotes carriers of the A allele. CONCLUSION: A new potential protective SNP (-135G>A) for TB is identified in the promoter of chemokine gene, CXCL-10. Interestingly, the exact same allele has been shown to enhance IP-10 transactivation and susceptibility to Hepatitis B virus infection in a recent publication. This SNP, located at 14bp upstream of a NF-kB binding site, might also account for the susceptibility to TB. Our results expanded the clinical significance of this SNP in CXCL-10 promoter.

In vitro postantibiotic effects of rifapentine, isoniazid, and moxifloxacin against Mycobacterium tuberculosis.

Postantibiotic effects (PAEs) of rifapentine, isoniazid, and moxifloxacin against Mycobacterium tuberculosis ATCC 27294 were studied using a radiometric culture system. Rifapentine at 20 mg/liter gave the longest PAE (104 h) among the drugs used alone. The combinations of rifapentine plus isoniazid, rifapentine plus moxifloxacin, and isoniazid plus moxifloxacin gave PAEs of 136.5, 59.0, and 8.3 h, respectively.