Rozprawy doktorskie na temat „Host-directed”

As a first step to producing a shape selective catalysts or enzyme mimic, two preorganized host molecules were synthesized. Binding studies of the two hosts with a variety of guests in three solvents demonstrated that an important driving force in the association was the formation of C-H???X-R hydrogen bonds (X = halogen). A deuterated host was utilized to further examine the formation of the C-H???X-R hydrogen bonds. In an effort to place functionality in the hydrophobic pocket of these hosts, two methods were developed. The first utilized directed ortho metallation to place electrophiles above and/or directed into the cavity. Perlithiation of the host could lead to sixty-nine products but reaction conditions and host rigidity limited product formation. This reaction technique led to the placement of carboxylic acid groups onto the host and the isolation of twelve products. Two different positions of the carboxylic acids (endoand exo-) direct the orientation of the guest. 1D- and 2D-NMR were utilized to examine how the was orientated inside the host. The second method employed to place functionality on the host, sited a tripodal zinc binding ligand on the side of the hydrophobic pocket of the host. The synthesized host was able to bind zinc strongly and in a 1:1 manner.

Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNy-dependent autophagy, respectively. Indeed, sustained expression and activation of PKR reduced the intracellular survival of Mtb, an effect that could be enhanced by IFNy treatment. We further demonstrate that the enhanced anti-mycobacterial activity of THP-PKR macrophages is mediated by a mechanism dependent on selective autophagy as indicated by increased levels of LC3-II that colocalize with intracellular Mtb. Consistent with this mechanism, inhibition of autophagolysosome maturation with bafilomycin A1 abrogated the ability of THP-PKR macrophages to limit replication of Mtb, whereas pharmacological activation of autophagy enhanced the anti-mycobacterial effect of PKR overexpression. As such, PKR represents a novel and attractive host target for development of HDT for TB, and our data suggest value in the design of more specific and potent activators of PKR.

Tuberculosis (TB), caused by Mycobacterium tuberculosis is the leading infectious disease epidemic that claims over 1.6 million lives, while 10 million fell ill in 2017. South Africa is burdened with the third highest global incidences following India and China with high rates of co-infections with HIV and highest numbers of multi-drug resistant (MDR) and extremely resistant (XDR) TB per capita. The current treatment regimen is decades old and requires a prolonged period of 6 months. The lack of efficient TB therapy and the emergence of MDR and XDR TB, there is an urgent need to find new drug targets for TB therapy through understanding the complex host-pathogen interactions. This may then lead to pathogen, host-directed therapies (HDT) or adjunct therapies as well as the development of effective drugs and drug formulations for the treatment of TB. Here we aimed to investigate potential targets for pathogen-and host-directed therapies for TB. We screened the anti-mycobacterial activity of 172 minor groove binder (MGB) compounds that selectively bind to AT-rich regions of the minor groove of bacterial DNA with the helical structure matching that of DNA in Mtb culture. Of the 172 total compounds screened 17 hits were identified, of which 2, MGB 362 and MGB 364 displayed intracellular mycobactericidal activity against Mtb HN878 at an MIC50 of 4.09 and 4.19 μM, respectively, whilst being non-toxic. Encapsulation of MGBs into non- ionic surfactant vesicles (NIVs) demonstrated a 1.6- and 2.1-fold increased intracellular mycobacterial activity, similar to that of rifampicin when compared with MGB alone. Treatment with MGB 364 or MGB 364 formulation did not cause DNA damage in murine infected macrophages as displayed by low expression of γ-H2Ax compared to H2O2 and DMSO. Intranasal administration of MGB 364 and MGB-NIV 364 formulation showed one log reduction in bacterial burden with improved pathology and immune cytokine production when in formulation. However, intranasal administration of 10 mg/kg MGB 362 together with rifampicin had no effect on bacterial loads. In summary, the data demonstrate the potential of MGB as a novel class of drug/chemical entity in anti-TB therapy and NIVs as an effective delivery system in a novel anti-TB formulation. Using deep CAGE and small RNA (CHIP-seq) technologies, International Center for Genetic Engineering and Biotechnology’s Cytokines and Diseases lab in collaboration with the RIKEN Center for Integrative Medical Sciences (Yokohama, Japan) performed a novel transcriptomics study approach by conducting a genome-wide transcriptional analyses of RNA transcripts from classically activated macrophages (caMph) and alternatively activated macrophages (aaMph) during Mtb infection. We identified host target genes that may play a role in host immune subverting mechanism by Mtb to hide away from host effector functions providing a possible target for host-directed therapy for tuberculosis. It is postulated that Mtb modulates the transcriptional landscape of IL-4/IL13 alternatively activated macrophages (aaMph) to escape killing by reactive nitrogen intermediates (NO) and reactive oxygen species (ROS) functions by IFN-γ stimulated classically activated macrophages (caMph). Here we report on the immunoregulatory role of IL-4i1, a candidate gene that was upregulated in aaMph during Mtb infection. IL-4i1 is a secreted L-amino oxidase with antibacterial properties. The enzyme converts Phenylalanine (Phe) into phenylpyruvate releasing toxic products ammonia and hydrogen peroxide (H2O2) which in-turn cause immunosuppression of effector T-cells by directly inhibiting polarization, proliferation and function or by promoting the generation of Foxp3 T-regulatory cells. Thus suggesting that IL-4i1 is involved in immune-regulatory mechanisms and may be implicated in immune evasion mechanisms by the pathogen. Here we report on the role of IL-4i1 on tissue localized T-cell activation and proliferative status thus maintaining immune local immune homeostasis. Thus showing that the absence of IL-4i1 could cause autoimmunity. To determine the functional role of IL-4i1 during Mtb infection, IL-4i1 deficient mice and wild-type littermate controls were infected with H37Rv and hypervirulent HN878 Mtb strain. IL-4i1 deficient mice were highly resistant to both strains of Mtb at 12- and 21-days post-infection as denoted by significant reduction in bacterial loads, reduced inflammation, reduced tissue iNOS expression reduced recruitment of interstitial macrophages, pro-inflammatory cytokines showed a trend for reduction. Interestingly there was a significant increase in NO production in infected tissues. There was an increase in M1-like macrophages that correlated with increased pro-inflammatory cytokines and chemokines. These data suggested that IL-4i1 regulates macrophage-mediated inflammatory responses during acute Mtb infection thus showing potential as an immunomodulatory target for TB HDT therapy. The study thus provides a framework for new drug targets for the development of new effective drugs and vaccines for TB therapy.

La tuberculosi (TB) és una epidèmia global causada per Mycobacterium tuberculosis (Mtb) amb 8,6 milions de malalts i 1,3 milions de morts cada any. El tractament actual amb antibiòtics és molt llarg, car, i presenta efectes adversos. Quan una persona s’infecta amb Mtb pot controlar la infecció en el 90% dels casos (infecció latent), desenvolupant només lesions microscòpiques al pulmó: granulomes de 0,5mm de diàmetre, invisibles en una radiografia. En el 10% restant la infecció no es controla i es desenvolupen lesions més grans, típicament cavitats d’uns 20mm en adults immunocompetents. La clau per comprendre la patogènesi de la TB activa és la formació de grans cavitats a partir de granulomes de 0,5mm. En aquesta tesi s’ha desenvolupat un model murí, mitjançant la infecció endovenosa de ratolins C3HeB/FeJ amb la soca virulenta de Mtb H37Rv, que desenvolupa lesions amb necrosi granulomatosa central i liqüefacció molt similars a les lesions prèvies a la cavitació en humans. Les lesions creixen de forma exponencial en part degut a la infiltració neutrofílica massiva, i en part degut a la coalescència de les lesions properes. Els estudis comparatius amb la soca resistent C3H/HeN i l’ús d’antiinflamatoris no esteroideus (AINEs) en el model han confirmat que la inflamació és un factor clau en el desenvolupament de la TB activa, i també que els AINEs podrien utilitzar-se com a tractament coadjuvant en la TB pulmonar en adults immunocompetents, ja que en frenar la inflamació ajuden a controlar la malaltia. D’altra banda s’ha desenvolupat un mètode profilàctic que mitjançant l’administració oral de dosis baixes de micobacteris inactivats indueix tolerància al Mtb, i en conseqüència una resposta immunitària més equilibrada, amb contenció de la resposta Th17, resultant en una millora de la supervivència, la càrrega bacil·lar i la histopatologia dels ratolins. Conclusions: S’ha desenvolupat un model murí de TB activa, s’ha caracteritzat el paper que té la inflamació en el desenvolupament de cavitats, concretament la infiltració massiva de neutròfils, s’ha proposat l’ús d’AINEs com a tractament coadjuvant de la tuberculosi activa en adults immunocompetents, i s’ha desenvolupat un nou mètode profilàctic que podria evitar la malaltia mitjançant la inducció de tolerància oral al Mtb que s’aconsegueix amb l’administració oral de dosis baixes de micobactèries inactivades.
La tuberculosis (TB) es una epidemia global causada por Mycobacterium tuberculosis (Mtb) con 8,6 millones de enfermos y 1,3 millones de muertes cada año. El tratamiento actual con antibióticos es muy largo, caro i presenta efectos adversos. Cuando una persona se infecta con Mtb puede controlar la infección en el 90% de los casos (infección latente), desarrollando solamente lesiones microscópicas en el pulmón: granulomas de 0,5mm de diámetro invisibles en una radiografía. En el 10% restante la infección no se controla y se desarrollan lesiones mayores, típicamente cavidades de unos 20mm en adultos inmunocompetentes. La clave para comprender la patogénesis de la TB activa es el paso de granulomas de 0,5mm a cavidades de gran tamaño. En esta tesis se ha desarrollado un modelo murino mediante la infección endovenosa de ratones C3HeB/FeJ con la cepa virulenta H37Rv de Mtb, que desarrolla lesiones con necrosis granulomatosa central y licuefacción, muy similares a las lesiones previas a la cavitación en humanos. Las lesiones crecen de forma exponencial debido en parte a la infiltración neutrofílica masiva, y en parte a la coalescencia de las lesiones vecinas. Los estudios comparativos con la cepa resistente C3H/HeN y el uso de antiinflamatorios no esteroideos (AINEs) en el modelo han confirmado que la inflamación es un factor clave en el desarrollo de la TB activa, y también que los AINE podrían utilizarse como tratamiento coadyuvante en la TB pulmonar en adultos inmunocompetentes, dado que en frenar la inflamación ayudan a controlar la enfermedad. Por otro lado se ha desarrollado un método profiláctico que mediante la administración oral de dosis bajas de micobacterias inactivadas induce tolerancia al Mtb, y en consecuencia una respuesta inmunitaria más equilibrada, conteniendo la respuesta Th17, resultando en una mejora de la supervivencia, la carga bacilar y la histopatología de los ratones. Conclusiones: Se ha desarrollado un modelo murino de TB activa, se ha caracterizado el papel de la inflamación en el desarrollo de cavidades, concretamente de la infiltración masiva de neutrófilos, se ha propuesto el uso de AINEs como tratamiento coadyuvante para la tuberculosis activa en adultos inmunocompetentes, y se ha desarrollado un nuevo método profiláctico que podría evitar la enfermedad mediante la inducción de tolerancia oral al Mtb que se consigue con la administración oral de bajas dosis de micobacterias inactivadas.
Tuberculosis (TB) is a global epidemic caused by Mycobacterium tuberculosis (Mtb). In 2012 an estimated 8,6 million of people developed TB and 1,3 million died from the disease. The current treatment with antibiotics is expensive, long-lasting and presents adverse effects. When people are infected with Mtb the infection is controlled in the 90% of the cases, developing microscopic lesions in the lungs, 0,5mm of size granulomas, invisibles to the X-rays. In the other 10% the infection is not controlled and bigger lesions are developed: in immunocompetent adults the most characteristic lesion is a cavity sized about 20mm of diameter. The clue to understand active TB pathogenesis must be the development of 20mm cavities from 0,5mm granulomas. In this work a murine model has been developed through the endovenous infection of C3HeB/FeJ mice with H37Rv virulent strain of Mtb, which develops lesions presenting central granulomatous necrosis and further liquefaction, very similarly to the lesions previous to cavity formation in human patients. The lesions grow exponentially due to massive neutrophilic infiltration and coalescence of neighbour lesions. The comparative studies with the resistant mice strain C3H/HeN and the use of non-steroidal anti-iflammatory drugs (NSAIDs) in the model confirmed that inflammation is clue in the active TB development, and also that NSAIDs could be use as adjunctive therapy in the treatment of pulmonary TB in immunocompetent adults, through control of excessive inflammation. On the other hand, a prophylactic method has been developed consisting on induction of tolerance to Mtb through oral administration of low doses of heat-killed mycobacteria, driving to a more balanced immune response, limiting Th17 development and resulting in a better outcome of mice in terms of survival, histopathology and bacillary load in lungs. Conclusions: A murine active TB model has been developed, and the role of inflammation in cavity formation characterized, namely the role of massive neutrophilic infiltration. The use of NSAIDs has been proposed as an adjuvant treatment of active TB in immunocompetent adults, and a new prophylactic method has been developed that could avoid the disease by induction of oral tolerance to Mtb through the administration of heat killed micobacteria at low doses.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
106
Some bacteria pathogens that can reside in host cells, for example: Salmonella spp. and will lead to chronic infection. These pathogens will invade host cells to evade external antibiotics’ attack and continue to survive in the cell, thus they are difficult to completely eradicated with antibiotics and often lead to drug resistance. Host-directed therapies have been proposed in many studies for the treatment of intracellular bacterial infections. AR-12 (a.k.a. OSU-03012), was originally used as an anti-cancer drug and have been showed in our previous studies that AR-12 combined with aminoglycoside antibiotics can significantly clear intracellular bacteria, making AR-12 as a potential drug for the treatment of intracellular bacterial infections. As the half-lethal dose (IC50) of AR-12 is close to half-effective concentration, we decided to conduct AR-12 structure optimization. In addition, we previously found that an antipsychotic drug - loxapine in combined with aminoglycoside antibiotics showed antimicrobial efficacy and significantly cleared intracellular bacteria with a high half-lethal dose (IC50), however, loxapine cannot prolong the survival of mice. Therefore, we hope to obtain potential drugs by synthesis of loxapine derivatives. In this study, we obtained 167 AR-12 derivatives and 5 loxapine derivatives. We tested the anti-bacterial activity against intracellular Salmonella Typhimurium of all compounds and simultaneously tested toxicity towards host cells to identify more effective and low toxicity drugs. At the same time, we used loxapine as the representative to explore its antibacterial mechanism. First, we performed AR-12 derivative screening. We found that seven compounds have lower toxicity and equal antibacterial activity to AR-12. Another seven compounds showed better antibacterial activity but equal toxicity to AR-12. We further evaluated the efficacy of these 14 compounds in combination with aminoglycoside antibiotics for the treatment of intracellular S. Typhimurium. The results of the cell infection experiments showed that 4 of 14 compounds had no significant antibacterial activity. Subsequent test of the three compounds with highest selectivity for antibacterial activity against multidrug-resistant strains also demonstrated the same effect. In the cell infection test and toxicity test of loxapine, three compounds showed better antibacterial ability than loxapine, but with high toxicity, thus there was no high selectivity. At present, the synthesis and test of AR-12 derivatives and loxapine derivatives are ongoing. The above findings demonstrated that the antibacterial activity of AR-12 can be dissociated from its antiproliferative activity via structure optimization and that hit compounds are potential to be served as antibiotic adjuvants for MDR Salmonella Typhimurium infection.

Mobile group II introns are retroelements that are found in prokaryotes, archaea, and the organelles of plants and fungi, but not in the nuclear genomes of eukaryotes. They consist of a catalytically active RNA and intron-encoded reverse transcriptase, which together promote site-specific integration into DNA sites in a mechanism called retrohoming. The group II intron Ll.LtrB has been developed into a programmable, DNA-targeting agent called "targetron", which is widely used in bacteria and an attractive technology for gene targeting in eukaryotes. However, group II intron genome targeting in human cells has not been equivocally shown. This dissertation focuses on the hypothesis that the low Mg2+-concentrations found in higher eukaryotes present a natural barrier to group II introns. First, I studied E. coli host proteins that aid group II intron retrohoming and found that synthesis of a second DNA-strand relies on host replication restart proteins. Next, I demonstrated that mutations in the distal stem of the catalytic core domain V (DV) improve Ll.LtrB retrohoming in a low Mg2+-concentration E. coli mutant and in biochemical assays. These results suggest that DV is involved in an RNA-folding step that becomes rate limiting at low Mg2+. Subsequently, I performed directed evolution of the intron RNA by injecting in vitro prepared mutant intron libraries into Xenopus laevis oocyte nuclei. The mutations were analyzed using Roche 454 sequencing to generate an intron fitness landscape, which revealed conserved positions and potentially beneficial mutations, enabling enhanced retrohoming in Xenopus oocytes. Finally, I used a hybrid Pol II/T7 Ll.LtrB eukaryotic expression system to show that high exogenous MgCl2 in the growth media enables retrohoming into plasmids and genomic DNA in human cells. In vivo directed evolution and mutation analyses using PacBio RS circular consensus sequencing indicated that only a few mutations may improve intron activity in human cells. This dissertation provides evidence that efficient group II intron retrohoming in human cells is limited by low Mg2+-concentrations and develops new approaches for overcoming this limitation to enable use of group II introns for gene targeting in higher organisms.
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Despite the numerous interventions employed in the past few decades, malaria remains one of the most lethal diseases affecting millions of people worldwide. This is partly due to the emergence of resistance to the current parasite-targeted antimalarials. In contrast, erythrocytic genetic mutations have been conferring malaria protection in humans for thousands of years without losing their effectiveness. This presents a new therapeutic approach to mimic these genetic mutations to treat malaria, known as host-directed therapy (HDT), which requires further understanding of host-parasite interactions to identify potential HDT drug targets. One such HDT target is the erythrocytic cytoskeleton, which parasites rely on for their survival. Ankyrin-1 (Ank-1) is one of erythrocytic cytoskeleton proteins, which has been associated with hereditary spherocytosis (HS) in humans. This thesis investigates the roles of Ank-1 in malaria infections using mouse models and blood from HS patients. Mice with Ank-1 mutations were found to exhibit phenotypes similar to human HS patients and are protected against malaria via multiple mechanisms, suggesting that Ank-1 plays a complex role in malaria infections. These mechanisms are heavily influenced by the nature of Ank-1 mutations, which is further confirmed in human HS erythrocytes. This thesis also explores the possibility of using the ankyrin-spectrin interaction as a HDT target. Results show that the disruption of this interaction has little effect on the health of the mice, while conferring significant resistance towards malaria, thus enabling the use of high throughput screening (HTS) for drug discovery. To summarise, this thesis highlights the complex interactions between the erythrocyte cytoskeleton and malarial parasites, as well as providing insights into the heterogeneous protective role of Ank-1 in mediating malaria resistance. It also raises the possibility of using erythrocytic cytoskeletal proteins as HDT drug targets, which could potentially yield novel therapies for malaria in the future.

Malaria is a lethal disease caused by the Plasmodium parasite. The current arsenal of antimalarial therapies targets the parasite, thereby selecting for mutant, resistant parasites. New antimalarials are desperately needed and a potential clue for a new therapeutic strategy has been provided by so-called "natural genetic antimalarials". Host genetic changes to red cell genes have offered millennia of stable protection to individuals living in endemic regions. By imitating natural resistance, this thesis proposes novel host-directed antimalarial pharmacologic therapies through the targeting of erythrocyte molecules required by the parasite for growth and survival. Work in this thesis investigated several enzymes from the haem biosynthetic pathway as potential targets for a host-directed therapy (HDT). Here, multiple experimental approaches were used to investigate and validate d-aminolevulinate dehydratase (ALAD), ferrochelatase (FECH) and uroporphyrinogen-III synthase (UROS) as targets for a novel host-directed antimalarial therapy. Firstly, it was demonstrated that host ALAD, FECH and UROS were localised in Plasmodium during intraerythrocytic growth. Moreover, the host enzymes were demonstrated to be required for normal parasite development as Plasmodium growth in vitro was impaired in UROS and FECH deficient red cells. This was shown using genetic models of human and mouse haem synthetic enzyme deficiency. Finally, the HDT strategy was validated with several inhibitors of ALAD and FECH demonstrating in vitro and in vivo anti-plasmodial activity. Host ALAD was specifically inhibited with succinylacetone (SA), a non-competitive irreversible ALAD inhibitor, demonstrating parasite growth inhibition in a P. Jalciparum in vitro assay with an IC\(_{50}\) of 2.5 μM. The antimalarial activity of SA was also demonstrated in vivo with SA treated mice demonstrating a significant reduction in P. chabaudi infection and increased survival compared to untreated controls. The competitive FECH inhibitor N-methylprotoporphyrin (NMPP) demonstrated anti-plasmodial activity in vitro with an IC\(_{50}\) of 25 nM, a figure comparable with many current antimalarials today. Griseofulvin, a second FECH inhibitor, is an antifungal agent, approved for use for over 50 years with an anti-FECH side effect, mediated through NMPP. Griseofulvin inhibited P. falciparum growth in an in vitro growth inhibition assay, with an IC\(_{50}\) between 10 and 50 μM on both chloroquine resistant and susceptible parasites. As griseofulvin is FDA and TGA-approved for human use, work in this thesis investigated parasite growth capacity in red cells from individuals taking pharmacologic doses of griseofulvin. It was demonstrated that griseofulvin concentrates in red cells and that parasites were unable to grow in red cells collected from human volunteers eight-hours after taking a clinically relevant dose of griseofulvin. Together, this data suggests that griseofulvin may be a useful antimalarial drug with a novel mode of action, potentially avoiding parasite resistance. Overall, work in this thesis has demonstrated that the parasite requires several host haem enzymes for growth and has provided proof-of-principle that targeting these enzymes as a HDT is a potentially effective antimalarial strategy. As griseofulvin is FDA and TGA approved for human use, it is quite possible that griseofulvin may be an "off the shelf' next generation antimalarial. The ultimate outcome from this work is a new generation of antimalarial therapies that may target the host and not the parasite, potentially limiting the development of drug resistance.

Dissertação de mestrado em Genética Molecular
A xylanase (pXyl) isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis is being commercialized worldwide for use in the baking industry. pXyl is a highly active coldadapted enzyme belonging to glycoside hydrolase family 8. It maintains high activity at low temperatures, is resistant to xylanase inhibitors and is highly specific for long chain unsubstituted xylan. However, a low stability and activity at acidic pHs, due mainly to precipitation, prevents the use of this enzyme in other industrial activities. In this study, the protein engineering techniques of random mutagenesis and site directed mutagenesis were investigated in an attempt to overcome the instability and loss of activity of pXyl at acidic pHs. Initially, random mutagenesis was investigated as this offers a powerful tool for identification of novel positive mutations in the absence of any knowledge of the protein involved. A critical limitation with this approach is the availability of a high throughput screening method for facile identification of positive mutants. We investigated an in-house strain of Saccharomyces cerevisiae for use in genetic complementation screening. This host has been engineered to enable growth on xylose and here it was attempted to develop this for growth on xylan by inclusion of both a β-xylosidase gene and the gene for pXyl. Unfortunately, following multiple attempts we were unable to isolate a strain capable of growth with xylan as the sole carbon source. We next turned our attention to site-directed mutagenesis. It is proposed that the strong precipitation of pXyl at low pHs may be related to its highly hydrophobic surface, as well as to negative surface amino acids which become protonated and more hydrophobic and hence more prone to precipitation at these lower pHs. Therefore, the highly exposed negative residues which do not participate in stabilising interactions in pXyl were identified (D70, E94, E95, E342 and E377) and mutated to the highly polar residue serine in an attempt to reduce acidic pH precipitation. Only E94S and E342S were successfully produced, purified and characterised. Both mutants showed minor increases in solubility at both acidic and basic pHs as compared to the wild-type enzyme, as well as an improvement in thermal stability, albeit being accompanied by alterations in the conformation. This preliminary study indicates the potential of the approach used in overcoming pXyl precipitation at acidic pHs.
A enzima xilanase (pXyl) isolada da bactéria antártica Pseudoalteromonas haloplanktis está mundialmente a ser comercializada como aplicação na indústria de panificação. A pXyl é uma enzima extremamente ativa e adaptada a baixas temperaturas, pertencendo à glycoside hydrolase family 8. Esta mantém uma elevada atividade a baixas temperaturas, é resistente a inibidores de xilanases e é específica para xilanos de cadeia longa não substituídos. No entanto, a baixa estabilidade e atividade a pHs ácidos, devido principalmente à sua precipitação nesta condição, impede a utilização desta enzima noutras áreas industriais. Neste estudo, as técnicas de engenharia de proteínas, mutagénese aleatória e mutagénese dirigida, foram utilizadas com o objetivo de superar a instabilidade e a perda de atividade da pXyl a pHs ácidos. Inicialmente, foi investigada a mutagénese aleatória, uma vez que esta técnica oferece uma estratégia poderosa para a identificação de novas mutações positivas nos casos em que a proteína envolvida não é totalmente conhecida. Uma limitação preponderante no uso desta abordagem é a disponibilidade de um método de rastreio de alto rendimento para a fácil identificação de mutantes positivos. Por isso, uma estirpe de Saccharomyces cerevisiae construída no nosso laboratório foi investigada para o seu uso no rastreio de complementações genéticas. Neste trabalho, este hospedeiro, previamente geneticamente manipulado para crescer em xilose, foi também adaptado para poder crescer em xilano. Para isso, os genes que codificam para uma β - xilosidase e para a pXyl foram expressos nesta estirpe. Infelizmente, após várias tentativas, não foi possível isolar uma estirpe capaz de crescer em xilano como única fonte de carbono. A segunda parte do trabalho focou-se na mutagénese dirigida. Estudos relacionados propõe que a forte precipitação da pXyl a pH baixos pode estar relacionada com a sua superfície altamente hidrofóbica, assim como com os aminoácidos negativos superficiais que se tornam protonados e mais hidrofóbicos e, portanto, mais propensos à precipitação nestes pHs. Por conseguinte, os resíduos negativos mais expostos ao solvente que não participam em interações de estabilização na pXyl foram identificados (D70, E94, E95, E342 e E377) e mutados por serina, um resíduo fortemente polar, numa tentativa de reduzir a precipitação a pH ácido. Apenas os mutantes E94S e E342S foram produzidos, purificados e caraterizados com sucesso.Ambos os mutantes apresentaram um ligeiro aumento na solubilidade tanto a pHs ácidos como básicos em comparação com a enzima selvagem assim como um aumento na estabilidade térmica, embora acompanhados por alterações na conformação. Este estudo preliminar indica o potencial da abordagem utilizada na diminuição da precipitação da pXyl a pHs ácidos.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how eROS levels are controlled is essential yet remains uncharacterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining non-toxic levels of eROS in Mtb. Integrating redoxosome with a global network of protein-protein interactions and transcriptional regulators revealed a hypothetical protein (rv0158) as a top node managing eROS and redox homeostasis in Mtb. RNA sequencing, seahorse XF flux measurements, and lipid analysis indicate that rv0158 is required to balance the deployment of fatty acid substrates between lipid anabolism and oxidation. Disruption of rv0158 perturbed redox balance in a carbon-source-specific manner, promoted killing in response to anti-TB drugs, reduced survival in macrophages, and lowered persistence in mice. We describe a novel pathogen response to moxifloxacin. Mtb, unlike Escherichia coli, decreases respiration in response to moxifloxacin. Nevertheless, cells were killed, as ROS increased due to NADH-dependent reductive stress. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with a ROS scavenger (thiourea), and an iron chelator (bipyridyl), indicating ROS is part and not a consequence of death processes. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Thus, redox and bioenergetic imbalance contribute to the moxifloxacin-mediated killing of Mtb. These results provide a way to make fluoroquinolones more effective anti-tuberculosis agents. We have previously reported that Mtb H37Rv sets up a gradient of mycothiol redox potential: EMSH-oxidized (-240 mV) to EMSH-reduced (-320 mV) inside macrophages, where the EMSH -reduced Mtb subpopulation are significantly more tolerant to anti-TB drugs. Therefore, one of the keys to subverting drug-tolerance is to impede the emergence of EMSH -reduced subpopulation by inducing overwhelming oxidative stress. In this study, we exposed THP1-macrophages infected with Mtb H37Rv expressing Mrx1-roGFP2-biosensor, to a library of FDA-approved drugs (Enzo Life Sciences; BML-2842) and scored for the oxidative shift in the Mtb- EMSH at 24 hours post infection. Based on their activity to trigger oxidative stress inside the bacterium, non-cytotoxicity to host, and inhibition of bacterial growth inside macrophages, C5 molecule emerged as the top hit. Pre-treatment with C5 potentiated killing of Mtb by all tested antibiotics (isoniazid, rifampicin, and moxifloxacin) and reduced drug-tolerance.
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