Identification and characterisation of proteases in Mycobacterium tuberculosis

Virulence determinants of M. tuberculosis remain largely unknown. Of key interest has been the ability of the bacterium to survive intracellularly within its host cell, the macrophage, and its ability to cause extensive tissue necrosis. Exported proteases are commonly associated with virulence in bacterial pathogens, yet their role in Mycobacterium tuberculosis has virtually not been studied. Preliminary experiments showed M. tuberculosis culture filtrates contained a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca²⁺, features typical of some serine proteases, notably subtilisins, and possibly metalloproteases. Purification attempts were unsuccessful. A family of five genes that encode putative, secreted, serine proteases has recently been described in M. tuberculosis. These proteases share 36-47% sequence identity and are all encoded with putative signal peptides, suggesting that they are translocated across the cytoplasmic membrane. One member, mycP1, was selected for further study. The gene product, mycosin-1, was 30-35% identical to bacterial subtilisin-like serine proteases and contained the classic catalytic triad and oxyanion hole. Mycosin-1 also contained a typical signal peptide, a likely propeptide, and a Cterminal hydrophobic sequence with a high transmembrane potential. Topology analyses predicted mycosin-1 to be a type I ectoprotein. Consistent with this, expression of mycosin-1 in M. tuberculosis and in Mycobacterium smegmatis transformed with mycP1 (M. smegmatis-P1) was limited strictly to the cell envelope, as seen by Western blotting, and immunogold electron microscopy. Only full-length, 50-kDa mycosin-1 was observed by Western blotting in broth-grown M. tuberculosis and M. smegmatis-P1 lysates, whereas a 40-kDa species was detected in 6-week M. tuberculosis culture filtrates. A similar 40-kDa immunoreactive band was also observed in lysates of macrophages infected with M. tuberculosis, consistent with robust transcription of the mycP 1 gene during growth in macrophages. Since putative mature mycosin-1 has a molecular weight of 38.6 kDa, the 40-kDa protein may represent activated mycosin-1 after propeptide cleavage. In conclusion, mycosin-1 is an exported, cell envelopeassociated subtilisin homolog that is expressed during growth of M. tuberculosis in vitro and in macrophages

Sensory neuropathy and metabolic risk factors in human immune deficiency virus infected South Africans receiving protease inhibitors

BackgroundProtease inhibitors (PI)s have been associated with distal sensory polyneuropathy (DSP) and metabolic complications in high-income countries. No data exist in Africans where second-line antiretroviral therapy (ART) often include PIs.MethodWe performed a cross-sectional study to assess the DSP frequency and metabolic risk factors in community-based South Africans taking ritonavir-boosted lopinavir as PI. Examination findings categorized subjects as having DSP (≥1 neuropathic sign) or symptomatic DSP [DSP with symptom(s)]. Fasting-state glucose and lipid profiles were assessed. We compared the ritonavir/lopinavir-group to a nested group on first-line ART [dideoxy-nucleoside reverse transcriptase inhibitors (d-drugs)] selected from a dataset collected at the same time and matched for d-drug exposure.ResultsThe ritonavir/lopinavir-group (n=86) consisted predominantly of women (84%) with a median age of 36years (IQR 32–41). The median current CD4+ count was 489cells/μL (IQR 291–665). The median exposure time to ritonavir/lopinavir was 18months (IQR 10–26) and to d-drugs, 24months (IQR 16–38). DSP was present in 78% and symptomatic DSP in 48%; symptoms were most frequently of moderate intensity. Only age independently associated with DSP and symptomatic DSP (p=0.08 and p=0.04, respectively). None of the metabolic syndrome components showed associations with DSP or symptomatic DSP despite a trend towards hypertriglyceridemia overall. The ritonavir/lopinavir-group had less DSP compared to the d-drug only group (p=0.002) but the frequency of symptomatic DSP was similar (p=0.49).ConclusionRitonavir-boosted lopinavir did not add additional risk to developing DSP in this community-based African cohort after a median of 18months on second-line ART.Electronic supplementary materialThe online version of this article (doi:10.1186/s12981-015-0073-8) contains supplementary material, which is available to authorized users

How Well Can We Measure Galaxy Dust Attenuation Curves? The Impact of the Assumed Star-dust Geometry Model in Spectral Energy Distribution Fitting

One of the most common methods for inferring galaxy attenuation curves is via spectral energy distribution (SED) modeling, where the dust attenuation properties are modeled simultaneously with other galaxy physical properties. In this paper, we assess the ability of SED modeling to infer these dust attenuation curves from broadband photometry, and suggest a new flexible model that greatly improves the accuracy of attenuation curve derivations. To do this, we fit mock SEDs generated from the Simba cosmological simulation with the Prospector SED fitting code. We consider the impact of the commonly-assumed uniform screen model and introduce a new non-uniform screen model parameterized by the fraction of unobscured stellar light. This non-uniform screen model allows for a non-zero fraction of stellar light to remain unattenuated, resulting in a more flexible attenuation curve shape by decoupling the shape of the UV attenuation curve from the optical attenuation curve. The ability to constrain the dust attenuation curve is significantly improved with the use of a non-uniform screen model, with the median offset in UV attenuation decreasing from $-0.30$ dex with a uniform screen model to $-0.17$ dex with the non-uniform screen model. With this increase in dust attenuation modeling accuracy, we also improve the star formation rates (SFRs) inferred with the non-uniform screen model, decreasing the SFR offset on average by $0.12$ dex. We discuss the efficacy of this new model, focusing on caveats with modeling star-dust geometries and the constraining power of available SED observations.Comment: Submitted to ApJ. 15 pages, 10 figure

Anti-retroviral therapy increases the prevalence of dyslipidemia in South African HIV-infected patients

Purpose Data on the prevalence of dyslipidaemia and associated risk factors in HIV-infected patients from sub-Saharan Africa is sparse. We performed a cross-sectional analysis in a cohort of HIV-infected South African adults. METHODS: We studied HIV-infected patients who were either antiretroviral therapy (ART)-naive or receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-based or protease inhibitor (PI)-based ART. Evaluation included fasting lipograms, oral glucose tolerance tests and clinical anthropometry. Dyslipidemia was defined using the NCEP ATPIII guidelines. RESULTS: The median age of the participants was 34 years (range 19-68 years) and 78% were women. The prevalence of dyslipidemia in 406 ART-naive and 551 participants on ART was 90.0% and 85%, respectively. Low HDL-cholesterol (HDLC) was the most common abnormality [290/406 (71%) ART-naïve and 237/551 (43%) ART- participants]. Participants on ART had higher triglycerides (TG), total cholesterol (TC), LDL-cholesterol (LDLC) and HDLC than the ART-naïve group. Severe dyslipidaemia, (LDLC> 4.9 mmol/L or TG >5.0 mmol/L) was present in <5% of participants. In multivariate analyses there were complex associations between age, gender, type and duration of ART and body composition and LDLC, HDLC and TG, which differed between ART-naïve and ART-participants. CONCLUSION: Participants on ART had higher TG, TC, LDLC and HDLC than those who were ART-naïve but severe lipid abnormalities requiring evaluation and treatment were uncommon

Final Summary Report on Truck Route Access Evaluation

This report summarizes the evaluation of truck route access between the National Highway System and 46 truck generating sites, including intermodal sites, throughout Kentucky (includes 81 routes and 800 miles of highway). Routes were evaluated quantitatively for nine highway features, assigned an overall route rating and recommendations for routine maintenance and re-construction were made. The level and quality of truck access to the NHS varies dramatically throughout the state. Some facilities are between 25 and 50 miles from the NHS. No significant difference in route ratings or other measures was found by geographic location. The different typography throughout the state contributed to differences in grade and curvature quality. Very few non-NHS truck routes have 12-foot lanes along their whole length. The intermodal facility routes in general were found to be of better quality than routes leading to truck only sites. Trucks are not always using the routes they should. Ten of the routes were considered the best and required no improvements at this time. Improvements on other routes varied from routine maintenance to the need for complete reconstruction. Routes were prioritized by length and amount of truck traffic to recommend the most critical routes for improvement

Cardiovascular risk factors in patients with Addison's disease: a comparative study of South African and Swedish patients

BACKGROUND: Patients with Addison's disease (AD) in Scandinavia have an increased risk for premature death due to cardiovascular disease (CVD). Serum lipids are important risk factors for CVD and vascular mortality. Replacement doses of hydrocortisone have historically been higher in Sweden than South Africa. The primary aim was to study the lipid profiles in a large group of patients with AD with the hypothesis that the lipid profile in patients in Sweden would be worse than in South Africa. METHODS: In a cross-sectional study, 110 patients with AD (55 from South Africa, 55 from Sweden) matched for age, gender, ethnicity and BMI were studied. Anthropometric measures, blood pressure, lipids, highly sensitive C-reactive protein (hs-CRP) and adiponectin were studied. RESULTS: All patients were Caucasian and the majority were women N = 36 (65.5%). Mean (standard deviation; SD) ages of the Swedish and South African patients were 52.9 (13.0) and 52.6 (14.4) years and BMI 25.3 (3.2) and 25.8 (4.1) kg/m 2 , respectively. The mean total daily hydrocortisone dose was greater in the Swedish patients than the South African patients, [33.0 (8.1) versus 24.3 (8.0) mg; p<0.0001]. South African patients had higher median (interquartilerange; IQR) triglycerides (TG) [1.59 (1.1-2.46) versus 0.96 (0.74-1.6) mmol/l; p<0.001], total cholesterol (TC) [6.02(1.50) versus 5.13 (0.87) mmol/l; p<0.001], LDL-C [4.43 (1.44) versus 2.75 (0.80) mmol/l; p<0.001] and median hs-CRP [2.15 (0.93-5.45) versus 0.99 (0.57-2.10) mg/L; p<0.003] and lower HDL-C [0.80 (0.40) versus 1.86 (0.46) mmol/l; p<0.001] than the Swedish patients. Approximately 20% of the patients in both cohorts had hypertension and diabetes mellitus. CONCLUSIONS: South African patients with AD have worse lipid profiles and higher hs-CRP compared to their matched Swedish patients, despite lower doses of hydrocortisone. It is uncertain at this time whether these are due to genetic or environmental factors

Tumor-induced osteomalacia: a case report

Background Tumor-induced osteomalacia is a rare, acquired paraneoplastic syndrome, including hypophosphatemia, high serum alkaline phosphatase, reduced active vitamin D, suboptimal bone mineral density, bone pain, fragility fractures, and muscle weakness. Case presentation We report a case of 74–year–old male of mixed ancestry with hypophosphatemia resistant to treatment despite optimal compliance, associated with profound reduction of bone mineral density and multiple nontraumatic fractures, including bilateral rib fractures, lower-thoracic (T11, T12) vertebrae, and two fractures involving the surgical and anatomical neck of the right humerus. We discuss an approach to identifying the underlying cause of hypophosphatemia associated with fragility fractures, and options for management of this rare condition. Conclusion Although rare, tumor-induced osteomalacia can be diagnosed if a logical stepwise approach is implemented. Surgery could be curative if the tumor is properly located and is resectable

Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages

BACKGROUND: Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism. RESULTS: Full-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca(2+), features typical of the subtilisins. CONCLUSIONS: Mycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates