In vitro SCREENING ANTIBACTERIAL ACTIVITY OF Bidens pilosa LINNé AND Annona crassiflora MART. AGAINST OXACILLIN RESISTANT Staphylococcus aureus (ORSA) FROM THE AERIAL ENVIRONMENT AT THE DENTAL CLINIC

Currently multiresistant Staphylococcus aureus is one common cause of infections with high rates of morbidity and mortality worldwide, which directs scientific endeavors in search for novel antimicrobials. In this study, nine extracts from Bidens pilosa (root, stem, flower and leaves) and Annona crassiflora (rind fruit, stem, leaves, seed and pulp) were obtained with ethanol: water (7:3, v/v) and their in vitro antibacterial activity evaluated through both the agar diffusion and broth microdilution methods against 60 Oxacillin Resistant S. aureus (ORSA) strains and against S. aureus ATCC6538. The extracts from B. pilosa and A. crassiflora inhibited the growth of the ORSA isolates in both methods. Leaves of B. pilosa presented mean of the inhibition zone diameters significantly higher than chlorexidine 0.12% against ORSA, and the extracts were more active against S. aureus ATCC (p < 0.05). Parallel, toxicity testing by using MTT method and phytochemical screening were assessed, and three extracts (B. pilosa, root and leaf, and A. crassiflora, seed) did not evidence toxicity. On the other hand, the cytotoxic concentrations (CC₅₀ and CC₉₀) for other extracts ranged from 2.06 to 10.77 mg/mL. The presence of variable alkaloids, flavonoids, tannins and saponins was observed, even though there was a total absence of anthraquinones. Thus, the extracts from the leaves of B. pilosa revealed good anti-ORSA activity and did not exhibit toxicity.     

Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive β-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.     

Population genomics of Mycobacterium tuberculosis in the Inuit

Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain.The tubercule bacillus, Mycobacterium tuberculosis, is a highly successful, medically important human-adapted pathogen. Studies of diverse strain collections reveal a geographic aggregation of the principal M. tuberculosis lineages (1) consistent with a dissemination of this organism around the world with the paleo migration (2). Ancient DNA studies also support the notion that M. tuberculosis has caused disease in humans for thousands of years. Thus, it can be inferred that M. tuberculosis has evolved in step with its human host, successfully responding to changes in the host and its environment that could affect the capacity to cause transmissible disease.In contrast to the global diversity of M. tuberculosis strains (1–3), we have previously observed limited genetic diversity in the Nunavik region of Québec (4). One possible explanation is a founder strain, wherein genetic similarity is due to a single recent introduction of a bacterium and may not necessarily represent ongoing spread between communities. In this scenario, isolates might have indistinguishable genotypes by conventional genotyping modalities (restriction fragment length polymorphism, mycobacterial interspersed repetitive units, spoligotyping) but distinct genotypes when assessed using a higher-resolution method, namely whole-genome sequencing (WGS) (5). An additional explanation is that a single clone of M. tuberculosis is currently spreading both within and between villages; however, the great distances between these communities that are not linked by roads make intervillage spread less likely. These possible explanations need not be mutually exclusive.To evaluate these possibilities, we conducted WGS on M. tuberculosis isolates from Nunavik isolated over 23 y. Estimation of the divergence date of the most recent common ancestor (MRCA) provided evidence that tuberculosis (TB) was introduced into this region in the early 20th century, following which time there has been substantial ongoing transmission, predominantly within villages. This setting provides a unique opportunity to study the genomic characteristics of an epidemiologically successful strain of M. tuberculosis over time.     

The ever-expanding association between rheumatologic diseases and tuberculosis

We summarized most of the rheumatologic manifestations of tuberculosis (TB) and the occurrence of Mycobacterium tuberculosis disease associated with rheumatologic diseases. We established 4 different categories: (1) direct musculoskeletal involvement of M. tuberculosis, including spondylitis, osteomyelitis, septic arthritis, and tenosynovitis; (2) M. tuberculosis as an infectious pathogen in rheumatologic diseases, particularly with the use of newer agents such as tumor necrosis factor-α inhibitors; (3) antimycobacterial drug-induced rheumatologic syndromes, including tendinopathy, drug-induced lupus, and others; and (4) reactive immunologic phenomena caused by TB, such as reactive arthritis, erythema nodosum, and others. In addition, Bacille-Calmette-Guérin vaccination used for the prevention of TB or as a chemotherapeutic agent for bladder carcinoma also may be associated with musculoskeletal adverse events. We conclude that M. tuberculosis can directly or indirectly affect the musculoskeletal system.     

Phytochemical screening and antioxidant capacity of the aerial parts of Thymelaea hirsuta L.

Objective

To assess antioxidant activities of different aerial parts of Thymelaea hirsuta (T. hirsuta) from west Algeria, and to search for new sources of safe and inexpensive antioxidants.

Methods

Samples of leaves, stems and flowers from T. hirsuta were tested for total phenolic content, flavonoids content, and evaluation its total antioxidant activity, were done using the spectrophotometric analyses.

Results

Results of preliminary phytochemical screening of leaf, flower and stem of T. hirsuta revealed the presence of tannins, alkaloids, steroids, saponins, coumarins, reducteurs compound and anthraquinones. The total phenolics and flavonoids were estimated. The aqueous extracts of the aerial parts of T. hirsuta showed potent in vitro antioxydant activities using various models viz, DPPH scavenging assay, ferric reducing antioxidant power (FRAP) and ABTS radical scavenging activity.

Conclusions

On the basis of the results obtained, T. hirsuta extracts are rich sources of natural antioxidants appears to be an alternative to synthetic antioxidants and this justifies its therapeutic usage.     

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM: To investigate the utility of immunohistochemical (IHC) staining with an antibody to Mycobacterium tuberculosis (M. tuberculosis) for the diagnosis of intestinal tuberculosis (TB).METHODS: We retrospectively identified 10 patients (4 males and 6 females; mean age = 65.1 ± 13.6 years) with intestinal TB. Clinical characteristics, including age, gender, underlying disease, and symptoms were obtained. Chest radiograph and laboratory tests, including sputum Ziehl-Neelsen (ZN) staining, M. tuberculosis culture, and sputum polymerase chain reaction (PCR) for tubercle bacilli DNA, as well as Tuberculin skin test (TST) and QuantiFERON-TB gold test (QFT), were examined. Colonoscopic records recorded on the basis of Sato’s classification were also reviewed, in addition to data from intestinal biopsies examined for histopathological findings, including hematoxylin and eosin staining, and ZN staining, as well as M. tuberculosis culture, and PCR for tubercle bacilli DNA. For the present study, archived formalin-fixed paraffin-embedded (FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M. tuberculosis complex. These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS: From the clinical data, we found that no patients were immunocompromised, and that the main symptoms were diarrhea and weight loss. Three patients displayed active pulmonary TB, six patients (60%) had a positive TST, and 4 patients (40%) had a positive QFT. Colonoscopic findings revealed that all patients had type 1 findings (linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules), all of which were located in the right hemicolon and/or terminal ileum. Seven patients (70%) had concomitant healed lesions in the ileocecal area. No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples, and both M. tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples. The histopathological data revealed that tuberculous granulomas were present in 4 cases (40%). IHC staining in archived FFPE samples with anti-M. tuberculosis monoclonal antibody revealed positive findings in 4 patients (40%); the same patients in which granulomas were detected by hematoxylin and eosin staining. M. tuberculosis antigens were found to be mostly intracellular, granular in pattern, and primarily located in the CD68⁺ macrophages of the granulomas.CONCLUSION: IHC staining with a monoclonal antibody to M. tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.     

Sporulation in mycobacteria

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette–Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.     

Essential roles for imuA′- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2–type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.     

Application status of MALDI-TOF mass spectrometry in the identification and drug resistance of Mycobacterium tuberculosis

Characterizing Mycobacterium tuberculosis (MTB) and detecting its drug resistance are challenging for clinical laboratory diagnosis, largely due to its slow growth and higher rate of genetic mutation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a revolutionary technique for the routine identification of microorganisms. In this review, we discuss the application status of mass spectrometry in the identification and drug resistance of M. tuberculosis.     

EFFECTS OF SINGLE,BINARY AND TERTIARY COMBINATIONS WITH Jatropha gossypifolia AND OTHER PLANT-DERIVED MOLLUSCICIDES ON REPRODUCTION AND SURVIVAL OF THE SNAIL Lymnaea acuminata

The effect of sub-lethal doses (40% and 80% of LC₅₀/24h) of plant derived molluscicides of singly, binary (1:1) and tertiary (1:1:1) combinations of the Rutin, Ellagic acid, Betulin and taraxerol with J. gossypifolia latex, leaf and stem bark powder extracts and their active component on the reproduction of freshwater snail Lymnaea acuminata have been studied. It was observed that the J. gossypifolia latex, stem bark, individual leaf and their combinations with other plant derived active molluscicidal components caused a significant reduction in fecundity, hatchability and survival of young snails. It is believed that sub-lethal exposure of these molluscicides on snail reproduction is a complex process involving more than one factor in reducing the reproductive capacity.     

Essential genes from Arctic bacteria used to construct stable,temperature-sensitive bacterial vaccines

All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA, which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 °C. One ligA gene was shown to render Francisella tularensis, Salmonella enterica, and Mycobacterium smegmatis temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis.     

A truncated lipoglycan from mycobacteria with altered immunological properties

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf–branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure–function relationship of TLR2 activation by mycobacterial lipoglycans.     

Association of Streptococcus bovis presence in colonic content with advanced colonic lesion

AIM:To prospectively examine the association between presence of Streptococcus bovis(S.bovis)in colonic suction fluid and the endoscopic findings on colonoscopy.METHODS:From May 2012 to March 2013,203 consecutivepatients who underwent colonoscopy for any reason were enrolled in the study.Exclusion criteria included:antibiotic use in the previous month,age younger than18 years,and inadequate preparation for colonoscopy.The colonoscopy was performed for the total length of the colon or to the occluding tumor.The endoscopic findings were registered.Samples were obtained proximal to the colonoscopic part of the suction tube from each patient and sent to the clinical microbiology laboratory for isolation and identification of S.bovis.Samples were incubated in enrichment media with addition of antibiotic disks for inhibition of growth of Gram-negative rods.The samples were seeded on differential growth media;suspected positive colonies were isolated and identified with Gram staining,catalase,and pyrrolidonyl arylamidase tests,and further identified using a VITEK2 system.Statistical analyses were performed using the Student’s t andχ2 tests.RESULTS:Of the 203 patients recruited,49(24%)patients were found to be S.bovis carriers;of them,the endoscopic findings included:17(34.7%)cases with malignant tumors,11(22.4%)with large polyps,5(10.2%)with medium-sized polyps,6(12.2%)with small polyps,4(8.1%)with colitis,and 6(12.2%)normal colonoscopies.Of 154 patients found negative for S.bovis,the endoscopic findings included:none with malignant tumors,9(5.8%)cases with large polyps,11(7.1%)with medium-sized polyps,26(16.9%)with small polyps,7(4.5%)with colitis,and101(65.6%)normal colonoscopies.S.bovis(Grampositive coccus)is considered part of the normal intestinal flora.There is an association between S.bovis bacteremia and colonic neoplasia.It is not well understood whether the bacterium has a pathogenetic role in the development of neoplasia or constitutes an epiphenomenon of colorectal neoplasms.There was a clear relationship between positivity for S.bovis in colonic suction fluid and findings of malignant tumors and large polyps in the colon.CONCLUSION:There is an association between S.bovis bacteremia and malignant colonic lesions;this should prompt for development of a reliable screening method for advanced colonic lesions.     

The autophagic machinery ensures nonlytic transmission of mycobacteria

In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.In recent years, our understanding of the interactions between the host autophagic machinery and intracellular pathogens has rapidly expanded. These interactions are complex; although, in many cases, the engagement of autophagy protects the host by capturing and destroying the pathogen, some bacteria actively subvert this pathway to promote their own survival (reviewed in ref. 1). Autophagy has also been suggested to promote cell-to-cell transmission of Brucella (2, 3), although the molecular mechanisms are unknown.Both Mycobacterium tuberculosis, which causes tuberculosis in humans, and the closely related species M. marinum have been shown to interact with the autophagy machinery of their host cell (4–7). After uptake by immune phagocytes, the bacteria arrest phagosomal maturation and convert their vacuole into a replication-permissive compartment. Both bacteria can translocate into the host cell cytosol dependent on an intact Region-of-Difference-1-locus (RD1) (8–11). The genomic RD1-locus encodes a secretion system, ESX-1 (Type-VII secretion system), which has been associated with mycobacterial virulence (ref. 12, reviewed in refs. 13 and 14). Once in the cytosol, M. marinum becomes ubiquitinated (4) likely recruiting adaptor proteins, such as members of the sequestosome-1 family (SQSTM1), which also bind LC3 (microtubule-associated proteins 1A/1B light chains 3A/LC3A and 3B/LC3B), here referred to as Atg8, on autophagosomal membranes. In this way, bacteria are normally targeted to autophagosomes and killed, but M. marinum efficiently escapes this fate, most probably by shedding the ubiquitinated material as a decoy (4). However, infection by M. tuberculosis can be overcome by stimulating the classic autophagic pathway (15) and autophagy can reduce the bacterial burden in vivo (7).It was previously thought that M. marinum and M. tuberculosis leave their host cell by inducing necrotic or apoptotic cell death (16). However, we recently showed that these bacteria also exit their host cell and spread via an F-actin structure, termed the ejectosome (17). This form of egress, which is common to M. tuberculosis and M. marinum in the amoeba Dictyostelium, is nonlytic for the host cell, even though its plasma membrane is perforated at the site of ejection. Previously, we showed that ejectosome formation is dependent on ESAT-6 (Early secretory antigenic target 6), a secreted virulence factor encoded in the RD1-locus, and the Dictyostelium small GTPase RacH. However, both the structure and mechanistic details of ejectosome function remain unknown.Using the Dictyostelium–M. marinum system (9, 17, 18) to further dissect the mechanism of ejectosome formation and function, we demonstrate an unexpected role for autophagic membranes in both mycobacteria egress and concomitant cell-to-cell transmission.     

EVALUATION OF THE MOLLUSCICIDAL POTENTIAL OF HYDROALCOHOLIC EXTRACTS OF Jatropha gossypiifolia Linnaeus, 1753 ON Biomphalaria glabrata (Say, 1818)

The action of extracts from the stem, leaves, and fruit of Jatropha gossypiifolia on Biomphalaria glabrata was studied by analyzing survival, feeding capacity and oviposition ability. The extracts were obtained by macerating the plant parts in 92% ethanol, which were then evaporated until a dry residue was obtained and phytochemically studied. The molluscicidal activity on B. glabrata was investigated using the procedures recommended by WHO (1965). The amount of food ingested and oviposition were measured during each experiment. The extract of leaves from J. gossypiifolia was shown to be a strong molluscicidal agent, causing 100% mortality of B. glabrata, even in the lowest concentration tested, of 25 ppm. Regarding the fruit extract, there was variation in the mortality, depending on the concentration used (100, 75, 50 and 25 ppm). The snails that were in contact with the fruit extract had significant reduction in feeding and number of embryos in comparison to the control. The stem extract did not present molluscicidal activity nor had any influence on the feeding and oviposition abilities of B. glabrata, in the concentrations tested. In conclusion, the extracts of leaves and fruits of J. gossypiifolia investigated in this work show molluscicidal effect and may be sources of useful compounds for the schistosomiasis control.     

Comparison of the effects of extracts from three Vitex plant species on Anopheles gambiae s.s. (Diptera: Culicidae) larvae

Acetone and methanol extracts of different parts of three Vitex species (leaves and stem bark of Vitex trifolia, leaves, stem bark and root bark of Vitex schiliebenii and stem and root bark of Vitex payos) were evaluated for their potential to control Anopheles gambiae Giles s.s. larvae (Diptera: Culicidae). The extracts gave different levels and rate of mortality of the larvae. Some (methanol extract of V. trifolia leaves, acetone extracts of stem bark and leaves of V. schiliebenii, acetone extract of root bark of V. payos) caused 100% mortality at 100 ppm in 72 h, with those of V. schiliebenii and V. payos showing faster rate of mortality (LT₅₀ = 8 h) than that of V. trifolia (LT₅₀ = 14 h). At lower doses of these extracts (≤50 ppm), most of the larvae failed to transform to normal pupae but gave larval–pupal intermediates between 4 and 14 days of exposure. Some pupated normally but the adults that emerged appeared to be weak and died within 48 h. Extracts of the stem bark of V. payos showed interesting effects on the larvae. Initially, the larvae were relatively hyperactive compared to those in control treatments. Later, the ones that did not transform to larval–pupal intermediates became stretched and inactive and died and floated in clusters on the surface. These observations suggest some interesting growth-disrupting constituents in the plants, with possible application in the practical control of mosquito larvae in aquatic ecosystems.     

The impact of alcohol on BCG-induced immunity against Mycobacterium tuberculosis

Background: Alcoholics are at heightened risk for developing active tuberculosis. This study evaluates chronic alcohol consumption in a murine model of vaccination with Mycobacterium bovis Bacille Calmette–Guèrin (BCG) and subsequent pulmonary infection with virulent Mycobacterium tuberculosis. Methods: BALB/c mice were administered the Lieber–DeCarli liquid ethanol diet or pair‐fed the liquid control diet for 3 weeks either before or after subcutaneous vaccination with M. bovis BCG. At least 3 weeks after BCG vaccination, groups of mice on the aforesaid diets were challenged with intratracheal infection with M. tuberculosis H37Rv. Lung mycobacterial burden, and lung and lung‐associated lymph node CD4⁺ lymphocyte production of tuberculosis‐specific interferon (IFN)‐γ were assayed. Popliteal lymph node lymphocytes from both dietary regimens undergoing BCG vaccination (in the absence of M. tuberculosis infection) were also evaluated for purified protein derivative–induced IFN‐γ production by ELISpot assay. Results: Mice begun on alcohol prior to vaccination with M. bovis BCG demonstrated impaired control of pulmonary challenge with virulent M. tuberculosis, as well as impaired lung CD4⁺ and popliteal lymph node T‐cell IFN‐γ responses. If BCG vaccination was delivered prior to initiation of alcohol feeding, the mice remained protected against a subsequent challenge with M. tuberculosis, and BCG‐induced immunity was not impaired in either the lung or the popliteal lymph nodes. Conclusions: Alcohol consumption blunts the development of the adaptive immune response to M. bovis BCG vaccination, which impairs the control of a secondary challenge with M. tuberculosis, but only if the alcohol exposure is begun prior to BCG vaccination. These results provide insight into mechanisms by which alcohol consumption impairs antimycobacterial immunity, including in response to vaccination and subsequent pathogenic challenge.     

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the l- and d-enantiomeric forms of Rv1738 enabled facile crystallization of the d/l-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing l- and d-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb), currently estimated to infect one-third of the world’s population (1). A major reason for this high rate of infection is the ability of Mtb to enter a dormant state known as nonreplicating persistence (2, 3). This occurs in response to engulfment of the bacterium by activated macrophages. A proportion of bacteria survives the immune system’s antimicrobial onslaught and persists in a dormant form, but can reemerge many years later as an active infection. In the nonreplicating persistent state, protein synthesis is drastically shut down (4), and the bacteria use host lipids, notably cholesterol, as a carbon source (5). Bacteria in this state are largely resistant to current drugs.Microarray studies have identified a number of genes that are highly up-regulated under conditions thought to trigger the onset of dormancy, including hypoxia (low oxygen concentration) and exposure to nitric oxide (6, 7). The most highly up-regulated gene in hypoxic conditions, and the second-highest in response to NO, is Rv1738, suggesting this gene to be a key element in the onset of persistence. Rv1738 is also classed as an essential gene in the genome-wide transposon mutagenesis study of Sassetti et al. (8). The Rv1738 gene encodes a predicted 94-residue protein of unknown function.In this research, we set out to determine the structure of the protein encoded by the ORF Rv1738 of Mtb H37Rv. Our hypothesis was that important clues to the function of the Rv1738 protein might be obtained from its 3D structure, as structure is much more highly conserved than sequence during evolution, and can reveal unsuspected functional and evolutionary relationships. There have been both failures and successes from this approach, but on discovering that Rv1738 could readily be expressed in soluble form in Escherichia coli, we initiated structural studies. Unfortunately, all attempts to crystallize recombinant Rv1738 failed, and the protein also proved unsuitable for structural analysis by NMR because of a tendency to aggregate.Because of our inability to obtain crystals using protein expressed in E. coli, we turned to racemic protein crystallography, a recently introduced approach that can facilitate crystallization of recalcitrant proteins (9). In this method, a racemic mixture comprising equal amounts of the d-protein and l-protein forms of a target molecule are used for crystallization. A protein racemate can crystallize in centrosymmetric space groups, and can thus access many additional space groups, including some predicted to be highly favored for protein crystallization (10). In contrast, natural proteins are chiral, built only from l-amino acids and the achiral amino acid glycine, and thus cannot be incorporated into crystal lattices that include mirror planes or inversion centers. Evidence is accumulating that racemic protein mixtures can be much easier to crystallize in cases in which the natural l-protein is refractory to crystallization (9). d-proteins can only be made by total chemical synthesis (11, 12), but as Rv1738 is a small protein, we considered it a good candidate for total chemical synthesis using modern ligation methods.Here we describe the total chemical synthesis of the d- and l-forms of Rv1738 and the successful crystallization of the {d-Rv1738 + l-Rv1738} protein racemate, in striking contrast to the complete failure of conventional crystallization approaches with the recombinant protein. The racemic crystals diffracted to good resolution, and the structure was solved using an ab initio approach that benefited from the quantized nature of centrosymmetric phases (13). Moreover, the structure of Rv1738 revealed a surprising similarity to a family of stress proteins known as hibernation-promoting factors (HPFs). This suggests that the functional role of the up-regulated Rv1738 protein in nonreplicating persistence of Mtb is to contribute to the shutdown of ribosomal protein synthesis.