Annelies Bogaerts

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal Findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.

Mass spectrometric profiling of (neuro)-peptides in the worker honeybee, Apis mellifera.

The honeybee is the economically most important beneficial insect and a model for studying immunity, development and social behavior. Hence, this species was selected for genome sequencing and annotation. An intensive interplay between bioinformatics and mass spectrometry (MS) resulted in the annotation of 36 neuropeptide genes (Hummon et al., 2006). Exactly 100 peptides were demonstrated by a variety of MS techniques. In this follow-up study we dissected and analysed separately all ganglia of the central nervous system (CNS) of adult worker bees in three repeats. The combined MALDI-TOF spectra enabled the accurate mapping of 67 peptides, encoded by 20 precursors. We also demonstrated the expression of an additional but already predicted peptide. In addition to putative bioactive peptides we also list and discuss spacer peptides, propeptides and truncated peptides. The majority of such peptides have a more restricted distribution pattern. Their presence provides some information on the precursor turnover and/or the location of neural cell bodies in which they are produced. Of a given precursor, the (neuro)-peptides with the widest distribution pattern are likely to be the best candidates to interact with receptors. The separate analysis of a neuroendocrine complex and the mushroom body yields suggestions as to which (neuro)-peptides might act as hormones and which neuropeptides might be involved in the complex spectrum of non-hormone driven honeybee behaviour, at these sites. Our data complement immunohistochemical studies of (neuro)-peptides in the honeybee, and form a reference for comparative studies in other insect or arthropod models, in particular in the light of recent or upcoming genome projects. Finally, they also form a firm basis for physiological, functional and/or differential peptidomics studies in the honeybee.

A differential proteomics study of Caenorhabditis elegans infected with Aeromonas hydrophila

The striking similarities between the innate defences of vertebrates and invertebrates as well as the amenability of Caenorhabditis elegans for genetic analysis, have made this free-living ground nematode a popular model system in the study of bacterial pathogenesis. Although genetic studies have brought new insights, showing the inducibility and pathogen-specificity of the immune response, there is still much to be discovered about the exact mechanisms underlying resistance to infection. In this paper a different angle was adopted to study host-pathogen interactions in C. elegans. We report the application of differential gel electrophoresis (DIGE), combined with mass spectrometry to search for proteins that are differentially synthesised in the worm after infection with the gram-negative bacterium Aeromonas hydrophila. Given the dynamic nature of an immune response, the proteome of C. elegans was investigated at three different time-points after infection. A total of 65 differential proteins were identified. This study confirms the involvement of galectins, C-type lectins and lipid binding proteins in the immunity of C. elegans. In addition a number of unknown proteins, which might represent important players of the worms defence system, were isolated and identified. This work gives a first indication of the complex changes that occur at the protein level during infection.

Proteome changes of Caenorhabditis elegans upon a Staphylococcus aureus infection

BackgroundThe success of invertebrates throughout evolution is an excellent illustration of the efficiency of their defence strategies. Caenorhabditis elegans has proven to be an appropriate model for transcriptome studies of host-pathogen interactions. The aim of this paper is to complement this knowledge by investigating the worms response to a Staphylococcus aureus infection through a 2-dimensional differential proteomics approach.ResultsDifferent types of growth media in combination with either E. coli OP50 or Staphylococcus aureus were tested for an effect on the worms lifespan. LB agar was chosen and C. elegans samples were collected 1 h, 4 h, 8 h and 24 h post S. aureus infection or E. coli incubation. Proteomics analyses resulted in the identification of 130 spots corresponding to a total of 108 differentially expressed proteins.ConclusionsExploring four time-points discloses a dynamic insight of the reaction against a gram-positive infection at the level of the whole organism. The remarkable upregulation after 8 h and 24 h of many enzymes involved in the citric acid cycle might illustrate the cost of fighting off an infection. Intriguing is the downregulation of chaperone molecules, which are presumed to serve a protective role. A comparison with a similar experiment in which C. elegans was infected with the gram-negative Aeromonas hydrophila reveals that merely 9% of the identified spots, some of which even exhibiting an opposite regulation, are present in both studies. Hence, our findings emphasise the complexity and pathogen-specificity of the worms immune response and form a firm basis for future functional research.ReviewersThis article was reviewed by Itai Yanai, Dieter Wolf and Torben Luebke (nominated by Walter Lutz).

The hemolymph proteome of the honeybee: Gel‐based or gel‐free?

The honeybee has an invaluable economic impact and is a model for studying immunity, development and social behavior. The recent sequencing and annotation of the honeybee genome facilitates the study of its hemolymph, which reflects the physiological condition and mediates immune responses. We aimed at making a proteomic reference map of honeybee hemolymph and compared gel‐free and gel‐based techniques. One hundered and four 2‐DE spots corresponding to 62 different proteins were identified. Eight identical 2‐DLC experiments resulted in the identification of 32 unique proteins. One repeat was clearly not representative for the potential of the given 2‐DLC setup. Only 27% of the identified hemolymph proteins were found by both techniques. In addition, we found proteins of three different viruses which creates possibilities for biomarker design. Future hemolymph studies will benefit from this work.

Intravenous multipotent adult progenitor cell treatment decreases inflammation leading to functional recovery following spinal cord injury.

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We investigated the capacity of adult bone marrow-derived stem cells, which exhibit immunomodulatory properties, to alter inflammation and promote recovery following SCI. In vitro, we show that human multipotent adult progenitor cells (MAPCs) have the ability to modulate macrophage activation, and prior exposure to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Using a contusion model of SCI, we found that intravenous delivery of MAPCs one day, but not immediately, after SCI significantly improves urinary and locomotor recovery, which was associated with marked spinal cord tissue sparing. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Our results demonstrate that MAPCs exert their primary effects in the periphery and provide strong support for the use of these cells in acute human contusive SCI.

Worker honeybee sterility: a proteomic analysis of suppressed ovary activation.

Eusocial behavior is extensively studied in the honeybee, Apis mellifera, as it displays an extreme form of altruism. Honeybee workers are generally obligatory sterile in a bee colony headed by a queen, but the inhibition of ovary activation is lifted upon the absence of queen and larvae. Worker bees are then able to develop mature, viable eggs. The detailed repressive physiological mechanisms that are responsible for this remarkable phenomenon are as of yet largely unknown. Physiological studies today mainly focus on the transcriptome, while the proteome stays rather unexplored. Here, we present a quantitative 2-dimensional differential gel electrophoresis comparison between activated and inactivated worker ovaries and brains of reproductive and sterile worker bees, including a spot map of ovaries, containing 197 identified spots. Our findings suggest that suppression of ovary activation might involve a constant interplay between primordial oogenesis and subsequent degradation, which is probably mediated through steroid and neuropeptide hormone signaling. Additionally, the observation of higher viral protein loads in both the brains and ovaries of sterile workers is particularly noteworthy. This data set will be of great value for future research unraveling the physiological mechanisms underlying the altruistic sterility in honeybee workers.

Dissection of the Human Multipotent Adult Progenitor Cell Secretome by Proteomic Analysis

Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in acute graft versus host disease clinical trials with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation‐related processes. Our previous studies documented that MAPCs secrete factors that play a role in regulating T‐cell activity. Here we expand our studies using a proteomics approach to characterize and quantify MAPC secretome components secreted over 72 hours in vitro under steady‐state conditions and in the presence of the inflammatory triggers interferon‐γ and lipopolysaccharide, or a tolerogenic CD74 ligand, RTL1000. MAPCs differentially responded to each of the tested stimuli, secreting molecules that regulate the biological activity of the extracellular matrix (ECM), including proteins that make up the ECM itself, proteins that regulate its construction/deconstruction, and proteins that serve to attach and detach growth factors from ECM components for redistribution upon appropriate stimulation. MAPCs secreted a wide array of proteases, some detectable in their zymogen forms. MAPCs also secreted protease inhibitors that would regulate protease activity. MAPCs secreted chemokines and cytokines that could provide molecular guidance cues to various cell types, including neutrophils, macrophages, and T cells. In addition, MAPCs secreted factors involved in maintenance of a homeostatic environment, regulating such diverse programs as innate immunity, angiogenesis/angiostasis, targeted delivery of growth factors, and the matrix‐metalloprotease cascade.

Multipotent Adult Progenitor Cells Enhance Recovery After Stroke by Modulating the Immune Response from the Spleen

Stem cell therapy modulates not only the local microenvironment of the brain but also the systemic immune responses. We explored the impact of human multipotent adult progenitor cells (MAPC) modulating splenic activation and peripheral immune responses after ischemic stroke. Hundred twenty‐six Long‐Evans adult male rats underwent middle cerebral artery occlusion. Twenty‐four hours later, they received IV MAPC or saline treatment. At 3 days after infusion, RNA was isolated from the injured cortex and spleen for microarray analysis. Spleen mass, splenocyte phenotype, and releasing cytokines were measured. Serum cytokines, MAPC biodistribution, brain lesion sizes and neurofunctional deficits were compared in rats treated with MAPC or saline with and without spleens. Stroked animals treated with MAPC exhibited genes that more closely resembled animals with sham surgery. Gene categories downregulated by MAPC included leukocyte activation, antigen presentation, and immune effector processing, associated with the signaling pathways regulated by TNF‐α, IL‐1β, IL‐6, and IFN‐γ within the brain. MAPC treatment restored spleen mass reduction caused by stroke, elevated Treg cells within the spleen, increased IL‐10 and decreased IL‐1β released by splenocytes. MAPC reduced IL‐6 and IL‐1β and upregulated IL‐10 serum levels. Compared with saline, MAPC enhance stroke recovery in rats with intact spleens but had no effects in rats without spleens. MAPC restores expression of multiple genes and pathways involved in immune and inflammatory responses after stroke. Immunomodulation of the splenic response by the intravenous administration of MAPC may create a more favorable environment for brain repair after stroke. Stem Cells 2017;35:1290–1302

C. elegans homologs of insect clock proteins: a tale of many stories

As a consequence of the Earths axial rotation, organisms display daily recurring rhythms in behavior and biochemical properties, such as hormone titers. The neuronal system controlling such changes is best studied in the fruit fly Drosophila melanogaster. In the nematode worm Caenorhabditis elegans, most homologs of these genes function in the heterochronic pathway controlling the (timing of) developmental events. Recent data indicate that in the worm at least one of the genes involved in developmental timing is also active in circadian rhythm control, thereby opening up new perspectives on a central (neuronal) timer interfering with many processes. Also, new neuropeptidergic clock homologs have been identified in nematodes, supporting the idea of a broad range of clock‐regulated targets. We will describe the current knowledge on homologous clock genes in C. elegans with a focus on the recently discovered pigment dispersing factor gene homologs. Similarities between developmental and daily timing are discussed.