Lipid metabolism plays a prominent role in the survival of Mycobacterium tuberculosis (MTB) in both the macrophage and mammalian hosts. A central question in the reconstruction of lipid metabolism in MTB is the key regulatory programs responsible for changes in this metabolism under different conditions. One of the most studied conditions is hypoxia (oxygen deprivation) as adaptations to hypoxia are thought to play an important role in MTB pathogenesis and latency. To identify temporal trends during a hypoxic time course and associate them with possible regulators, expression data was clustered into paths using DREM algorithm and available ChIP-Seq data for over 80 transcription factors (TFs). The degree to which expression patterns might reflect the direct action of transcription factors was assessed by evaluating the consistency between the path, the expression of each TF binding genes in the path, and the predicted regulatory role of the TF from the regulatory network. DosR was correctly identified as an activator of the path corresponding almost entirely to its regulon. Rv0081 was found to be a candidate high level regulator broadly predictive of the overall expression of sets of genes during hypoxia and re-aeration. An essential part of the continued survival of MTB in the host is due to its adaptation to the phagosomal compartment of the macrophage where the bacterium faces hypoxic as well as other stresses. Using the same approach and several time course expression data sets available for MTB in macrophage cultures, macrophage temporal expression models were built and compared to the hypoxia model. Similar trends were found in the expression of genes involved in respiration, cholesterol catabolism and methylcitrate cycle. In contrast, a group of genes responsible for the synthesis of complex cell wall lipids (PAT/DAT and SL-1) were up-regulated in the macrophage models and downregulated in the hypoxic model. PhoP was predicted as a potential main regulator of these genes as a result of pH change which takes place in the macrophage environment but not during hypoxia.

Tuberculosis is an infectious disease caused by the pathogen Mycobacterium tuberculosis which is still a public health threat. Due to the appearance of multidrug-resistant M. tuberculosis strains, it is urgent to find new drugs to fight against tuberculosis, especially against the extensively drug resistant tuberculosis. The unique impermeable mycobacteria cell wall, rich in lipids, is a key barrier to anti-tuberculosis drug uptake. One of these lipids, the phthiocerol dimycocerosate (PDIM), is related to mycobacteria virulence, drug resistance and mycobacteria cell wall integrity. Therefore, research focusing on PDIM biosynthesis potentially provides a new route for the development of new anti-tuberculosis drugs.In this context, we investigated two proteins, the thioesterase TesA and the chaperonin GroEL1, that are involved in the PDIM biosynthesis, and studied their structural and functional properties. Firstly, using various substrates, we demonstrated that TesA displays thioesterase and esterase activities and showed that TesA undergoes substrate-dimerization. A new TesA inhibitor, methyl arachidonyl fluorophosphonate (MAFP), was identified that irreversibly binds to the TesA catalytic serine residue. Furthermore, MAFP can increase the susceptibility to vancomycin in a TesA inhibition dependent manner while it can also affect the M. bovis BCG biofilm formation through broader action mechanisms. Secondly, we observed that the mycobacterial GroEL1, unlike the other chaperonins, E. coli GroEL and mycobacteria GroEL2, increases copper tolerance. We demonstrated that the copper can specifically bind to the GroEL1 with a strong affinity for its natural carboxyl terminal histidine-rich region. The classical feature of chaperonins is also increased in the presence of copper. These results suggest that GroEL1 could help mycobacteria to tolerate high bactericidal metal concentrations during macrophage infection, potentially through a metal storage mechanism. Last but not the least, we identified new mycobacterial GroEL1 and GroEL2 activities. In one word, the biochemical properties of two essential proteins, GroEL1 and TesA, involved in PDIM biosynthesis in M. tuberculosis have been further elucidated. The implications of these results are important not only to develop new antituberculous drugs but also to fight drug resistance. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Thesis (M. Sc. (Medical Sciences)) -- University of Limpopo, 2015 / Tuberculosis (TB) patients may have mixed infections with both drug-susceptible and drug-resistant Mycobacterium tuberculosis (MTB) strains. This phenomenon termed heteroresistance presents a challenge TB management and is considered a preliminary stage to full resistance. Heteroresistance is more likely to occur in high TB incidence areas and in chronic patients as they have more opportunity to become infected with various strains of TB and has been proven to occur in new cases, treatment failure and relapse. Methods: Sputum samples were collected from new consulting and hospitalised patients who were on treatment for MDR TB. A total of 231 samples were run on MTBDRplus to determine heteroresistance of Mycobacterium tuberculosis to isoniazid and rifampicin. To determine heteroresistance to second-line drugs, 91 samples were run on MTBDRsl. Nineteen (19) samples that were heteroresistant to 2nd line drugs were subjected to spoligotyping to determine the families/lineages they belonged to. Results: A total of 66 were confirmed as Mycobacterium tuberculosis complex by the line probe assays. Out of the 66 MTBC, rifampicin resistance was found in 22 (10%) and 44 (19%) were reported susceptible. Isoniazid resistance was found in 39 (17%) and 27 (12%) were reported susceptible. Of the 66 MTBC positive samples, moxifloxacin resistance was found in 33 (16%) and 14 (7%) were reported susceptible. Kanamycin resistance was found in 17 (8%) and 30 (14%) were reported susceptible. Ethambutol resistance was found in 25 (12%) and 22 (10%) were reported susceptible. Heteroresistance was evident in 22 (10%) samples for the first-line and in 23 (11%) for the second-line drugs. Results of a total of 19 heteroresistant samples subjected to spoligotyping when compared to those in the international spolDB4 database indicated that 4 of them matched existing shared spoligotype international types, 15 were unknown (orphans). Eighteen (18) of 19 heteroresistant samples subjected to spoligotyping were also MDR. Fourteen of the samples that were resistant to both RIF and INH were orphans. Of the 14 MDR, 3 samples belonged to clades T1, T-H37RvV817 and LAM 3 with SITs: 879, 568 and 2301, respectively. One sample with SIT 1196 had an unknown clade was resistant to RIF but susceptible to INH. Conclusion: This study has shown that heteroresistance remains an important phenomenon in clinical tuberculosis, especially in highly endemic areas. According to the current study, heteroresistance was associated more with recurrent cases who are on initiation or continuation phase than new cases and a larger percentage of heteroresistance was reported in second-line drugs than there is in first-line drugs. The T1 genotype was found to be predominant amongst recurrent cases. The LAM3 and T-H37RvV817 lineages were found amongst the new cases. In the present study there was no significant association between heteroresistance and the patient’s treatment history as indicated by a P-value of 0.473 and between heteroresistance and spoligotype families (P-value, 0.991). The predominance of orphan SITs and unknown clades followed by non-Beijing strains in the study may be due to the migration of carriers from the neighboring countries as the Limpopo Province is flanked by Botswana, Zimbabwe and Mozambique. Further studies with larger numbers of patients should focus on the prevalence to associate heteroresistance with patients‟ treatment history and establish the contributing MTBC strain lineages.

Objetivo: Al menos uno de los péptidos sintéticos muestra actividad inhibitoria para PknG Diseño: Esta investigación califica como un estudio in vitro, dado que los experimentos serán realizados bajo condiciones controladas fuera de un organismo vivo.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objectives: Diagnosis of tuberculous meningitis (TM) is a challenge in countries with a high burden of the disease and constrained resources and clinical prediction rules (CPRs) could be of assistance. We aimed at developing a CPR for diagnosis of TM in a Latin American setting with high tuberculosis incidence and a concentrated HIV epidemic. Methods: We enrolled adult patients with clinical suspicion of TM attending two hospitals in Lima, Peru. We obtained information on potential anamnestic, clinical and laboratory predictive findings that are easy to collect and promptly available. We independently diagnosed TM according to a composite reference standard that included a series of microbiological tests. We performed bivariate analysis and constructed a logistic regression model to select the predictive findings associated with TM. With the selected predictors included in the model, we developed a score-based CPR. We assessed its internal validity and diagnostic performance. Results: Of 155 analysed patients, 59 (38%) had TM. The CPR we derived includes three predictors: cough for 14 days or more, 10–500 cells in CSF and adenosine deaminase ≥ 6 U/l in CSF. It classifies patients into high-, moderate- or low-score groups and has an overall area under the ROC curve of 0.87. 59% of patients were assigned to either the high- or the low-score group, permitting prompt decision-making. In patients in the high-score group, it attains a positive likelihood ratio for TM of 10.6 and in patients with low scores, a negative likelihood ratio of 0.10. Bootstrap analysis indicated high internal validity. Conclusion: This CPR could support decision-making in patients with clinical suspicion of TM. External validation and further assessment of its clinical impact are necessary before application in other settings. / Revisión por pares

Bovine tuberculosis (bTB) was first diagnosed in the Kruger National Park (KNP) in 1990 and research has since focused primarily on the buffalo (Syncerus caffer) as the maintenance host and lion (Panthera leo) as a clinically affected species. However, little is known about the role that small predators might play in the tuberculosis epidemiology. The aim of this pilot study was to screen banded mongoose populations in the bTB high prevalence zone of the KNP for mycobacteria in general and for Mycobacterium bovis and other Mycobacterium tuberculosis complex members in particular to detect presence of infection. Faecal swabs, tracheal swabs and tracheal lavage of 76 banded mongooses caught in cage traps within a two kilometre radius of Skukuza Rest Camp in the KNP were submitted for culture, isolation and speciation of Mycobacterium as the gold standard of bTB diagnosis. Blood was collected and serologically analysed for M. bovis and Mycobacterium tuberculosis antibodies using the ElephantTB STAT-PAK® Assay (STAT-PAK) and the EnferplexTM TB Assay (Enferplex). DPP® VetTB Assay for elephants (DPP) was used on STAT-PAK positive samples. To complement the sample set obtained from live banded mongooses 12 animals were necropsied. Lesions and pooled lymph node samples together with a standard set of organ samples were submitted for culture and histopathology analysis. Two banded mongooses had developed well demarcated, irregularly margined, greyyellow nodules of up to 5 mm diameter located in the caudal lung lobes and/ or tracheo-bronchial, retropharyngeal or superficial cervical lymph nodes. These lesions were characterised by central necrosis in the one and calcification in the other animal. Histopathologically the lesions were described as caseating necrosis associated with epithelioid macrophages and necrogranuloma with calcified centre respectively. No acid fast bacteria were identified with Ziehl-Neelsen stain. M. bovis was isolated from lung, lymph node and liver samples as well as tracheal lavages and tracheal swab from the same two banded mongooses but not from any other study animal. No other Mycobacterium of the M. tuberculosis complex was isolated. However, a variety of environmental mycobacteria, the most frequent from the Mycobacterium avium complex, M. fortuitum group, M. simiae group and M. terrae group, were cultured. M. fortuitum group was only and M. terrae group predominantly isolated from tracheal and faecal samples whereas M. simiae group and M. avium complex were the most frequent species isolated from post mortem samples, including tissue lesions and lymph nodes. Serological analysis revealed 12 banded mongooses with a positive STAT-PAK result, confirmed with DPP. Enferplex was positive for MPB83 in four and MPB70 peptide in one animal. Only two banded mongooses, the ones with the strongest positive reaction on both STAT-PAK and DPP, reacted positively on all three serological assays. These were the same two animals that had developed granulomatous lesions and that M. bovis was cultured from ante and post mortem samples. In conclusion, this study has provided the first evidence of bTB infection in banded mongooses in the KNP and demonstrated their ability to shed M. bovis. This finding has opened the discussion around possible sources of infection and its significance at the human/ wildlife interface in and around Skukuza. / Dissertation (MMedVet)--University of Pretoria, 2014. / tm2015 / Production Animal Studies / MMedVet / Unrestricted

<p><i>Mycobacterium abscessus</i> is an emerging infectious pathogen capable of causing pulmonary disease similar to tuberculosis, but has many intrinsic and extrinsic properties making it more drug-resistant than <i>Mycobacterium tuberculosis</i>. Current treatments, including those used for <i>M. tuberculosis</i> infection, have had poor results. Although <i>in vitro</i> studies have shown promise with drug treatment for this microorganism, clinical trials have been mostly unsuccessful. An <i>in vitro </i>model that mimics the physiological stresses encountered within the human body is likely to enable the discovery of mechanisms of antibiotic resistance used by <i>M. abscessus</i> during infection. Therefore, we subjected <i>M. abscessus</i> to a combination of stresses thought to be encountered by mycobacteria inside the human body. We subjected the pathogen to low oxygen, low pH, and nutrient starvation. This is the first report on subjecting <i>M. abscessus</i> to such a combination of stresses. It is also the first to investigate the effect of the combination of stresses on the tolerance of the pathogen to antibiotics, and the effect of efflux pump inhibitors under such conditions. We found that under these conditions, <i>M. abscessus</i> entered a non-replicating state. We investigated whether the multiple-stressed <i>M. abscessus</i> displayed altered tolerance to antibiotics commonly used to treat infection, and whether efflux pump inhibitors affected the antibiotic resistance under such conditions.<i> </i>We found that when subjected to our multiple stress model, <i>M. abscessus</i> in the reactivating phase had higher tolerance to erythromycin in combination with efflux pump inhibitors verapamil and reserpine compared to non-replicating <i>M. abscessus</i>. Reactivating phase cells had a higher tolerance to antibiotic erythromycin than non-replicating cells. Reactivating phase cells also showed antibiotic tolerance in the presence of ATP. This physiologically-relevant experimental model for <i>M. abscessus</i> could potentially be used in discovering the mechanisms of antibiotic resistance in the pathogen.</p>

Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...

The group of Mycobacteria is one of the most intensively studied bacterial taxa, as they cause the two historical and worldwide known diseases: leprosy and tuberculosis. Mycobacteria not identified as tuberculosis or leprosy complex, have been referred to by ‘environmental mycobacteria’ or ‘Nontuberculous mycobacteria (NTM). Mycobacterium kansasii (M. kansasii) is one of the most frequent NTM pathogens, as it causes pulmonary disease in immuno-competent patients and pulmonary, and disseminated disease in patients with various immuno-deficiencies. There have been five documented subtypes of this bacterium, by different molecular typing methods, showing that type I causes tuberculosis-like disease in healthy individuals, and type II in immune-compromised individuals. The remaining types are said to be environmental, thereby, not causing any diseases. The aim of this project was to conduct a comparative genomic study of M. kansasii types I-V and investigating the gene expression level of those types. From various comparative genomics analysis, provided genomics evidence on why M. kansasii type I is considered pathogenic, by focusing on three key elements that are involved in virulence of Mycobacteria: ESX secretion system, Phospholipase c (plcb) and Mammalian cell entry (Mce) operons. The results showed the lack of the espA operon in types II-V, which renders the ESX- 1 operon dysfunctional, as espA is one of the key factors that control this secretion system. However, gene expression analysis showed this operon to be deleted in types II, III and IV. Furthermore, plcB was found to be truncated in types III and IV. Analysis of Mce operons (1-4) show that mce-1 operon is duplicated, mce-2 is absent and mce-3 and mce-4 is present in one copy in M. kansasii types I-V. Gene expression profiles of type I-IV, showed that the secreted proteins of ESX-1 were slightly upregulated in types II-IV when compared to type I and the secreted forms of ESX-5 were highly down regulated in the same types. Differentially expressed genes in types II-IV were also evaluated and validated by qPCR for selected genes. This study gave a general view of the genome of this bacterium and its types, highlighted some different aspects of its subtypes and supplemented by gene expression data.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis (M. tb.) is likely important in the establishment of a primary infection in the lung. M. tb. binds to a variety of phagocyte receptors, of which the mannose receptor and the complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a β₂ integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tb. to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesised complement, which are inherent in studies with mononuclear phagocytes. The surface expression and functional activity of CR3 were confirmed by rosetting with beads coupled to anti-CR3 monoclonal antibodies (MAbs) and with C3bi-coated microspheres, respectively. We found thatM. tb. binds 4-7-fold more avidly to CR3- expressing CHO cells than to wild-type cells, and importantly, that this binding is very similar in the presence of fresh or heat-inactivated human or bovine sera, or no serum. The binding of M. tb. to the transfected CHO cells is CR3-specific, as it is inhibited by anti-CDllb and anti-CD18 MAbs; interestingly, binding is not inhibited by a MAb (2LPM19c) specific for the C3bi-binding site on CDI lb. Electron micrographs of infected CR3-expressing CHO cells reveal the presence of intracellular bacteria enclosed in well-defined, membrane-bound vacuoles. We conclude that the binding of M. tb. to CR3 is nonopsonic and that the organism likely expresses a ligand that directly binds to CR3.