Role of a 21-kDa iron-regulated protein IrpA in the uptake of ferri-exochelin by Mycobacterium smegmatis.

AIMS: To characterize the 21-kDa iron-regulated cell wall protein in Mycobacterium smegmatis co-expressed with the siderophores mycobactin, exochelin and carboxymycobactin upon iron limitation. METHODS AND RESULTS: Mycobacterium smegmatis, grown in the presence of 0·02 µg Fe ml-1 (low iron) produced high levels of all the three siderophores, which were repressed in bacteria supplemented with 8 µg Fe ml-1 (high iron). Exochelin, the major extracellular siderophore was the first to rise and was expressed at high levels during log phase of growth. Carboxymycobactin, a minor component in log phase iron-starved M. smegmatis continued to rise when cultured for longer periods, reaching levels greater than exochelin. Iron-starved bacteria expressed a 21-kDa iron-regulated protein (IrpA) that was identified as Clp protease subunit (MSMEG_3671) and characterized as a receptor for ferri-exochelin. CONCLUSIONS: Ferri-exochelin is the preferred siderophore in M. smegmatis and this ferri-exochelin: IrpA machinery is absent in Mycobacterium tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Exochelin machinery is functional in M. smegmatis and the carboxymycobactin-mycobactin machinery is the sole iron uptake system in M. tuberculosis. The absence of the ferri-exochelin: IrpA system in the pathogen signifies the importance of the carboxymycobactin-mycobactin system machinery in M. tuberculosis.

Pathogen-specific leptospiral proteins in urine of patients with febrile illness aids in differential diagnosis of leptospirosis from dengue.

Leptospirosis and dengue are two commonly seen infectious diseases of the tropics. Differential diagnosis of leptospirosis from dengue fever is often difficult due to overlapping clinical symptoms and lack of economically viable and easy-to-perform laboratory tests. The gold standard for diagnosis is the microscopic agglutination test (MAT). In this study, the diagnostic potential of screening for pathogen-specific leptospiral antigens in urine samples is presented as a non-invasive method of disease diagnosis. In a study group of 40 patients, the serum was tested for anti-leptospiral antibodies by MAT and enzyme-linked immunosorbent assay (ELISA). Urine of these patients was screened for leptospiral antigens by ELISA using specific antibodies against LipL32, LipL41, Fla1, HbpA and sphingomyelinase. Group I patients (n = 23) were classified as leptospirosis-positive based on MAT and high titres of circulating IgM-specific anti-leptospiral antibodies. All of these patients excreted all five leptospiral antigens in the urine. The 17 MAT-negative cases included six patients with pyrexia of unknown origin (PUO; Group II) and 11 confirmed dengue patients (Group III). The latter tested negative for both serum anti-leptospiral antibodies and urinary leptospiral antigens. A salient outcome of this study was highlighting the usefulness of screening for urinary leptospiral antigens in disease diagnosis, as their presence confirmed leptospiral aetiology in two PUO patients. Immunoblots of urinary antigens identified well-defined bands corresponding to LipL32, HbpA and sphingomyelinase; the significance of the 42- and 58-kDa sphingomyelinase bands is discussed.

Randomized clinical trial of intraoperative endoscopic retrograde cholangiopancreatography versus laparoscopic bile duct exploration in patients with choledocholithiasis.

BACKGROUND: Various minimally invasive approaches exist for the management of choledocholithiasis at the time of laparoscopic cholecystectomy. The aim of this study was to compare endoscopic retrograde cholangiopancreatography (ERCP) with laparoscopic bile duct exploration (LBDE) and test the hypothesis that intraoperative ERCP is no different to LBDE in terms of rate of bile duct clearance or retained stones. METHODS: Eligible patients with choledocholithiasis undergoing emergency laparoscopic cholecystectomy were randomized to intraoperative ERCP or LBDE in a 1 : 1 ratio. The primary outcomes were rates of bile duct clearance and retained stones. Secondary outcomes were postprocedure complication rate, mortality rate, postoperative length of hospital stay, conversion to open surgery rate, procedural time and total duration of surgery. RESULTS: Some 104 patients were randomized, and 52 patients in each group were included in an intention-to-treat analysis. Duct clearance rates were 87 per cent for patients who had intraoperative ERCP and 69 per cent for those in the LBDE group (P = 0·057). The rate of retained stones was lower in the ERCP group than in the LBDE group: 15 versus 42 per cent respectively (P = 0·004). Median postoperative length of stay was shorter with ERCP (2 days versus 3 days for LBDE; P = 0·015). CONCLUSION: Intraoperative ERCP is more effective than LBDE in terms of minimizing the rate of retained stones in patients with choledocholithiasis undergoing emergency laparoscopic cholecystectomy. REGISTRATION NUMBER: ACTRN12613000761763 (http://www.anzctr.org.au/).

Highly immunoreactive antibodies against the rHup-F2 fragment (aa 63-161) of the iron-regulated HupB protein of Mycobacterium tuberculosis and its potential for the serodiagnosis of extrapulmonary and recurrent tuberculosis.

HupB is an iron-regulated protein in Mycobacterium tuberculosis that functions as a positive regulator of mycobactin biosynthesis. It is essential for the growth and survival of the pathogen inside macrophages. Previously, using the full-length rHupB of M. tuberculosis, we demonstrated high levels of anti-HupB antibodies in the serum of pulmonary tuberculosis (TB) and, interestingly, extrapulmonary TB patients with negligible levels in household contacts and healthy controls. Here, we used three antigenic fragments of HupB, namely the recombinant HupB-F1 (aa 1-71), HupB-F2 (aa 63-161) and HupB-F3 (aa 164-214), as antigens in enzyme-linked immunosorbent assay (ELISA) to screen serum from TB patients. HupB-F2 showed enhanced immunoreactivity with serum from patients with pulmonary TB (three groups consisting of new cases, defaulters and recurrent cases) and extrapulmonary TB, with negligible levels in normal healthy controls. The negative correlation of the anti-(HupB-F2) antibodies with serum iron was maximal, with a Pearson's correlation coefficient value of -0.415. The study, in addition to strengthening the diagnostic potential of HupB, reflected the superior performance of HupB-F2 as an antigen in screening pulmonary and extrapulmonary TB.

Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum.

Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 micrograms/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 micrograms/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 micrograms/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.

Studies on the tissue distribution of liposome-associated clofazimine, an antileprosy drug.

Clofazimine, a potent antimycobacterial drug, being highly lipophilic accumulates in fatty tissue and in the reticuloendothelial system causing dose-dependent side effects. In this study, the distribution of the free drug and liposome-associated drug was compared after intravenous administration in mice. Differences in the distribution of the drug were observed in the liver, spleen, kidney and lung tissues when injected as free drug and as liposome-associated drug. Following intravenous challenge with the free drug, the drug accumulated quickly and high concentrations of the drug were seen in the spleen, liver, kidney and lung even after 24 h, indicating poor clearance. However, with liposome-associated drug, increased levels were seen in liver, spleen and lung at 1 h with levels falling considerably at 24 h, with no accumulation in the kidney either at 1 h or 24 h after challenge. Clofazimine associated with neutral liposomes was preferentially targetted to spleen and lung, positively charged liposome-associated drug accumulated more in the lungs than in other tissues, while negatively charged liposome-associated drug was directed to liver and spleen. The results suggest that inclusion of clofazimine into liposome not only targets the drug to the organs concerned but also facilitates clearance of the drug, resulting in little accumulation. Also, renal accumulation is much lower as compared to the free drug. This suggests the potential usefulness of liposome as a carrier for clofazimine, thereby reducing the harmful side effects due to excessive accumulation of the drug.

Polymerase chain reaction in the diagnosis of tuberculosis.

A rapid, sensitive, specific and yet economical method for the diagnosis ofM. tuberculosis and other mycobacteria in clinical specimen is a desperate and urgent requirement of the day in the laboratory diagnosis and hence management of tuberculosis. This need is further accentuated by emerging diseases like multi drug resistant tuberculosis, tuberculosis in AIDS patients and opportunistic mycobacterial infections, which do not respond to conventional anti TB therapy. Molecular methods, particularly PCR based detection ofM. tuberculosis, has come a long way since it was first described about fifteen years ago. Several probes have been developed and some of them, particularly the IS6110 and TB400 have been validated on several clinical samples. The latter has been validated on a variety of clinical specimens along with a simple sample processing method. Polymerase chain reaction based diagnosis ofM. tuberculosis has been introduced as one of the routine/confirmatory tests in clinical microbiology laboratory in some countries like Canada, the United States and the United Kingdom several years ago. The possibility of introducing PCR based direct diagnosis of drug resistance is being explored in some laboratories, particularly for drugs like rifampicin. The evolution and application of PCR for diagnosis ofM. tuberculosis is being analysed and discussed in this review.

Identification of a LDL-receptor in the lymphatic filarial parasiteWuchereria bancrofti.

Little is known about host-parasite inter-relationship in the lymphatic filarial parasites. There is no information available about the ability of these parasites to acquire cholesterol, though it is known that in general, nematodes lack the ability to synthesise cholesterolde novo. In this study, we have shown that the filarial parasites also lack the ability to incorporate labelled acetate into cholesterol, indicating the absence of the machinery for cholesterol biosynthesis. We have further shown that they elaborate a 43 kDa surface receptor for acquiring LDL-bound cholesterol. We have shown by polymerase chain reaction the presence of a 860 bp fragment indicating the presence of the gene for LDL-related protein (LRP) in the human filarial parasiteWuchereria bancrofti in the genomic DNA. We have also shown that it is expressed as seen in the cDNA clones identified from an expression library.

Direct diagnosis ofMycobacterium tuberculosis in blood samples of HIV infected patients by polymerase chain reaction.

We have developed a simple, economical and reproducible method for processing blood samples from HIV infected patients for diagnosis of tuberculosis. The procedure was validated on 55 samples selected for tuberculosis based on clinical criteria. 52 patients had radiological changes indicative of pulmonary tuberculosis of which only 28 were positive for AFB in sputum (sensitivity 54%) and 27 for tuberculin (sensitivity 52%). 26 HIV positive patients who showed positive X-ray did not react to tuberculin. The genus PCR probe missed 3 samples (sensitivity 94%) compared to X-ray.M.tuberculosis was detected in the blood of all X-ray positive cases by PCR using TB400 probe (sensitivity 100%) and another probe forM. tuberculosis, IS6110, missed 6 of them (sensitivity 88% compared to X-ray and 89% compared to TB400). It is proposed that this simple sample processing method could be used to screen all blood samples quickly for mycobacteremia using the genus PCR and only those positive for mycobacteria need to be tested forM.tuberculosis. This would save the scarce resources and time by reducing significantly the number of samples to be screened for species confirmation.

Isolation, PCR based identification, and sensitivity pattern of environmental mycobacteria from leprosy and tuberculosis patients.

We have isolated and identified the biotype of environmental mycobacteria from the expectorate of leprosy patients, their contacts, their drinking water supply and also from the sputa samples of tuberculosis patients. 78% of the isolates from lepromatous leprosy patients and their contacts wereMycobacterium fortuitum- chelonae complex (MFC), 9%Mycobacterium avium complex (MAC), 9%Mycobacterium scrofulaceum and 4% wereMycobacterium smegmatis. Among the isolates from tuberculosis patients 63% belonged toM. fortuitum- chelonae complex, 19% toM. avium complex, 12% toMycobacterium Kansasii and 6% toM. smegmatis. All the isolates were multi-drug resistant when tested for sensitivity total of 21 drugs. TheMycobacterium fortuitum-chelonae complex organisms from leprosy contacts were more sensitive to rifampicin than those isolated from lepromatous leprosy and tuberculosis patients. Among 23 isolates from leprosy patients one isolate was resistant to 20 drugs, one isolate to 17 drugs and another isolate was resistant to 13 drugs. Among the 18 isolates from drinking water supply six showed resistance to more than 12 drugs. Polymerase Chain Reaction (PCR) and subsequent hybridisation with specific probes confirmed all the isolated strains as nontuberculous mycobacteria (Using genus primers and probe sensitivity 100%) and none asM. tuberculosis, suggesting that PCR could be used to rapidly identify mycobacteria at the genus level and to rule out tuberculosis in leprosy patients at an early stage to decide on appropriate course of therapy.

Structural analysis of the Leptospiral sphingomyelinases: in silico and experimental evaluation of Sph2 as an Mg-dependent sphingomyelinase.

BACKGROUND: Leptospiral sphingomyelinases are candidate virulence factors present only in pathogenic Leptospira spp. Leptospira interrogans serovar Lai encodes Sph1, Sph2, Sph3, Sph4 and SphH. Except for Sph4, they all possess the exo-endo-phosphatase domain that groups them under the DNase I superfamily. METHODS, RESULTS AND CONCLUSIONS: Modeling of exo-endo-phosphatase domains reveals high-level structural similarity of Sph2 with the crystal structure of SmcL and BC SMase sphingomyelinases from Listeria ivanovii and Bacillus cereus, respectively. A ß-hairpin loop, essential for host cell membrane interaction, is absent in leptospiral sphingomyelinases. Instead, several aromatic amino acids were oriented outward from the surface of these molecules and formed clusters of hydrophobic regions that possibly enables the anchoring of these molecules into the host cell membrane, as demonstrated in Sph2 and Sph3. Sph2 is unique and possesses the Mg(++)-binding Glu53 residue in the metal-binding site and two His residues (His151 and His286) in the catalytic site. We demonstrate experimentally the Mg(++)-dependent hemolysis of erythrocytes by rSph2 and its ability to cleave sphingomyelin to ceramide. Anti-Sph2 antibodies neutralized the hemolytic activity of Sph2. In conclusion, we provide evidence showing that Sph2 is a Mg(++)-dependent hemolysin with both sphingomyelinase and hemolytic activities.

T-cell recognition of iron-regulated culture filtrate proteins of Mycobacterium tuberculosis in tuberculosis patients and endemic normal controls.

BACKGROUND: Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are potential vaccine candidates. OBJECTIVE: The aim was to study the influence of iron levels on CFPs and assess the immuno-protective potential of defined antigenic fractions from high (8 µg Fe/mL) and low iron (0.02 µg Fe / mL) cultures of M. tuberculosis. MATERIALS AND METHODS: The CFPs of M. tuberculosis from high (CFP-high) and low (CFP-low) iron conditions were first compared to identify iron-regulated proteins and then fractionated to obtain ten antigen pools (CF-Ags H1- H5 and L1-L5) that were used to assess the immune response of TB patients and normal healthy controls. RESULTS: Iron limitation resulted in the up-regulation of two novel iron-regulated low-molecular-weight proteins Irp-1 (in CF-Ag L4) and Irp-2 (in CF-Ag L5) and repression of two ESAT proteins (identified with monoclonal antibody HYB 76.8). The median stimulation indices (SIs) against most of the CF-Ags were high in pulmonary TB patients. The CF-Ags L1 and L2 showed statistically significant SI (P values of 0.0027 and 0.0029 respectively); the % case recognition was high with these antigens as well as with L4 ( P = 0.0275). IFN-γ in response to these CF-Ags was significantly high in the endemic normals; maximal expression was seen with CF-Ag L5 (median value of 233 pg mL -1 ) that was higher than the corresponding H5 (140 pg mL -1 ) and H3 and L3 (205 and 206 pg mL -1 respectively). CONCLUSIONS: CF-Ags L5, H3 and L3 showed immuno-protective potential in this geographical location.

Serological evaluation of leptospirosis in Hyderabad Andhra Pradesh: a retrospective hospital-based study.

PURPOSE: Leptospirosis is a zoonotic disease with humans getting the infection either from rodent hosts or from domestic animals. Urine contaminated environment is the common source of infection. This is an under-reported disease in Andhra Pradesh. We report a retrospective hospital-based study on 55 patients with suspected leptospirosis. METHODS: A total of 55 serum samples were collected from patients with suspected leptospirosis and subjected to serological testing by LeptoTek Dri-dot, microscopic agglutination test (MAT) and IgM enzyme-linked immunosorbent assay (ELISA). Identification of the predominant infecting serotype was done using a panel of 12 serovars. RESULTS: MAT analysis of all the 55 samples identified all cases to be positive. The predominant serogroup was Icterohaemorrhagiae (68%) followed by Australis (22%), Autumnalis (8%) and Javanica (2%). LeptoTek Dri-dot showed a sensitivity of 96% as compared to MAT. IgM ELISA done on 32 samples showed a sensitivity of 86.7% compared to MAT. CONCLUSIONS: MAT helped to identify Icterohemorrhagiae as the predominant serovar in this study. Despite the small number of samples analyzed, the data obtained establishes a need for a prospective study in this region.

Iron-regulated envelope proteins of mycobacteria grown in vitro and their occurrence in Mycobacterium avium and Mycobacterium leprae grown in vivo.

Several iron-regulated envelope proteins (IREPs), 11-180 kDa, have been detected in preparations of walls and membranes of Mycobacterium smegmatis, in an armadillo-derived mycobacterium (ADM) and in M. avium. The same sized proteins from M. vacae appeared under both iron-deficient and iron-sufficient growth conditions. Two larger proteins, of 240 and 250 kDa, appeared in the membranes of M. smegmatis and M. avium only when grown iron-sufficiently but were constitutively present in both ADM and M. vaccae. The IREPs from M. smegmatis were not induced under zinc-deficient growth conditions. Three of the four IREPs (14, 21 and 29 kDa) recognized in M. avium grown in vitro were also recovered from membrane fractions of the same strain grown in mice. In addition, these membranes contained both the high-molecular-mass proteins associated with iron-sufficient growth conditions. Membranes of M. leprae, recovered from infected armadillos, showed the faint presence of a possible IREP at 29 kDa and wall preparations showed the presence of a 21-kDa protein. Membranes also contained the two larger proteins at 240 and 250 kDa. An explanation for the simultaneous occurrence of both low-iron-regulated and high-iron-regulated proteins is offered.

Iron-regulated proteins (IRPS) of Leptospira biflexa serovar Patoc strain Patoc I.

BACKGROUND: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. MATERIALS AND METHODS: Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N'-diacetic acid (EDDA) and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA) and the second method involved the addition of iron at 0.02 microg Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 microg Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase), periplasmic space (aqueous phase) and the protoplasmic cylinder (cell pellet). The proteins were subjected to SDS-PAGE for analysis. RESULTS: In the presence of the iron-chelators, four iron-regulated proteins (IRPs) of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 microg Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 microg Fe/mL. The blue CAS agar plates with top agar containing 0.02 microg Fe/mL showed a colour change to orange-red. CONCLUSION: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation was demonstrated in the non-pathogenic L. biflexa serovar Patoc.

Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake.

Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.