No description available.

For centuries medicinal plants have been used all over the world for the treatment and prevention of various ailments, particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years the use of and search for drugs derived from plants have been accelerated. Ethnopharmacologists, botanists, microbiologists, and natural-product chemists are trying to discover phytochemicals and “leads” which could be developed for the treatment of infectious diseases. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. The evaluation of these plants for biological activity is necessary, both to substantiate their use by communities, and also to discover possible new drug or herbal preparations. Twenty five plants selected through ethno-botanical surveys in Mozambique which are used to treat respiratory diseases, wounds, viruses, stomach ailments and etc., were collected and investigated for antimicrobial activity. Acetone extracts of selected plants were tested for antibacterial, antimycobacterial and anti-HIV-1 activity. Antibacterial activity was evaluated using the agar diffusion method. Five Gram positive (Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis) and five Gram negative (Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens) bacterial species were used in this study. The extracts of each plant were tested at concentrations ranging from 0.125 to 5.0 mg/ ml. Most of the plant extracts inhibited the growth of the Gram-positive microorganisms. The minimum inhibitory concentration of eight plants (Cassia abbreviata, Elephanthorrhiza elephantina, Hemizygia bracteosa, Hoslundia opposita, Momordica balsamina, Rhoicissus tomentosa and Salvadora australis) against Gram-positive bacteria was found to be 0.5 mg/ml. Gram-positive bacteria were found to be susceptible to extracts of Lippia javanica at concentration of 0.125 mg/ml. Among the 22 acetone extracts tested, two were found to have activity against Gram-negative bacteria at a concentration of 5.0 mg/ml (Adenia gummifera and Momordica balsamina). Rhoicissus revoilli inhibited E. cloacae, a Gram-negative strain, at a concentration of 2.5 mg/ml. To evaluate antimycobacterium activity ten plants species were tested against H37Rv, a drug-sensitive strain of Mycobacterium tuberculosis at concentrations ranging from 0.5 to 5.0 mg/ml using BACTEC radiometric method. Four of the plant species tested (Cassia abbreviata, Hemizigya bracteosa, Lippia javanica and Melia azedarach) were observed to be active against the H37Rv. (ATCC 27294) strain of TB at a concentration of 0.5 mg/ml which was the lowest concentration used in this study. Seventeen plant species, were screened for anti-HIV bioactivity in order to identify their ability to inhibit the enzymes glycohydrolase (á -glucosidase and â- glucuronidase) and eleven species were further tested against Reverse transcriptase. It was found that 8 plant species (Cassia abbreviata, Elephantorrhiza elephantina, Rhoicissus tomentosa, Pseudolachnostylis maprouneifolia, Lippia javanica, Litogyne gariepina, Maerua junceae and Momordica balsamina) showed inhibitory effects against á-glucosidase and â-glucuronidase at a concentration of 200 ìg/ml. The results of the tests revealed that the plant extracts of Melia azedarach and Rhoicissus tomentosa appeared to be active, showing 49 and 40% inhibition of the enzyme activity respectively. Lippia javanica was found to have the best activity exhibiting a minimum inhibitory concentration of 0.125 mg/ml. The extracts also showed positive activity against Mycobacterium tuberculosis at concentration of 0.5 mg/ml and HIV-enzyme glycohydrolase was (á-glucosidase and â-glucuronidase) inhibited by 62 % and 73 % respectively. Considering its medicinal use local for HIV and various infections, it was therefore, selected for identifying its bioactive constituents. In the initial screening of plants used in Mozambique Hoslundia oppositademonstrated good antitubercular activity. It was therefore, selected to identify its bioactive constituents. A Phytochemical investigation of L. javanica led to the isolation of eight compounds, 4-ethyl-nonacosane (1), (E)-2(3)-tagetenone epoxide (2), myrcenone (3), piperitenone (4), apigenin (5), cirsimaritin (6), 6-methoxyluteolin 4'-methyl ether (7), 6-methoxyluteolin and 3',4',7-trimethyl ether (8). Three known compounds, 5,7-dimethoxy-6-methylflavone (9), hoslunddiol (10) and euscaphic acid (11) were isolated from H. opposita. This is the first report of compounds (1), (2), (5-8) from L. javanica and of compound (9) from H. opposita. The compounds were tested against Mycobacterium tuberculosis and HIV-1 reverse transcriptase for bioactivity. It was found that compounds (2), (4) and (9) inhibited the HIV-1 Reverse transcriptase enzyme by 91%, 53% and 52% respectively at 100 ìg/ml. Of all the compounds tested against a drug-sensitive strain of Mycobacterium tuberculosis, euscaphic acid (11) was found to exhibit a minimum inhibitory concentration of 50 ìg/ml against this strain. The present study has validated scientifically the traditional use of L. javanica and H. opposita and a few other Mozambican medicinal plants to some extent. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted

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.

Includes bibliographical references / Tuberculosis (TB) is one of the major infectious diseases and epidemics in the world. It is responsible for severe morbidity and mortality rates, especially in poor and resource-deficient countries. According to the World Health Organization 2014 report, about one third of the world's population is infected with tuberculosis and about 10-15% is co-infected with HIV, which further complicates the TB epidemic. Tuberculosis claims 2-3 million lives every year and is one of the biggest social and financial burdens on many countries. The disease is treatable but has been hampered by the emergence of drug resistance in the causative bacterium, Mycobacterium tuberculosis (Mtb). Resistant strains of Mtb counter the efficacy of various anti-TB drugs via mechanisms that help it overcome the toxic and inhibitory effects of these drugs. These mechanisms include mutation, enzymatic drug degradation, target modification and drug efflux. Drug efflux by efflux pumps (EPs) is one of the major mechanisms responsible for the development of drug tolerance leading to the emergence of drug resistance. These efflux pumps are regulated by the house keeping proteins present in the cell membrane of Mtb and perform a pre-existing role of rescuing the Mtb from toxic agents. These EPs extrude structurally unrelated compounds from the cell including anti-TB drugs and reduce the drug concentration to sub-inhibitory levels and aid Mtb in developing resistance. Therefore, development of antimycobacterials that target EPs and reduce their activity can be a viable strategy to reduce the global TB burden and counter the emergence of resistance. Many strategies have been used to counter the EP-mediated resistance in Mtb. In this study, two strategies were employed: (i) the development of efflux pump inhibitors (EPIs) via structural modification of a known efflux pump inhibitor, verapamil (VER), and the development of hybrid efflux pump inhibitors (HEPIs) incorporating a VER motif; and (ii) the development of antimycobacterial agents based on covalent linking or attachment of efflux pump inhibitor moieties to an anti-TB drug. These agents are termed reversed anti-TB agents and are based on isoniazid for this study.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions.

No description available.

This anthropological research began with curiosity about human relationships with microbes. Inside the contained environment of a Biosafety Level 3 laboratory at a South African university-based tuberculosis research division, the fieldwork focused on the relationships between scientists and Mycobacterium tuberculosis − the pathogenic bacterium that causes the disease tuberculosis (TB). These deadly bacteria were cared for and nurtured by women scientists. This care extended to the cells and various species with which they worked. Moreover, this care moved beyond the scope of their immediate scientific research projects and well beyond the laboratory. Care was also central to how the participants conducted their scientific research and themselves in the world. This long-term, qualitative ethnographic research weaves together many layers of care in biomedical scientific research, highlighting that scientific research is a deeply personal, caring and subjective practice. The natural and the social are not − and can never be − mutually exclusive. Boundaries between mind/body, subject/object, human/nonhuman, researcher/researched, subjectivity/objectivity and science/society are porous. Acutely aware of the socio-political moment in which this research was embedded, these findings are put into conversation with South African student calls to decolonise science that emerged alongside the #RhodesMustFall student movement. In particular, the focus is on a 2016 meeting about decolonising science at the University of Cape Town where students argued for connection between the university and the community, science and society and the world of academia and the world of Africans. Implicit was the need for science to be relevant to Africans and deeply complex African social formations and problems. The care by women scientists that was observed inside the laboratory and beyond it speaks volumes to cultivating a more caring science and caring institutions of science that connect the laboratory to the world in which it exists in meaningful, relevant and impactful ways. I demonstrate how the participants embodied a decolonised science, and that what they cared about and how they acted upon those cares could serve as important guides for decolonising science and scientific institutions. This research provides important contributions to the field of science and technology studies (STS), to anthropological research on TB and to the conversation on decolonising science in South Africa.

Human immunodeficiency virus type 1 (HIV-1) and Mycobacterium tuberculosis (Mtb) coinfection remains a major global health threat. Both pathogens synergistically drive pathogenesis of the other. The risk of developing active tuberculosis (TB) is increased in people living with HIV-1, even in those receiving antiretroviral therapy (ART), whilst TB was responsible for 15 % of HIV-related deaths in 2020. Mtb co-infection increases the likelihood of transcriptionally activating HIV-1 replication potentially due to bioactive Mtb lipids engaging macrophage surface receptors, thus triggering signaling pathways which activate human transcriptional factors (hTF) and production of inflammatory cytokines capable of activating HIV-1 transcription. This work investigated the hypothesis that clinical Mtb strains with single nucleotide polymorphisms (SNP) in lipid-metabolizing genes, required for cell wall lipid biosynthesis, differentially affect HIV-1 replication and human macrophage inflammatory response during Mtb-HIV-1 co-infection in vitro. Monocyte derived macrophages (MDM) were the predominant model used to investigate this phenomenon. Infections, in the presence or absence of HIV-1 co-infection, were performed using either lineage 2 or lineage 4 clinical strains with non-synonymous SNP in polyketide synthase 2 (pks2) required for sulfolipid 1 (SL-1) biosynthesis and compared to control infections using phylogenetically close clinical strains without the SNP of interest and canonical lineage 2 and 4 laboratory strains (H37RvP1939/T605, CDC1551WT and HN878WT). Secreted cytokines and chemokines were measured in supernatant (SN) by Luminex. The effect of Mtb on HIV-1 viral production was assessed by measuring HIV-1 Gag p24 in the SN of co-infected MDM or SN of HIV-1 infected MDM incubated with conditioned media from Mtb-infected MDM. The influence of Mtb on HIV-1 transcriptional activity was measured using a transgenic cell line (TZM-bl) with Luciferase reporter under HIV-1 long terminal repeat (LTR) expression. The impact of incubating TZM-bl cells in Mtb-induced conditioned media before or after HIV-1 infection was assessed. One pair of phylogenetically close clinical strains with and without a pks2 SNP of interest (EX30Q1939/A605 and MRC16P1939/A605) with interesting lipid and inflammatory phenotypes, and H37RvP1939/T605 as a lineage 4 control, were subject to single nucleotide mutagenesis using recombineering to either revert SNP of interest to match the alleles of H37Rv or introduce the SNP of interest into the control strains. The wild-type and mutant strains were used in a trans-well assay to infect MDM in the presence of HIV-1 co-infection in the top chamber, while simultaneously mimicking the bystander effect of cytokine-mediated HIV-1 regulation in the bottom chamber which was only infected with HIV-1. Results demonstrate there was increased cytokine production by MDM infected with MRC16P1939/A605 in both the presence and absence of HIV-1 co-infection compared to its phylogenetically close paired strain EX30 Q1939/A605. The data shows that there was no difference in LTR activity in TZM-bl cells co-incubated with inflammatory environment between the strains of interests, however co-incubation of TZM-bl cells with Mtb-induced inflammatory environment generally increased LTR activity during HIV-1; a proxy for HIV-1 replication. In the trans-well co-infection assay, a significant positive association between production of HIVp24 and secretion of CCL2 was observed, whilst IL-1β secretion showed a significant negative relationship with the production of HIVp24, with donor variability in baseline cytokine production also associated with the extent of HIVp24, CCL2, IL-1β and IL-8 production. Introduction of the pks2 T605A SNP into H37RvP1939/T605 and reversion in EX30Q1939/A605T significantly modified their inflammatory phenotype. Together these results support the hypothesis that Mtb clinical stains with genetic variation in cell wall lipid biosynthesis impacts the inflammatory milieu and, subsequently, HIV-1 replication during co-infection. The outcome of Mtb-HIV co-infection is therefore not homogenous but contingent on the phenotype of infecting Mtb strain and individual.

Transmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission.

Made available in DSpace on 2014-07-29T15:04:41Z (GMT). No. of bitstreams: 1 dissertacao lilian.pdf: 281825 bytes, checksum: 488c9e5d58cb1a521b28a8592ee6de50 (MD5) Previous issue date: 2006-02-23 / According to the World Health Organization (WHO), an hundred million of individuals are infected by M. tuberculosis, annually. Health care workers play an important role to control of tuberculosis, but they are also at high risk for this infection. Then, the objectives of the present study were to evaluate the prevalence of M tuberculosis infection in nursing professionals from the Tropical Diseases Hospital in Goiânia City, State of Goiás, to analyze the factors associated to tuberculin skin test (TST) positivity and to determine the TB infection incidence density in susceptible professionals Initially, the prevalence and factors associated to TST were investigated in 128 eligible individuals. Further, susceptible professionals (n=32) were followed up during three years (2001-2004) to detect TST conversion. Of the total individuals investigated, 69.5% (IC 95%: 60.7-77.2) were positive to TST. Two occupational factors were independently associated to skin test positivity: duration of profissional activity longer than 5 years (Adjustd OR = 6.3; 95% CI: 1.5-26.2) and occupational contact with a person with pulmonary TB ≤ 2 years (Adjusted OR = 12.2; 95% CI: 1.2-106.3). Seven profissionals showed tuberculinic conversion during the three years of follow up, and an incidence density of 11.5 new conversions to 100 persons-year was detected. All of them had taken care of patients during the period of the study. Two individuals developed tuberculosis disease. The data of this study ratify the high risk of tuberculosis in nursing team, and highlight the importance of this infection as an occupational disease to nursing professionals of our region. / De acordo com a Organização Mundial de Saúde (OMS), cem milhões de pessoas são infectadas pelo M. tuberculosis, a cada ano. Os profissionais de saúde são importantes para o controle da tuberculose, mas também um grupo de risco elevado para esta infecção. Assim, o presente estudo teve como objetivos avaliar a prevalência da infecção causada pelo M. tuberculosis em profissionais de enfermagem de uma instituição especializada em doenças infecciosas, em Goiânia Go, analisar os fatores associados à positividade à prova tuberculínica nesta população e determinar a densidade de incidência da infecção pelo M. tuberculosis, nos profissionais susceptíveis. Inicialmente, verificou-se a prevalência e os fatores associados à positividade à PT. A seguir, os profissionais suscetíveis à infecção (n=32) foram acompanhados, por três anos (2001-2004), para detecção de conversão tuberculínica. Do total de profissionais investigados, 69,5% (IC 95%: 60,7- 77,2) foram positivos à PT. Dois fatores ocupacionais foram independentemente associados à positividade à PT: tempo de atividade profissional > 5 anos (OR ajustado = 6,3; IC 95%: 1,5-26,2) e último contato laboral com alguém com TB ≤ 2 anos (OR ajustado = 12,2; IC95%: 1,2-106,3). Sete profissionais apresentaram viragem tuberculínica, resultando em uma densidade de incidência de 11,5 novas conversões por 100 pessoas/ano. Todos desenvolviam atividades assistenciais, durante o período do estudo. Duas profissionais desenvolveram tuberculose doença. Os resultados, deste estudo, ratificam o elevado risco de tuberculose nos profissionais de enfermagem, e evidenciam a importância desta infecção como doença ocupacional para equipe de enfermagem de nossa região.