Proteomic response of the marine ammonia-oxidising archaeon Nitrosopumilus maritimus to iron limitation reveals strategies to compensate for nutrient scarcity

Dissolved iron (Fe) is vanishingly low in the oceans, with ecological success conferred to microorganisms that can restructure their biochemistry to maintain high growth rates during Fe scarcity. Chemolithoautotrophic ammonia-oxidising archaea (AOA) are highly abundant in the oceans, constituting ~30% of cells below the photic zone. Here we examine the proteomic response of the AOA isolate Nitrosopumilus maritimus to growth-limiting Fe concentrations. Under Fe limitation, we observed a significant reduction in the intensity of Fe-dense ferredoxins associated with respiratory complex I whilst complex III and IV proteins with more central roles in the electron transport chain remain unchanged. We concomitantly observed an increase in the intensity of Fe-free functional alternatives such as flavodoxin and plastocyanin, thioredoxin and alkyl hydroperoxide which are known to mediate electron transport and reactive oxygen species detoxification, respectively. Under Fe limitation, we found a marked increase in the intensity of the ABC phosphonate transport system (Phn), highlighting an intriguing link between Fe and P cycling in N. maritimus. We hypothesise that an elevated uptake of exogenous phosphonates under Fe limitation may either supplement N. maritimus' endogenous methylphosphonate biosynthesis pathway - which requires Fe - or enhance the production of phosphonate-containing exopolysaccharides known to efficiently bind environmental Fe.     

A comprehensive proteome map of the lipid-requiring nosocomial pathogen Corynebacterium jeikeium K411

Corynebacterium jeikeium is a lipid-requiring pathogen that is considered as part of the normal microflora of the human skin and associated with severe nosocomial infections. Systematic reference maps of the cytoplasmic, cell surface-associated, and extracellular proteome fractions of the clinical isolate C. jeikeium K411 were examined by 2-DE coupled with MALDI-TOF MS. A sum total of 555 protein spots were identified by PMF, corresponding to 358 different proteins that were classified into functional categories and integrated into metabolic pathways. The majority of the proteins were linked to housekeeping functions in energy production and translation and to physiological processes in amino acid, carbohydrate, nucleotide, and lipid metabolism. A complete enzymatic machinery necessary to utilize exogenous fatty acids by beta-oxidation was detected in the cytoplasmic proteome fraction. In addition, several predicted virulence factors of C. jeikeium K411 were identified in the cell surface-associated and extracellular subproteome, including the cell surface proteins SurA and SurB, the surface-anchored pilus subunits SapA and SapB, the surface-anchored collagen adhesin CbpA, the cholesterol esterase Che, and the acid phosphatase AcpA.     

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.Microglia, along with astrocytes, form the backbone of the immune response in the brain. Microglia, in particular, comprise 10–15% of the brain, varying by region and predominating in areas of the midbrain such as the hippocampus and substantia nigra (1). Separated from the systemic immune system by the blood-brain barrier, the brain''s immune response relies on the ability of microglia to act as a multifaceted immune cell; microglia are able to sense pathogens, toxins, injury, and cytokine levels, as well as respond in a neurotrophic or neurotoxic manner similar to the macrophage in the systemic immune system (2).Microglia can respond to insult and injury in a neurotoxic manner (3, 4) where activated microglia are able to induce pro-inflammatory cytokines to recruit other microglia and astrocytes in response to bacterial infection and produce a wide and varied array of factors including reactive oxygen species (ROS)¹, and reactive nitrogen species (RNS), cytokines and lipid mediators as well as remove cellular debris as a post-infection response through phagocytosis (5). As such, microglia protect themselves from their own toxic products through a series of antioxidant proteins regulated through the actions of nuclear factor, erythroid 2-like 2 protein (NFE2L2) (6). Microglia have been implicated in a growing number of CNS-associated diseases; classically activated microglia have been found in brain regions afflicted with Parkinson''s disease, Alzheimer''s disease, and AIDS-related dementia (7–9). Microglial activation has also been reported to play a role in brain injury because of chronic alcohol exposure (10–13).Raivich et al. described microglia response and phases as a linear set of stages that microglia pass through in response to injury, pathogens, or antibodies from the systemic immune system that have crossed the blood-brain barrier (14). The first stage is a quiescent resting state, followed by an alert stage characterized by increased expression of integrin-binding proteins, or cell adhesion molecules, such as CD11b. The homing stage of activation that follows is characterized by increased cell mobility and adhesion as microglia target sites of injury or invasion. The fourth stage is a phagocytic stage that is often termed the classical microglia response, characterized by production of neurotoxic factors such as ROS through a cell membrane-bound NADPH oxidase complex and RNS through the action of inducible nitric oxide synthase, iNOS, as well as phagocytosis of cellular debris. The final stage, known as the bystander activation stage, potentiates the microglia response by activating additional microglia through the production and release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-6 (IL-6).Our understanding of the role of microglia has broadened in recent years to include neurotrophic as well as neurotoxic features (15, 16). The presence of activated microglia does not always correlate to an inflammatory state in the local brain region, implying a noninflammatory or possibly neurotrophic role for these microglia. Microglia that display multiple activation states have been observed in the brains of Alzheimer''s patients (17). It has been suggested that microglia that enter an inflammatory neurotoxic state first change into a neurotrophic healing response prior to returning to their quiescent resting phase (1). As such, a new schema to describe microglia phenotype was required. M1 phase, which can be triggered in vivo and in vitro by lipopolysaccharide (LPS) and inflammatory cytokines, has been established to describe classically activated microglial cells that are similar to those found in the fourth and fifth stages of Raivich''s microglial hierarchy. Microglia do not return to a resting state without first receiving anti-inflammatory triggers that are released by other microglia. These additional stages have been classified as alternative activation and have multiple healing responses. Microglia can be induced into the first alternative activation stage, M2a, through treatment with interleukin-4 (IL-4), and/or interleukin-13 (IL-13). M2a is a healing phase typified by tissue repair and growth stimulation through the actions of various extracellular matrix factors. Most importantly, M2a microglia act as an anti-inflammatory counterpart to M1 phase microglia by competing for arginine, a nitrogen pool for the production of RNS during M1 phase; M2a phase microglia compete for this pool through the production of arginase-1 (ARG1) which converts arginine into ornithine (18). M2b phase is a mixed activation state that responds to viral infection and activated antibodies characterized by the production of the pro-inflammatory cytokines, TNFα and IL-6, in addition to reduction of IL-12 and increased production of IL-10 (19). M2b phase microglia can be reproduced, in vitro, by treating with IL-1β and LPS concurrently or activated IgA complexes, which bind to Fcγ receptors. M2c phase microglia can be induced through IL-10 exposure in vivo and in vitro, and the emergence of M2c microglia shuts down microglial immune response.In order to study microglia in a laboratory setting, enriched ex vivo microglia, primary microglia, or immortalized cell lines are required. BV2 immortalized mouse microglia have been described as producing 41% of the cytokines and chemokines produced by ex vivo cells as compared with 96% coverage by primary microglia. However, Wilcock et al. showed that BV2 cells were successful at producing the classical activators for all four microglia activation stages as measured by real-time polymerase chain reaction (17). In addition, proteomic analysis of pathway level changes may be able to smooth over the lack of full expression through high levels of accurate protein quantification.Because of their importance in immune response and possible role in multiple disease states, a thorough investigation of the differential proteomic expression in the various microglial activation states is required. Using SILAC-labeled immortalized BV2 microglial cells treated with activators of the various activation stages, a proteome profile that includes the major canonical microglial pathways across all four activation states, providing crucial information as to where in these pathways of various states diverge, was established. In addition, using the differential protein expression data, a novel marker of microglia activation, DAB2, was identified and confirmed in primary mouse microglia through Western blot analysis. The abundance of this protein, as well as other differentially expressed proteins identified in this study, may prove as novel indicators in differentiating and categorizing activated microglia in the brain.     

Zinc differentially regulates mitogen-activated protein kinases in human T cells

Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.     

Using partial genomic fosmid libraries for sequencing complete organellar genomes

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.     

CD8+ cells protect mice against reinfection with the intestinal parasite Eimeria falciformis

We investigated cellular immune responses of mice infected with the apicomplexan parasite Eimeria falciformis in order to characterise protective immune mechanisms and effector functions. Adoptive transfer experiments with mesenterial lymph node cells (MLNC) from immune donor mice were performed, and the oocyst output monitored after challenge infection. Phenotypical analysis by fluorescence cytometry and T cell proliferation assay showed that already from day four post infection E. falciformis-specific lymphocytes were present in the MLN. The frequency of parasite-specific, IFN-γ producing CD4+ and CD8+ cells increased in this period by 9.8% and 16.4%, respectively. Infection experiments with IFN-γ deficient mice revealed that IFN-γ is involved in resistance to primary and secondary infection. Transfer of total MLNC from immune donors reduced the oocyst output by 65–74%, as compared to the oocyst output of animals transferred with cells from naïve donors. Transfer of CD8+ cells inhibited parasite development resulting in a reduction of oocyst numbers by 42–64%, whereas CD4+ cells showed no influence on resistance to reinfection.     

Noninvasive Monitoring of Respiratory Viruses in Wild Chimpanzees

To diagnose respiratory disease among wild great apes, there is a need for noninvasive diagnostic methods. Therefore, we analyzed fecal samples from habituated chimpanzees from Taï National Park, Côte d’Ivoire. Samples had been collected during four distinct outbreaks: two with known aetiology (March 2004 and February 2006) and two with unknown aetiology (October 2004 and August 2005). Fecal samples were screened by polymerase chain reaction (PCR) for the presence of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV), two paramyxoviruses previously found in lung tissue of chimpanzees that died due to respiratory disease. In the March 2004 outbreak, 72% of the tested individuals were positive for HMPV, and during the 2006 epidemic, 25% tested HRSV-positive. In the outbreaks where no causative pathogen was previously known, fecal samples tested positive for either HRSV or HMPV, showing that reinfection occurred. Virus sequences were generated and compared with sequences previously found in tissue; nearly identical virus sequences in both tissue and fecal samples were found. These results demonstrate that fecal samples collected during outbreak times can be used for the diagnostic and phylogenetic analysis of HMPV and HRSV. Using such diagnostic tools, systematic noninvasive disease investigation of respiratory outbreaks in wild great apes becomes possible. The methods presented here may also be applied for the investigation of further acute diseases in great apes and other species.     

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.