Bacteriophage ms2 l protein: genetic and biochemical characterization

In order to release progeny, bacteriophages must lyse the host cell by compromising the peptidoglycan layer. There are two known strategies of lysis: the holin-endolysin system and single gene lysis (SGL), which are dependent on the genome size. Large phages encode multiple proteins, including a holin and endolysin, for lysis. In contrast, small ssRNA phages (Leviviridae and Alloleviviridae) and ssDNA phages (Microviridae) do not encode a muralytic enzyme and accomplish lysis with a single gene. The cellular target of the lysis gene E from the prototypic microvirus, φX174, and A2 from the prototypic allolevivirus, Qβ, has been elucidated. In both cases, these proteins were demonstrated to inhibit specific enzymes within the peptidoglycan biosynthetic pathway and infected cells lyse as a result of septal catastrophes. The prototype Levivirus MS2 encodes L, a 75 aa polypeptide that effects lysis without inhibiting murein synthesis. The purpose of the work described in this dissertation was to characterize MS2 L using genetic and biochemical strategies. Using a genetic approach, PcnB was shown to be important to the entry of the MS2 RNA into the cytoplasm. L accumulation during infection was quantified by comparison to purified, oligohistidine-tagged L. Biochemical experiments demonstrated the L protein behaved as a periplasmic, membrane-associated protein. The morphologies of cells undergoing L-mediated lysis are significantly different from cells lysing due to A2 expression, since L-lysing cells do not show septally localized membrane protrusions

Lysis strategy of Streptococcus pneumoniae bacteriophages : mechanisms and host implications

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2011Bacteriophages (phages), the most abundant entities in the biosphere, play a central role in the shaping of natural populations of bacteria. Phages have also been the focus of several studies due to their potential as tools for therapeutic purposes. Notably, detailed analysis carried out in different bacterial species established that phages have a prominent influence in virulence. The abundance of lysogenic phages in Streptococcus pneumoniae isolates associated with infection was suggested some years ago, and recently, it has been proposed that lysogens account for as much as 76% of the samples analyzed. However, the role of pneumococcal prophages in the pathogenic potential of its host remains so far unknown. Bacterial lysis promoted by the major autolysin LytA has been implicated in the capacity of pneumococcus to cause infection, essentially due to the release of proinflammatory cell wall compounds and intracellular virulence factors. Even if no phage-encoded virulence factors were ever found, prophage-mediated host lysis by itself may contribute significantly to pneumococcal pathogenesis. Therefore, investigating the phage lysis system is clearly important in furthering our understanding of this effect. This work explores the exact mechanism underlying the lysis strategy of S. pneumoniae phages to release their progeny and also the implications of lysogeny, particularly due to induced cell lysis, in the host ability to form biofilms, a bacterial lifestyle associated with pneumococcal human infections. Pneumococcal phages lyse their bacterial hosts, and consequently release the newly formed phage particles, at the end of the vegetative cycle through the combined action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. The powerful lytic activity of the S. pneumoniae autolysin raised the possibility that it could play an important role in this process. By deleting the bacterial and phage lysins in both lysogenic and lysogenized strains, the contribution of LytA to phage release was evaluated based on bacterial culture lysis monitoring and phage plaque assays. It was found that, independently of the host genetic background, the bacterial autolysin is activated during phage-mediated lysis. Flow cytometry assessment of the membrane integrity after phage induction revealed that LytA triggering results from holininduced membrane disruption, similarly to the activation of the phage lysin. These results provide evidence that the energy status of the membrane may be involved in autolysin regulation at the cell surface. 3 We were able to demonstrate that, in the absence of the phage lytic enzyme, LytA by itself mediates extensive bacterial lysis, accompanied by the release of a large amount of fully functional phages capable of completing their life cycle since phage plaques were clearly detected. The overwhelming majority of phages of other bacterial species are absolutely incapable of bacterial lysis, trapping the phage progeny within the host cell, when the genes encoding lysins are deleted. Moreover, those rare mutants that bring about lysis depend only on phage-encoded factors. Nevertheless, exclusive dependence on the autolysin delayed the lysis timing and reduced the lysis extent. Accordingly, phage plaques were detected later than those in the presence of both host and phage lysins and a significant decrease on the number of virions released was observed. Therefore, lysis strictly dependent on LytA can lead to phage fitness impairment by retaining phage progeny longer within the host and reducing the amount of particles that actually escapes from entrapment. Nonetheless, under normal conditions, it was found that the concurrent activation of LytA with the phage lysin increases the total number of phages that exit the cell when the infective cycle is completed. Hence, pneumococcal phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy and are unique among lysin-equipped phages in their dependence on bacterial lytic factors to achieve such task. Although the function of holin and phage lysin is characterized, the interplay between them to achieve lysis in S. pneumoniae was never fully determined. It has been shown that pneumococcal phage lysins are structurally and functionally similar to LytA, thus, they may share the same cellular localization and control mechanisms. Our finding that holin-induced membrane lesions trigger the bacterial cell wall autolysin prompted a deeper study of the pneumococcal lysis strategy. For this purpose, deletions of the holin and autolysin were performed in a lysogenic strain, in which the resident phage has a typical holin-lysin cassette. In the absence of these functions, western blot analysis and the effect of membrane permeabilizing and proton motive force (pmf)-dissipating agents on culture lysis allowed concluding that pneumococcal phage lysins accumulate with time across the lytic cycle and are continuously targeted to the cell wall. The phage lysin remains inactive associated with the choline residues within this structure. Therefore, the access of pneumococcal phage lysins to the bacterial surface is holin independent, hence they can be classified as exolysins. These findings are in contrast to what is observed in the large majority of holin-lysin phages where endolysins accumulate in the cytoplasm since they lack an intrinsic secretory signal sequence and consequently depend on holins to reach the peptidoglycan target. In addition, the involvement of the host Sec pathway in the phage lysin export was investigated. We assessed the cell wall localization of the phage 4 lysin by the same experimental procedures after culture treatment with the Sec inhibitor sodium azide. It was found that the phage lytic enzyme is possibly exported by the Sec system of pneumococci in spite of the striking absence of a signal sequence that could target it to the extracytoplasmic environment. This may constitute the first evidence, on phages encoding only holins and lysins in their lytic cassettes, of an exolysin without a secretion signal that is translocated through the membrane by the host Sec machinery. Furthermore, since the cell wall located autolysin also lacks obvious motifs or signals for an external localization, these results may provide clues for its transport mechanism. Dependence exclusively on the pmf-dissipating agent for complete host lysis, together with the previous observation of holin’s permeabilizing effect, showed that collapse of the cytoplasmic membrane electrochemical gradient achieved by the holins is the triggering signal to activate the phage lysin. In this study, it was further confirmed that activation of the externalized bacterial autolysin LytA, previously shown to contribute to phage progeny release, is also equally related to perturbations on the energized membrane. Thus, these results demonstrate that in S. pneumoniae phages, holin is not required for lysin export but is crucial to trigger the phage and bacterial lysins already residing in the cell wall by pmf dissipation upon formation of lesions on the membrane. In this regard, holins are the timing device that dictates when lysis takes place. After the characterization of the phage lytic mechanism, the contribution of lysis mediated by lysogenic phages to pneumococcal biofilms was investigated. S. pneumoniae lysogens are associated with human infections and pneumococcal biofilms have been implicated both in colonization and infection. It was explored if prophage spontaneous induction and consequent bacterial lysis enhance pneumococcal biofilm development providing a source of extracellular DNA (eDNA), a major factor in the biofilm matrix. Monitoring biofilm growth of lysogens and nonlysogenic bacteria by biomass quantification, viable cell counts and confocal laser scanning microscopy (CLSM), indicated that lysogenic bacteria are more prone to form biofilms, yielding structures with higher biomass and cell viability and showing denser biofilms in CLSM. Spontaneous phage induction was demonstrated to occur continuously as phages could be detected throughout biofilm formation through measurement of the total number of PFUs (plaque forming units) at specific time points. When comparing biofilm development between wild-type lysogens and those deleted in the phage lysin, bacterial autolysin LytA or both lysins, it was observed that phagemediated lytic events influence positively the biofilm structure. These results established that prophage promotes biofilm development due to bacterial lysis upon spontaneous induction. In 5 agreement, lysis inside biofilms also occurs in other bacterial species and it might be related to increased biofilm fitness. However, the effects created by the ablation of either the phage or bacterial lysins were overcome by the addition of external DNA. Additionally, in independent experiments, it was found that treatment with DNase I resulted in sparser and thinner biofilms while supplementation with DNA resulted in a thicker and more densely packed structure, confirming the important role of eDNA in pneumococcal biofilms. The quantification of eDNA released within these structures by real-time PCR also supported that lytic events promoted by phage are an important source of this matrix component, as biofilms of lytic strains contained more eDNA than those of nonlytic strains. Therefore, limited phage-mediated host lysis constitutes an important source of eDNA in S. pneumoniae biofilms favoring biofilm formation by lysogenic strains. Interestingly, massive phage induction leading to a high proportion of lysis was observed to disrupt severely biofilms of pneumococcal lysogens with a significant decrease in biofilm mass confirmed by CLSM visualization. These findings corroborate previous studies that show the potential use of lytic phages to destroy bacterial biofilms. The presented results and conclusions are of great value not only to directly increase our knowledge on phage biology and their relationship with the host bacteria, but ultimately to uncover the role of lysogeny in pneumococcal virulence. In this context, massive prophageinduced lysis of the host could mimic the major bacterial autolysin by releasing factors known to contribute to the course of infection. On the other hand, lysis due to spontaneous levels of induction, characteristic of prophage carriage, may have an impact in pathogenesis by enhancing S. pneumoniae biofilm formation, which has been implicated in the processes of colonization and disease. A deeper understanding of the mechanisms underlying pneumococcal infection is of vital significance to manage this important human pathogen.Os bacteriófagos (fagos) são entidades extremamente abundantes na natureza que desempenham um papel central na modulação das populações bacterianas. Devido ao seu potencial como ferramentas para fins terapêutico, os fagos têm sido alvo de diversos estudos. A análise detalhada realizada em diferentes espécies bacterianas permitiu estabelecer que os fagos têm uma influência marcante na virulência. A abundância de estirpes lisogénicas de Streptococcus pneumoniae responsáveis por infecção foi sugerida há alguns anos e, recentemente, foi proposto que correspondem aproximadamente a 76%. No entanto, o papel dos profagos no potencial infeccioso dos pneumococos é ainda desconhecido. A lise bacteriana promovida pela principal autolisina LytA foi implicada na capacidade de S. pneumoniae causar infecção sobretudo por promover a libertação de componentes da parede celular com actividade pro-inflamatória e factores de virulência intracelulares. Mesmo que, até à data, não tenham sido identificados factores de virulência nos genomas fágicos, a lise bacteriana mediada pelos profagos pode por si só contribuir significativamente para a patogenicidade do pneumococo. Desta forma, é importante estudar o sistema de lise dos fagos. O presente trabalho explora os mecanismos da estratégia de lise adoptada pelos fagos de S. pneumoniae para libertar a descendência fágica e as implicações da lisogenia, particularmente devido à indução de lise, na capacidade do hospedeiro formar biofilmes, uma forma de crescimento bacteriano associada com infecções pneumocócicas. Os fagos de S. pneumoniae lisam as células hospedeiras, e consequentemente libertam as partículas fágicas recém-formadas, no final do ciclo lítico através da acção conjunta de holinas que formam lesões na membrana citoplasmática e lisinas que degradam o peptidoglicano da bactéria. No entanto, como a autolisina de S. pneumoniae é caracterizada por uma extensa actividade lítica, é possível que possa desempenhar um papel importante neste processo. A contribuição de LytA na libertação dos fagos foi avaliada através da eliminação das actividades das lisinas fágica e bacteriana em estirpes lisogénicas e lisogenizadas, subsequente acompanhamento da lise das culturas bacterianas e realização de ensaios de placas fágicas. Foi determinado que, independentemente do contexto genético do hospedeiro, a autolisina bacteriana é activada durante a lise mediada pelo fago. A avaliação da integridade da membrana por citometria de fluxo após indução do fago revelou que a activação de LytA resulta, tal como a activação das lisinas fágicas, dos danos na membrana induzidos pelas holinas. Estes resultados sugerem que o estado energético da membrana está envolvido na regulação da autolisina na superfície celular. 3 Na ausência da enzima lítica do fago, demonstrou-se que LytA medeia uma lise bacteriana extensa acompanhada da libertação de uma grande quantidade de fagos funcionais capazes de completar o ciclo infeccioso, uma vez que foram claramente detectadas placas fágicas. A grande maioria dos fagos que infectam outras espécies bacterianas é absolutamente incapaz de causar lise bacteriana, aprisionando a descendência fágica dentro da célula hospedeira, quando são eliminados os genes que codificam as suas lisinas. Além disso, os poucos mutantes capazes de lisarem as bactérias hospedeiras dependem unicamente de factores codificados por si próprios. No entanto, a dependência exclusiva na autolisina adiou o momento da lise e reduziu a sua extensão. Em concordância, as placas fágicas foram detectadas mais tarde do que as observadas na presença de ambas as lisinas (bacteriana e fágica) e observou-se uma diminuição significativa no número de viriões libertados. Assim, a lise estritamente dependente de LytA pode influenciar negativamente o “fitness” do fago ao reter a descendência fágica durante mais tempo no interior do hospedeiro e ao reduzir a quantidade de partículas que de facto escapam ao aprisionamento. No entanto, em circunstâncias normais de infecção bacteriana, verificou-se que a activação de LytA em simultâneo com a da lisina fágica aumenta o número total de fagos libertos da célula hospedeira uma vez completo o ciclo infeccioso. Logo, os fagos de S. pneumoniae utilizam a ubíqua autolisina bacteriana para optimizar a sua estratégia de libertação da descendência sendo, entre os fagos equipados com lisinas, os únicos que dependem de factores líticos bacterianos para a libertação óptima. Embora as funções da holina e da lisina fágica estejam caracterizadas, a interacção entre estas proteínas para alcançar a lise em S. pneumoniae não foi integralmente determinada. Como as lisinas fágicas são estrutural e funcionalmente similares a LytA, é possível que partilhem a mesma localização celular e mecanismos de regulação. A observação de que as lesões na membrana provocadas pelas holinas activam a autolisina bacteriana localizada na parede celular, incitou a um estudo mais detalhado sobre a estratégia de lise do pneumococo. Para isso, foram eliminadas as funções da holina e da autolisina numa estirpe lisogénica em que o profago contém uma cassete holina-lisina típica. Na ausência destas actividades, a análise por “western blot” e a avaliação na lise das culturas do efeito de agentes que permeabilizam a membrana e dissipam a força motriz protónica (fmp) permitiram concluir que as lisinas fágicas de S. pneumoniae acumulam-se ao longo do tempo durante o ciclo lítico e são continuamente transportadas para a parede celular, onde permanecem inactivas associadas aos resíduos de colina. Assim, o acesso das lisinas fágicas à superfície bacteriana é independente das holinas, podendo ser caracterizadas como exolisinas. Estes resultados diferem do que se observa na grande maioria dos fagos dependentes do sistema holina-lisina. Nesses casos, as endolisinas acumulam-se no citoplasma, uma vez que são desprovidas de uma 4 sequência sinal secretória intrínseca e, consequentemente, dependem das holinas para alcançarem o peptidoglicano. Foi também estudado o envolvimento do sistema Sec do hospedeiro na exportação da lisina fágica. A localização da lisina fágica na parede celular foi avaliada pelos mesmos procedimentos experimentais após tratamento das culturas com azida de sódio, um inibidor desta via de transporte. Demonstrou-se que a enzima lítica fágica é possivelmente exportada pelo sistema Sec de S. pneumoniae apesar da ausência de sequências sinal que a possam dirigir para o compartimento extracitoplasmático. Estes resultados podem constituir a primeira evidência experimental, nos fagos que codificam nas suas cassetes líticas apenas as funções de holina e lisina, de uma exolisina sem sinal de exportação que é translocada através da membrana pelo sistema Sec do hospedeiro. Além disso, uma vez que a autolisina bacteriana localizada na parede celular também não apresenta motivos nem sequências de sinalização que justifiquem uma localização externa, estas observações podem ajudar a elucidar o seu mecanismo de transporte. A lise completa devido exclusivamente ao agente que dissipa a fmp, conjuntamente com a observação anterior do efeito permeabilizante das holinas, demonstrou que o colapso do gradiente electroquímico da membrana citoplasmática provocado pelas holinas constitui o sinal para activar a lisina fágica. Neste estudo, foi também confirmado que a activação da autolisina bacteriana externalizada, que se tinha verificado anteriormente contribuir para a libertação da descendência fágica, está igualmente relacionada com perturbações no estado energético da membrana. Logo, estes resultados demonstram que nos fagos de S. pneumoniae, a holina não é necessária para a exportação da lisina mas é crucial para activar tanto a lisina fágica como a bacteriana residentes na parede celular por dissipação da fmp aquando da formação de lesões na membrana, determinando o momento da lise. Após a caracterização do mecanismo fágico de lise, foi investigada a contribuição da lise mediada por fagos lisogénicos nos biofilmes do pneumococo. Em S. pneumoniae, as estirpes lisogénicas estão associadas com a infecção e os biofilmes foram implicados em ambos os processos de colonização e infecção. Foi estudado se a indução espontânea dos profagos e a consequente lise bacteriana favorece o desenvolvimento de biofilmes de S. pneumoniae por ser uma fonte de DNA extracelular (eDNA), um factor importante na matriz dos biofilmes. O desenvolvimento de biofilmes de estirpes lisogénicas e não lisogénicas foi analisado por quantificação da biomassa, determinação da viabilidade celular e por CLSM (“confocal laser scanning microscopy”). Observou-se que as estirpes lisogénicas são mais propensas a formar biofilmes, os quais se caracterizam por maior biomassa e viabilidade celular relacionadas com a maior densidade observada por CLSM. Foi demonstrado que a indução espontânea do fago ocorre continuamente uma vez que se detectaram partículas fágicas durante todo o 5 desenvolvimento do biofilme por medição do número total de UFPs (unidades formadoras de placas) a tempos específicos. Por comparação da formação do biofilme entre estirpes lisogénicas com e sem a lisina fágica, a autolisina bacteriana ou ambas, foi observado que eventos líticos mediados pelo fago influenciam positivamente a estrutura do biofilme. Estes resultados estabeleceram que o profago promove o desenvolvimento do biofilme através da lise bacteriana aquando da indução espontânea. Em concordância, a lise em biofilmes também ocorre noutras espécies bacterianas e parece estar relacionada com o aumento do crescimento do biofilme. No entanto, os efeitos da eliminação quer da lisina fágica quer da lisina bacteriana, foram anulados pela adição de DNA. Em experiências independentes, verificou-se que o tratamento com DNase I resultou em biofilmes menos compactos e densos enquanto a suplementação com DNA originou estruturas mais robustas, confirmando o papel importante de eDNA nos biofilmes de S. pneumoniae. A quantificação de eDNA presente nestas estruturas por PCR em tempo real também demonstrou que eventos líticos promovidos pelo fago são uma fonte importante deste componente da matriz pois biofilmes de estirpes capazes de lise continham mais eDNA do que as estirpes sem lisinas. Desta forma, a lise limitada mediada pelo fago constitui uma fonte importante de eDNA nos biofilmes de S. pneumoniae favorecendo o seu desenvolvimento no caso de estirpes lisogénicas. Interessantemente, a indução substancial do fago, levando a uma elevada proporção de lise, destrói gravemente os biofilmes de estirpes lisogénicas diminuindo significativamente a massa do biofilme como confirmado por CLSM. Estas observações corroboram estudos anteriores que demonstram o potencial do uso de fagos líticos para eliminar biofilmes bacterianos. Os resultados e conclusões apresentados são importantes não só por alargarem directamente o conhecimento acerca da biolo

RecD2 helicase Limits Replication Fork Stress in Bacillus subtilis.

DNA helicases have important roles in replication, recombination, and repair. RecD2 has been identified for study by homology to the Escherichia coli RecD helicase. E. coli RecD has been well studied as a component of the RecBCD helicase-nuclease enzyme important for double-strand break repair. Bacillus subtilis and many other bacteria lack RecB and RecC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism, RecD2 helicases have been shown to provide resistance to a broad range of DNA damaging agents while also contributing to the mismatch repair pathway. I have investigated the importance of B. subtilis RecD2 to genome integrity. Biochemical characterization of RecD2 indicates it is a 5´ to 3´ helicase and that it directly binds single-stranded DNA binding protein. I found that loss of RecD2 sensitized cells to a variety of DNA damaging agents including mitomycin C and the DNA break-inducing peptide phleomycin. I showed that deletion or overexpression of recD2 conferred a modest increase in spontaneous mutagenesis and induced the SOS response, and overexpression resulted in cell death. Further characterization included measurement of replication fork progression in vivo and showed that fork movement was slowed in ∆recD2 cells as well as during recD2 overexpression, supporting the hypothesis that normal levels of RecD2 are important for efficient replication fork movement. I identified specific variants of RecD2 that mitigate ectopic expression-induced toxicity while also conferring sensitivity to DNA damaging agents at low levels of expression. Together, these results highlight novel roles for RecD2 in DNA replication, by helping to maintain replication fork integrity during normal growth and following DNA damage, while also demonstrating that dysregulation of RecD2 is toxic.PhDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116783/1/bwwalsh_1.pd

Molecular genetic analysis of extracellular enzyme secretion by Erwinia carotovora

Erwinia carotovora subsp. carotovora (Ecc) secretes a variety of extracellular enzymes, namely pectinases (Pel), cellulases (Cel) and proteases (Prt). Some of these extracellular enzymes are considered to be the major pathogenicity determinants of this bacterium. Using the chemical mutagen ethyl methyl sulphonate (EMS), a range of Ecc mutants defective in extracellular enzyme production have been generated. One class was found to be pleiotropically defective in the production of Pel and Cel but unaffected for Prt production. Pel and Cel were still synthesised in this class of mutant but both enzymes accumulated within the periplasm. Mutants of the Pel-, Cel-, Prt+ class have been termed Out- mutants. A single Out- mutant, RJP190, was partially resistant to infection by two Ecc bacteriophages. Using a cosmid library of wild-type Ecc, 12 of the 14 Out- mutants were complemented to Out+. Further analysis of the complementing cosmids led to the identification of at least six out loci. A 3.7 kb region of DNA containing out genes was sequenced. This fragment of DNA overlapped with other out genes sequenced In this laboratory. The contiguous DNA (5.7 kb) encoded four proteins, OutD, OutE, OutF and OutG, which were visualised using a T7 directed expression system. The predicted Out proteins were found to share homology with other eubacterial proteins involved in macromolecular trafficking. Accumulated findings strongly suggest that this Out-type system is the major pathway used by Gram-negative bacteria for secreting proteins to the extracellular milieu

Mapping the proteins of the herpes simplex virus type 1 capsid

The aims of the work presented in this thesis were to use a variety of mutagenesis techniques to investigate the proteins of the HSV-1 capsid. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. Insertional and deletional mutagenesis was carried out on VP19C and the effects of the mutations on virus growth and capsid assembly were examined. Insertional mutagenesis showed that VP19C can be divided into three regions with respect to their ability to tolerate five amino acid insertions, with two regions of approximately 100 amino acids at the N- and C-terminal regions of the protein being more tolerant of such insertions than a ~350 amino acid central region. The N-terminal ~100 amino acids of the protein, which are particularly insensitive to insertional mutagenesis, correspond to a region that is poorly conserved among herpesviruses. Some, but not all, severely disabled mutants were compromised in their ability to bind VP23 and VP5. Analysis of deletional mutants revealed the presence of an unusual nuclear localisation signal (NLS) near the N-terminus of VP19C. This was mapped to a 33 amino acid region by fusion of specific sequences to a green fluorescent protein (GFP) marker. By replacing the endogenous NLS with that from the simian virus 40 (SV40) large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in the formation of aberrant capsids and prevented virus growth, suggesting that the poorly-conserved N-terminal sequences have some as-yet-unidentified function

Colorectal Cancers: From Present Problems to Future Solutions

The scientific community has made significant progress in our molecular understanding of sporadic and hereditary colorectal carcinogenesis and progression. Thie pertains to, e.g., the discovery of (mutated) oncogenes and tumor suppressor genes, microsatellite instabilities, modifications in DNA repair, cellular aging, signaling cascades, genomic, epigenetic, transcriptional, translational, and protein modifications, as well as microbiotic factors and further parameters. Progression and metastasis have been more intensively studied, especially during recent years, leading to an intensified knowledge on molecular protagonists and microenvironmental interactions contributing to invasion, dissemination, and metastasis; still, more concerted efforts need to be made to better understand issues such as metastasis to different sites or the metastatic heterogeneity of single cells. Nevertheless, based on actual discoveries, personalized medicine, together with highly interdisciplinary therapeutic strategies combining advanced levels of surgical techniques, oncology, and radiation in neoadjuvant, adjuvant, or palliative settings, has started to improve the clinical prognosis of individual patients with colorectal cancer. The present Special Issue features articles of excellent international experts with the latest data in the fields mentioned. With this Special Issue, we aim to deepen discussions amongst colleagues in all kinds of disciplines working on this disease and to intensify interdisciplinary collaborations aimed at an ultimate understanding of strategies to defeat and prevent, colorectal cancer, and its progression

Subcellular localization of lethal lysis proteins of bacteriophages lambda and phiX174.

The gene products of the lethal lysis genes S and E of the bacteriophages lambda and phiX174, respectively, were shown to be associated primarily with inner membrane material by isopycnic sucrose gradient centrifugation of lysates of infected cells. A small amount of each polypeptide appeared to be in the outer membrane fraction

Qualitative and Quantitative Assessment of the 'Dangerous Activities' Categories Defined by the CISSM Controlling Dangerous Pathogens Project

The Controlling Dangerous Pathogens Project of the Center for International Security Studies at Maryland (CISSM) outlines a prototype oversight system for ongoing microbiological research to control its possible misapplication. This so-called Biological Research Security System (BRSS) foresees the creation of regional, national, and international oversight bodies that review, approve, or reject those proposed microbiological research projects that would fit three BRSS-defined categories: Potentially Dangerous Activities (PDA), Moderately Dangerous Activities (MDA), and Extremely Dangerous Activities (EDA). It is the objective of this working paper to assess these categories qualitatively and quantitatively. To do so, published US research of the years 2000-present (early- to mid-2005) will be screened for science reports that would have fallen under the proposed oversight system had it existed already. Qualitatively, these selective reports will be sorted according to the subcategories of each individual Dangerous Activity, broken down by microbiological agent, and year. Quantitatively, institutes and researchers, which conducted research that would have fallen under review by BRSS, will be listed according to category and year. Taken together, the results of this survey will give an overview of the number of research projects, institutes, and researchers that would have been affected had the new proposed system existed, and thus should allow estimating the potential impact of BRSS on US microbiological academic and industrial research in the future. Furthermore, this working paper might aid refining the proposed system

The role of mismatch repair in mediating cellular sensitivity to cisplatin : the Escherichia coli methyl-directed repair paradigm

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references (v. 2, leaves 195-258).The anticancer drug cisplatin is in widespread use but its mechanism of action is only poorly understood. Moreover, human cancers acquire resistance to the drug, which limits its clinical utility. A paradox in the field is how loss of mismatch DNA repair leads to clinical resistance to this widely used drug. The phenomenon of cisplatin tolerance in mismatch repair deficient cells was initially discovered in E. coli, where methylation deficient dam mutants show high sensitivity to cisplatin and dam mutants with an additional mutation in either of the mismatch repair genes mutS or mutL show near wildtype levels of resistance. A prevalent explanation for this observation is the abortive repair model, which proposes that in dam mutants, where the strand discrimination signal is lost, mismatch repair attempts futile cycles of repair opposite cisplatin-DNA adducts. Previous findings have supported this model to the extent that MutS, the E. coli mismatch recognition protein, specifically recognizes DNA modified with cisplatin. However it has recently been shown that MutS binding to cisplatin adducts may contribute to toxicity by instead preventing the recombinational repair of a cisplatin-modified substrate, and we have previously shown that recombination is an essential mechanism for tolerating cisplatin damage.(cont.) In the present study, we examined the global transcriptional responses of wildtype, dam, dam mutS, and mutS mutant E. coli after treatment with a toxic dose of cisplatin. We also determined any dose-response at the transcriptional level of several SOS response genes and other genes involved in DNA repair by real time RT-PCR. Furthermore, we performed single-cell electrophoresis in order to determine the effect of mismatch repair on the level of double-strand break formation in cisplatin-treated cells. Our results show that Dam-deficient strains exhibit unique gene regulation that may be due to mismatch-repair induced DNA damage in the absence of adenine methylation. In addition, cisplatin treatment induces double-strand break formation and the SOS response in a dose-dependent manner, and both break formation and the SOS response are greatest in the hypersensitive dam mutant strain. The higher level of cisplatin-induced double-strand breaks in the dam mutant may be dependent on functional mismatch repair.by Jennifer L. Robbins.Ph.D