The crystal structure of a family GH25 lysozyme from Bacillus anthracis implies a neighboring-group catalytic mechanism with retention of anomeric configuration

Lysozymes are found in many of the sequence-based families of glycoside hydrolases (www.cazy.org) where they show considerable structural and mechanistic diversity. Lysozymes from glycoside hydrolase family GH25 adopt a (α/β)₅(β)₃-barrel-like fold with a proposal in the literature that these enzymes act with inversion of anomeric configuration; the lack of a suitable substrate, however, means that no group has successfully demonstrated the configuration of the product. Here we report the 3-D structure of the GH25 enzyme from Bacillus anthracis at 1.4 Å resolution. We show that the active center is extremely similar to those from glycoside hydrolase families GH18, GH20, GH56, GH84, and GH85 implying that, in the absence of evidence to the contrary, GH25 enzymes also act with net retention of anomeric configuration using the neighboring-group catalytic mechanism that is common to this ‘super-family’ of enzymes.     

Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10⁷-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD₅₀ of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.     

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

Background

Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.

Methods and Findings

A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient''s peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.

Conclusions

This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.     

Metabolic Differentiation in Biofilms as Indicated by Carbon Dioxide Production Rates

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO₂ production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O₂ flux, with CO₂ production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO₂ production rate of cells from this region increased within 0.25 h from 9.45 ± 5.40 fmol of CO₂·cell⁻¹·h⁻¹ to 22.6 ± 7.58 fmol of CO₂·cell⁻¹·h⁻¹ when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.Spatial differences in biofilm cohesiveness have been observed after the application of increased shear forces. Coufort et al. (8) subjected both aerobic and anaerobic biofilms, cultivated on ethanol or wastewater, to increased shear stress and found that the biofilm layer at the bulk liquid interface was removed by slight increases in shear rates (0.2 Pa), whereas the middle and base biofilm layers were able to resist removal when exposed to up to 2 Pa and 13 Pa, respectively (8). Total organic carbon (TOC) analyses indicated that the sensitive top layer of the biofilm contained approximately 60% of the total biofilm biomass while the remaining two layers each represented approximately 20%. In a follow-up study, biofilms grown under similar conditions exhibited comparable degrees of heterogeneity in the susceptibility of the various biofilm layers to shear and abrasion (9). It was also indicated that the basal biofilm layer contained active microorganisms, as characterized by oxygen uptake rates, but no details were provided on the methodology or time lapse after the removal of the less-cohesive upper biofilm layers.Spatial differentiation in metabolic activity in biofilms has also been noted. Most experimental strategies to determine biofilm activity have been centered on microscopy in combination with fluorescent reporter genes or probes that target various indicators of physiological activity in the cell. Several fluorescent stains have been applied previously, such as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) (15) and acridine orange (27) as well as the commercially available BacLight viability kit (17). Reporter gene expression is another means to evaluate physiological activity in a biofilm. Alkaline phosphatase activity correlated well with oxygen penetration into the upper layers (30 μm) of 117- to 151-μm-thick biofilms (28).Although all of the above approaches have been shown to be effective, most suffer from inherent disadvantages (26), including incomplete penetration of fluorescent stains and the production of artifacts, and, perhaps most significantly, generally allow only end point analysis due to cellular toxicity. Reporter gene technologies may circumvent this problem but require prior genetic manipulation, and it is unknown what, if any, changes in cell physiology may occur as a result of expression of the reporter gene. The need for genetic manipulation further constrains analysis to pure culture studies.The basis for spatial heterogeneity in biofilm physiological activity is widely accepted, as previously reviewed (25, 26). Limited diffusion of nutrients and oxygen into the biofilm from the bulk liquid and waste products from a multilayered biofilm are among the simplest explanations since the absence of a complete exchange with the environment, in concert with microbial activity, leads to the formation of chemical gradients in the biofilm. The bacteria in the biofilm respond to the gradients, likely by altering gene expression patterns as determined by global regulators. The remarkable recalcitrance of biofilms toward many antimicrobials may in part be due to the insensitivity of dormant cells in the regions of the biofilm where limited diffusion reduces metabolic activity.The effect of air bubbles on biofilm stability has mostly been studied in a dental context, where biofilm removal is the goal. Gomez-Suarez et al. (11) utilized a single bubble to investigate the strength of bacterial cell adhesion to various surfaces (11). According to the authors, the probability of cell detachment due to the movement of an air bubble over an attached cell is determined by several factors, namely, collision efficiency, bubble-bacteria attachment efficiency, and the stability of the bubble-bacteria aggregate. For a bubble spanning the entire width of a flow chamber, the collision efficiency is expected to be equal to 1 although the velocity of the bubble may also influence the detachment efficiency since a rapidly moving bubble will result in a thicker liquid film surrounding the bubble, which in turn decreases the collision efficiency. Bacterium-substratum adhesion forces of ∼10⁻⁹ N were estimated, which is significantly smaller than the detachment force of a bubble moving over an attached cell (up to 10⁻⁷ N).Liquid flow in most environments—in nature, industry, and clinical or dental settings—typically shows much variation. It can be expected that microbial biofilms have evolved to manage this variability and even to utilize the resulting differences in flow to optimize activity (e.g., the prevention of excessive biomass accumulation for the maintenance of optimum gradients of nutrients and gases) or to relocate to more favorable environments.Furthermore, increased shear is a recognized strategy to remove unwanted microbial growth from surfaces; therefore, methods to measure the effect of shear on biofilms, including biofilm recovery after partial shear-induced removal, should contribute to our overall understanding of this important form of microbial existence. We developed an approach that measures CO₂ production as an indication of biofilm activity in real time and combined this method with confocal laser scanning microscopy (CLSM) and cell yield measurements to study activity-structure relationships in biofilms. This approach is an extension of the one we described in 2009 (18) and enables us to comment on differences in metabolic activity of the whole biofilm versus that of the shear-susceptible biofilm region and to compare biofilm-derived planktonic cells with those growing in batch culture.     

Mucolipidosis type IV and the mucolipins

MLIV (mucolipidosis type?IV) is a neurodegenerative lysosomal storage disorder caused by mutations in MCOLN1, a gene that encodes TRPML1 (mucolipin-1), a member of the TRPML (transient receptor potential mucolipin) cation channels. Two additional homologues are TRPML2 and TRPML3 comprising the TRPML subgroup in the TRP superfamily. The three proteins play apparently key roles along the endocytosis process, and thus their cellular localization varies among the different group members. Thus TRPML1 is localized exclusively to late endosomes and lysosomes, TRPML2 is primarily located in the recycling clathrin-independent GPI (glycosylphosphatidylinositol)-anchored proteins and early endosomes, and TRPML3 is primarily located in early endosomes. Apparently, all three proteins' main physiological function underlies Ca(2+) channelling, regulating the endocytosis process. Recent findings also indicate that the three TRPML proteins form heteromeric complexes at least in some of their cellular content. The physiological role of these complexes in lysosomal function remains to be elucidated, as well as their effect on the pathophysiology of MLIV. Another open question is whether any one of the TRPMLs bears additional function in channel activity.     

Chemosystematic evaluation of Aloe section Pictae (Asphodelaceae)

The chemosystematic value of UV-absorbing leaf constituents was considered in previously uncharacterised representatives of Aloe section Pictae, the problematic maculate species complex. Comparative data indicate that the anthrone C-glycoside, 6′-malonylnataloin (7-hydroxychrysaloin 6′-O-malonate) is typical of maculate species in East Africa, but is unconvincing as a synapomorphy for section Pictae. A naphthalene derivative found widely in Aloe, plicataloside, was detected in Aloe greatheadii. Biogeographical trends were observed in the occurrence of the flavonoids isoorientin (luteolin-6-C-glucoside) and isovitexin (apigenin 6-C-glucoside). Isoorientin is a common constituent of tropical and sub-tropical species of Aloe, whereas isovitexin is restricted to a few southern African species. Isoorientin and isovitexin co-occur in the southern African maculate species Aloe parvibracteata, and the disjunct West African maculate species, Aloe macrocarpa. This is the first report of isoorientin and isovitexin in maculate species of Aloe; the presence of flavonoids in section Pictae is of taxonomic interest.     

Pronounced Effect of the Nature of the Inoculum on Biofilm Development in Flow Systems

Biofilm formation renders sessile microbial populations growing in continuous-flow systems less susceptible to variation in dilution rate than planktonic cells, where dilution rates exceeding an organism''s maximum growth rate (μ_max) results in planktonic cell washout. In biofilm-dominated systems, the biofilm''s overall μ_max may therefore be more relevant than the organism''s μ_max, where the biofilm μ_max is considered as a net process dependent on the adsorption rate, growth rate, and removal rate of cells within the biofilm. Together with lag (acclimation) time, the biofilm''s overall μ_max is important wherever biofilm growth is a dominant form, from clinical settings, where the aim is to prevent transition from lag to exponential growth, to industrial bioreactors, where the aim is to shorten the lag and rapidly reach maximum activity. The purpose of this study was to measure CO₂ production as an indicator of biofilm activity to determine the effect of nutrient type and concentration and of the origin of the inoculum on the length of the lag phase, biofilm μ_max, and steady-state metabolic activity of Pseudomonas aeruginosa PA01 (containing gfp), Pseudomonas fluorescens CT07 (containing gfp), and a mixed community. As expected, for different microorganisms the lengths of the lag phase in biofilm development and the biofilm μ_max values differ, whereas different nutrient concentrations result in differences in the lengths of lag phase and steady-state values but not in biofilm μ_max rates. The data further showed that inocula from different phenotypic origins give rise to lag time of different lengths and that this influence persists for a number of generations after inoculation.Microbial growth in batch cultures has been studied for a long time, and the observed phases have been designated the lag phase, the acceleration phase, the exponential phase, the retardation phase, the stationary phase, and the phase of decline although not each culture displays all of the mentioned phases (16). In contrast to batch cultures and static (no flow) biofilms (e.g., those that form in 96-well plates), the increase in biofilm cells in a flowing environment is a net process that is dependent on the irreversible adsorption rate of cells to the surface, the growth rate of the microorganisms, and the removal rate of cells lost to the bulk flow (18). There are numerous benefits for the cells in biofilms, e.g., protection against antimicrobials and the opportunity for and proliferation by continuous cell dispersion. There is also a possible competitive advantage if cells colonize surfaces at multiple sites and grow in such a manner that the resulting three-dimensional architecture exposes the maximum biofilm surface area to surrounding nutrients. The most successful colonizers would therefore be the cells with the ability to adhere to the surface (and stay adhered) and to start multiplying at maximum rate. The process of events from being free-floating cells to the so-called permanently surface-attached phase involves early steps including reversible attachment and a phenotypic change in the cells from a planktonic state to a sessile state, with concomitant changes in gene expression; these steps contribute to a lag phase that will occur before maximal growth/biofilm development can take place (23). Clearly, the ability to progress from the lag phase to a fast-growing phase, as well as the duration of the lag phase, is an important determinant of biofilm function and has an impact in a diverse range of environments, often with implications for infection or contamination control, as well as in industrial processes.At the cell level, an extended lag phase and slower growth create the risk that the cells will be displaced by faster-growing microcolonies, as was demonstrated by Klayman et al. (13) in dual species biofilms. A microorganism''s competence in dominating a surface area can therefore be evaluated by comparing the lag phases and maximal growth rates (μ_max) of a biofilm growth curve. Knowing a bacterial population''s specific growth rate is a requirement for its cultivation at optimum rates in a chemostat or other continuously fed bioreactor. A key assumption for this type of cultivation is that wall growth has a negligible effect, which is in stark contrast to systems where surface-associated growth dominates. Indeed, while dilution rates exceeding an organism''s μ_max results in cell washout in a conventional chemostat setting, biofilm formation enables microbial populations to persist at dilution rates much higher than the organism''s μ_max.Biofilm growth rates have been determined by various techniques, such as fluorescence in situ hybridization (FISH) (12, 31), measuring the incorporation of radioactive substances like [³H]thymidine and ³²P (8, 9), microscopy (3, 13, 17), measuring the total increase in biofilm mass (both cells and extracellular polymeric substances) (24, 28), colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H- tetrazolium hydroxide (XTT) assays (26), or measurement of amide II bands, as determined by attenuated total reflectance-Fourier transform infrared spectroscopy (6). Some of the above-mentioned techniques suffer the drawback that the biofilms have to be sacrificed with sampling or that the measured increases do not distinguish between live and dead matter in the biofilm (i.e., increases measured might not represent an accurate increase in viable cell numbers).In this study a carbon dioxide evolution measurement system (CEMS) (15) was used to track the biofilm development rate in real time. The advantage of using this system is that the measured rates represent the metabolic activity of the active cell mass and can be done nondestructively for any biofilm-forming microorganism. In the past, measurement of oxygen uptake rates has been used for determination of growth rates in batch cultures (19) and of the localized growth rate in biofilms (32). CO₂ measurements by a gas chromatograph have been used to determine growth rates in batch systems (4), but to our knowledge this is the first time that CO₂ measurements have been used to determine whole-biofilm specific growth rates. We applied this technique to compare the biofilm μ_max values for two well-described pseudomonads and a mixed microbial community when the organisms are grown on different nutrients and to test the premise that the origin of the inoculum has an impact on early biofilm development.