Orientador: Prof. Dr. Luciano Puzer / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / Myxobacterias sao bacterias Gram-negativas com uma peculiaridade no ciclo de vida em comparacao a outros procariotos. Em resposta a periodos de baixa disponibilidade de nutrientes, inicia-se um processo de morfogenese cooperativa que culmina na formacao de corpos de frutificacao, um agregado de celulas que contem estruturas de resistencia caracteristicas. Subtilisinas sao serino proteases da familia S8A, inicialmente encontrada no Bacillus subtilis e atualmente identificada em uma ampla variedade de organismos procarioticos e eucarioticos. Essas enzimas apresentam um grande mercado de aplicacoes biotecnologicas e industriais. O objetivo deste trabalho foi clonar aqualisina, uma subtilisina extracelular da Myxococcus xanthus, uma especie de myxobacterias que preda outros microrganismos, e submete-la a um sistema de expressao heterologa em celulas E. coli BL2 (DE3). A proteina permaneceu na fase insoluvel apos as lises; portanto, foi necessario otimizar protocolos de solubilizacao e purificacao da aqualisina dos corpos de inclusao da celula, para que fosse possivel a caracterizacao cinetica. A solubilizacao utilizando tampao 0,1M de tris e pH 12 mostrou-se a mais eficiente em solubilizar os corpos de inclusao, o que permitiu obter os valores cineticos Km de 0,6 e 1,3 ¿ÊM, para aqualisina purificada (AQN) e para nao retida pela coluna (FT), respectivamente, que demonstram uma alta afinidade pelo substrato utilizado. No entanto, valores obtidos de kcat, e kcat/Km apontam a presenca de duas populacoes de proteinas, nas quais a populacao inativa esta presente em maior quantidade. Embora o sistema de expressao da aqualisina tenha ocorrido com sucesso, a solubilizacao dos corpos de inclusao sem utilizacao de agentes desnaturantes, e posterior purificacao ainda necessitam ser otimizadas, a fim de aumentar a recuperacao de enzima ativa, possibilitando que sua caracterizacao cinetica seja realizada sem a influencia desta populacao de enzima inativa. / Despite its status as one of the oldest registered diseases, rabies remains a public health challenge. This zoonosis affects the central nervous system (CNS) of all mammals and is usually considered fatal. However, some cases in which the disease was reportedly cured have been registered worldwide, leading to renewed interest in researching potential antiviral mechanisms against the rabies virus (RABV). The aim of this work was to evaluate the antiviral activity of the hydroethanolic plant extract Dalbergia variabilis against different genetic lineages of RABV. In the first step, the maximum tolerated concentration (MTC) of plant extract in murine neuroblastoma cells (N2a) was determined to be 1.56 mg/ml. The antiviral activity of plant extract Dalbergia variabilis was evaluated to determine the difference presence and absence of the plant extract in four samples of RABV (IP4005- sample of RABV with the genetic lineage characteristic of haematophagous bat, Desmodus rotundus; IP964- sample of RABV with the genetic lineage characteristic of insectivorous bat, Eptesicus furinalis; IP4871- sample of RABV with the genetic lineage characteristic of wild dog, Cerdocyon thous; and a standard sample of RABV, "Pasteur vírus" - PV). Since there was a difference in the infectious viral titer between the RABV samples analysed in the presence of the plant extract, subsequent experiments were carried out to better understand the performance of this plant extract against RABV. The antiviral activity of plant extract Dalbergia variabilis against samples of RABV was analysed, and the interference in viral replication was more significant against samples of RABV with genetic lineage characteristic of bats.

No description available.

Myxococcus xanthus Dif proteins are chemotaxis homologues that regulate exopolysaccharide (EPS) biogenesis. Previous genetic studies suggested that Dif protein might interact with one another as do the chemotaxis proteins in enterics. The interactions among Dif proteins were since investigated with the yeast two-hybrid (Y2H) system. The results indicate that DifC interacts with both DifA and DifE. Using a modified Y2H system, DifC was shown to be able to bring DifA and DifE into a protein complex. Further Y2H experiments demonstrated that the different conserved domains of DifE likely function as their counterparts of CheA-type kinases because the putative P2 domain of DifE interacts with DifD, P5 with DifC and the dimerization domain P3 with itself. Similarly, DifA can interact with itself through its C-terminal region. In addition, DifG was found to interact with the CheY homologue DifD. These findings support the notion that Dif proteins constitute a unique chemotaxis-like signal transduction pathway in M. xanthus. In addition, KapB, a TPR (Tetratricopeptide repeats) protein, was identified as an interacting partner of DifE byY2H library screening. Further analysis demonstrated that the N-terminal half of KapB interacted with the putative P2 domain of DifE. KapB had been previously reported to interact with several Serine/Threonine (Ser/Thr) kinase pathways including the Pkn4-Pfk pathway. This pathway is implicated in glycogen metabolism in M. xanthus by a previous report. In this study, kapB as well as pfkn deletion mutants were found to overproduce EPS. It was also found that the Dif pathway is involved in glycogen metabolism because the glycogen level is altered in dif mutants. These results indicate EPS biogenesis and glycogen metabolism may be coordinately regulated. This coordination of the Dif-regulated EPS production and the Pkn4-regulated glycogen metabolism appears to involve KapB. This is the first example of a TPR protein mediating the interplays of a histidine kinase pathway and a Ser/Thr kinase pathway. / Master of Science

Myxococcus xanthus, a Gram-negative bacterium with a developmental cycle, displays a type IV pili (TFP) mediated surface motility known as social (S) gliding. Beside the polarly localized TFP, the fibril or extracellular polysaccharide (EPS) is also required for S-motility to function. It is proposed that S-motility, along with the related bacterial twitching motility in other species, is powered by TFP retraction. EPS is proposed to anchor and trigger such retractions in M. xanthus. EPS production is known to be regulated by TFP and the Dif signal transduction pathway. Two genetic screens were performed previously to identify additional genes important for EPS production. The first was for the isolation of pilA suppressors, the second for the identification of mutants underproducing EPS in a difA suppressor background. Both screens identified transposon insertions at the stk locus. In particular, StkA, a DnaK homolog, was identified as a possible negative regulator of EPS production by a stkA transposon insertion that suppressed a pilA mutation. A stkB transposon insertion was found to have diminished EPS production in a difA suppressor background. In this study, in-frame deletion mutants of the five genes at the stk locus, stkY, stkZ, stkA, stkB and stkC, were constructed and examined. In addition, mutations of rbp and bskL, two genes downstream of the stk locus, were constructed. Like transposon insertions, the stkA in-frame deletion resulted in overproduction of EPS. The stkB and to a less extent the stkC mutants underproduced EPS. Mutations in the other genes had no obvious effects on EPS production. Genetic epistasis suggests that StkA functions downstream of TFP and upstream of the Dif sensory proteins in EPS regulation in M. xanthus. Epistasis analysis involving stkB was inconclusive. It is unresolved whether StkB plays a role in the biosynthesis or the regulation of EPS production in M. xanthus. / Master of Science

La bactérie Myxococcus xanthus forme des structures multicellulaires appelées corps fructifères pour résister à des conditions de carence nutritive. La formation de ces structures implique un système chimiotactique particulier, le système Frz, qui régule le changement de direction des cellules, provoqué par la relocalisation simultanée des deux appareils de motilité (A) et (S) d’un pôle à l’autre de la cellule. Au cours de ma thèse, j’ai travaillé sur la connexion entre le système chimiotactique Frz et ses protéines cibles MglAB dans le contrôle de l’inversion de la polarité. L’axe de polarité des cellules est établi par MglA, une petite protéine G de la famille Ras, qui constitue un embranchement vers la régulation des deux appareils de motilité au pôle avant, et son inhibiteur MglB localisé au pôle arrière. Nous avons montré qu’en interagissant directement et spécifiquement avec le cytosquelette, MglA contrôle l’assemblage et le désassemblage de la machinerie de motilité A. Par une approche évolutive, nous avons élucidé l’architecture modulaire du système Frz et l’implication de quatre domaines régulateurs pour connecter le système Frz aux protéines MglAB, filtrer et amplifier le signal. Nous proposons un mécanisme d’inversion de la polarité dans lequel l’action indépendante de deux RRs à chaque pôle de la cellule perturbe les interactions entre une petite protéine G et son inhibiteur apparenté pour convertir un axe de polarité stable en un oscillateur biochimique. La régulation de la direction de mouvement chez M. xanthus pourrait donc constituer un cas émergent de couplage entre des régulateurs de type procaryotes et eucaryotes. / The bacterium Myxococcus xanthus forms multicellular structures called fruiting bodies to resist to starvation conditions. Fruiting body formation implies a chemosensory-like system, the Frz system which regulates directional changes through the simultaneous pole-to-pole relocalization of two motility systems, (A) and (S). During my PhD, I have worked on the connection between the Frz chemosensory-like system and the downstream regulators MglA and MglB in the control of polarity inversion. The cell polarity axis is established by (i) a Ras-like small G protein, MglA, which constitutes a branch node in the regulation of A and S motility systems at the leading cell pole, and (ii) its cognate inhibitor MglB that localizes at the lagging cell pole. We showed that MglA interacts directly and specifically with the cytoskeleton to promote assembly and disassembly of the A-motility machinery. Using an evolutionary approach, we elucidated the modular architecture of the Frz system and the implication of four regulatory domains to (i) connect the Frz system to the MglAB proteins, (ii) filter and (iii) amplify the signal. We now propose a mechanism for polarity inversion in which the independent action of two response regulators at each cell pole perturbs the interactions between a small-G-protein and its cognate inhibitor to trigger the conversion of a stable polarity axis into a biochemical oscillator. The regulation of directional movement in M. xanthus is an interesting emergent coupling between prokaryotes and eukaryotes regulators.

The surface gliding motility of Myxococcus xanthus is required for a multicellular developmental process initiated by unfavorable growth conditions. One form of the M. xanthus surface motility, social (S) gliding, is mediated by the extension and retraction of polarly localized type IV pili (Tfp). Besides Tfp, exopolysaccharides (EPS), another cell surface associated component, are also required for M. xanthus S motility. Previous studies demonstrated that the Dif chemotaxis-like signal transduction pathway is central to the regulation of EPS production in M. xanthus. Specifically, difA, difC and difE mutants were found to be defective in EPS production and S motility. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis proteins (MCPs), CheW and CheA, respectively, are therefore positive regulators of EPS. This study, undertaken to better understand the regulation of EPS production, led to a few major findings. First, DifD and DifG, homologous to CheY and CheC, respectively, were found to be negative regulators of EPS production. Both DifD and DifG likely function upstream of the DifE kinase in EPS regulation. DifB, which has no homology to known chemotaxis proteins, was found not to be involved in EPS production. Secondly, this study led to the recognition that Tfp likely function upstream of the Dif pathway in the regulation of EPS production. Extracellular complementation experiments suggest that Tfp may act as sensors instead of signals for the Dif chemotaxis-like pathway. We propose a regulatory feedback loop that couples EPS production with Tfp function through the Dif signaling proteins. Lastly, we sought to identify additional genes involved in EPS production. Our efforts identified a mutation in a separate chemotaxis gene cluster as a suppressor of difA mutations, suggesting potential cross-talks among the multiple chemotaxis-like pathways in M. xanthus. In addition, we identified twenty-five previously uncharacterized genes that are predicted to be involved in M. xanthus EPS production. These genes appear to encode additional EPS regulators and proteins with biosynthetic function. / Ph. D.

The Myxococcus xanthus Dif and Frz chemosensory pathways play important roles in the regulation of gliding motility. The Dif system regulates the production of exopolysaccheride (EPS), which is essential for social motility and fruiting body formation. The Frz pathway controls reversal frequency, which is fundamental for directed movement by this surface-gliding bacterium. In addition, both pathways are involved in the chemotactic response towards several phosphatidylethanolamine (PE) species such that the Dif pathway is required for excitation while the Frz pathway is essential for adaptation. In this study we addressed three crucial questions regarding the signal processing of these two chemosensory pathways by focusing on DifA and FrzCD, the MCP homologs from their respective pathways. First, the receptor protein in the Dif pathway, DifA, lacks a perisplasmic domain, the typical signal-sensing structure. To examine whether DifA shares similar transmembrane signaling mechanism with typical transmembrane sensor proteins (MCPs and sensor kinases), we constructed a chimeric protein that is composed of the N-terminus of NarX (nitrate sensor kinase) and the C-terminus of DifA. This NarX-DifA chimera restores the DifA functionality (EPS production, agglutination, S-motility and development) to a "difA mutant in a nitrate-dependent manner, suggesting DifA shares a similar transmembrane signaling mechanism with typical MCPs and sensor kinases despite its unorthodox structure. Second, the M. xanthus chemotaxis is still controversial. It has been argued that the taxis-like response in this slowly gliding bacterium could result from physiological effects of certain chemicals. To study motility regulation by the Frz pathway, we constructed two chimeras between the N-terminus of NarX and C-terminus of FrzCD, which is the receptor protein of the Frz pathway. The two chimeras, NazDF and NazDR, are identical except that NazDR contains a G51R mutation in the otherwise wild-type NarX sensory module. This G51R mutation was shown to reverse the signaling output of a NarX-Tar chimera to nitrate. We discovered that nitrate specifically decreased the reversal frequency of NazDF-expressing cells and increased that of NazDR-expressing cells. These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazDR strain failed to adapt to nitrate in temporal assays, as did the wild type to known repellents. Therefore, the lack of temporal adaptation to negative stimuli is an intrinsic property in M. xanthus motility regulation. Third, the Dif and Frz pathways are both involved in the chemotactic response towards certain PE molecules such that the Dif pathway is required for excitation and while the Frz system is essential for adaptation. In addition, 12:0 PE, known to be sensed by DifA, results in increased FrzCD methylation. These findings suggested that in the regulation of PE response, two pathways communicate with each other to mediate adaptation. Here we provided evidence to indicate that DifA does not undergo methylation during EPS regulation and PE chemotaxis. On the other hand, using mutants expressing the NarX-DifA chimera, it was found that signal transduction through DifA, DifC (CheW-like) and DifE (CheA-like) modulates FrzCD methylation. Surprisingly, the attractant 12:0 PE can modulate FrzCD methylation in two ways distinguishable by the dependency on DifA, DifC and DifE. The DifACE-independent mechanism, which may result from specific sensing of 12:0 PE by FrzCD, increases FrzCD methylation as expected. Unexpectedly, 12:0 PE decreases FrzCD methylation with the DifACE-dependent mechanism. This "opposite" FrzCD methylation by DifACE-dependent signaling was supported by results from NafA-expressing mutants because nitrate, which acts as a repellent, increases FrzCD methylation. Based on these findings, we proposed a model for chemotaxis toward 12:0 PE (and 16:1 PE). In this model, DifA and FrzCD both sense the same signal and activate the pathways of excitation (Dif) and adaptation (Frz) independently. The two pathways communicate with each other via methylation crosstalk between DifACE and FrzCD in such a way that processes of excitation and adaptation can be coordinated. / Ph. D.

Bacterial DNA replication can stall at DNA lesions, leading to cell death if the damage fails to be repaired. To circumvent this, bacteria possess a mechanism called translesion DNA synthesis (TLS) to allow DNA damage bypass. The ImuABC TLS mutasome comprises the RecA domain-containing protein ImuA, the inactive polymerase ImuB, and the error-prone polymerase ImuC. ImuA and ImuB are necessary for the mutational function of ImuC that can lead to antimicrobial resistance (AMR) as seen in high-priority pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis. Understanding how ImuA and ImuB contribute to this function can lead to new targets for antimicrobial development. This research aims to discover the molecular functions of ImuA and ImuB homologs from Myxococcus xanthus through structural modelling and biochemical analyses. ImuA was discovered to be an ATPase whose activity is enhanced by DNA. Based on predicted structural models of the ATPase active site, I identified the critical residues needed for ATP hydrolysis, and found that the ImuA C-terminus regulates ATPase activity. Further, ImuA and ImuBNΔ34 (a soluble truncation of ImuB) display a preference for longer single-stranded DNA and overhang DNA substrates, and their affinity for DNA was quantified in vitro. To better understand how ImuA and ImuB assemble in the TLS mutasome, bacterial two-hybrid assays determined that ImuA and ImuB can self-interact and bind one another. Mass photometry revealed that ImuA is a monomer and ImuBNΔ34 is a trimer in vitro. ImuA and ImuBNΔ34 binding affinity was quantified in vitro at 1.69 μM ± 0.21 by microscale thermophoresis, and removal of the ImuA C-terminus weakens this interaction. Lastly, ImuA and ImuBNΔ34 secondary structures were quantified using circular dichroism spectroscopy, and ImuA was modified to enable crystallization for future structural studies. Together, this research provides a better understanding of ImuABC-mediated TLS, potentially leading to novel antibiotics to reduce the clinical burden of AMR. / Thesis / Master of Science (MSc) / The antimicrobial resistance (AMR) crisis is fueled by the emergence of multi-drug resistant microbes, posing a major threat to global health and disease treatment. Bacteria can develop resistance to antibiotics through mutations in the genome. When the genome becomes damaged, bacteria can acquire these mutations by an error-prone replication mechanism called translesion DNA synthesis (TLS). In some bacteria, TLS involves a specialized enzyme complex, consisting of proteins ImuA, ImuB and ImuC, allowing replication past bulky DNA damage and lesions. The goal of this thesis is to investigate how the ImuA and ImuB proteins contribute to the functioning of this mistake-making machinery. I used biochemical and biophysical methods to identify ImuA and ImuB interactions with each other and themselves. I discovered that ImuA is an enzyme that uses energy to enhance its binding to DNA, and determined the specific amino acids involved in this function.