• Hepatic Transcriptome Analysis Identifies Divergent Pathogen-Specific Targeting-Strategies to Modulate the Innate Immune System in Response to Intramammary Infection.

      Heimes, Annika; Brodhagen, Johanna; Weikard, Rosemarie; Seyfert, Hans-Martin; Becker, Doreen; Meyerholz, Marie M; Petzl, Wolfram; Zerbe, Holm; Hoedemaker, Martina; Rohmeier, Laura; et al. (Frontiers, 2020-04-29)
      Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.
    • Heparin 2.0: A New Approach to the Infection Crisis.

      Seffer, Malin-Theres; Cottam, Daniel; Forni, Lui G; Kielstein, Jan T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Karger AG, 2020-07-02)
      In April 2020, the US Food and Drug Administration granted emergency use authorization for certain medical devices to be used in patients with coronavirus disease 2019 (CO-VID-19). This included extracorporeal blood purification devices. This narrative review will give a brief overview regarding some of the extracorporeal devices that could be used to treat COVID-19 patients, including the Seraph® 100 Microbind® Affinity Blood Filter, produced by ExThera Medical (Martinez, CA, USA), first licensed in the European Economic Area in 2019. The Seraph® 100 contains ultrahigh molecular weight polyethylene beads with end point-attached heparin and is approved for the reduction of pathogens from the bloodstream either as a single agent or as an adjunct to conventional anti-infective agents. Bacteria, viruses, fungi, and toxins have been shown to bind to the immobilized heparin in a similar way to the interaction with heparan sulfate on the cell surface. This binding is nonreversible and as such, the pathogens are removed from the bloodstream. In this review, we describe the pathophysiological basis and rationale for using heparin for pathogen removal from the blood as well as exploring the technology behind the adaptation of heparin to deprive it of its systemic anticoagulant activity. In addition, we summarize the in vitro data as well as the available preclinical testing and published clinical reports. Finally, we discuss the enormous potential of this technology in an era of increasing antibiotic resistance and high mortality associated with sepsis and consider the application of this as a possible treatment option for COVID-19.
    • Clearance of chloroquine and hydroxychloroquine by the Seraph® 100 Microbind® Affinity blood filter - approved for the treatment of COVID-19 patients.

      Seffer, Malin-Theres; Martens-Lobenhoffer, Jens; Schmidt, Julius J; Eden, Gabriele; Bode-Böger, Stefanie M; Kielstein, Jan T; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-06-19)
      On April 17th 2020 the US Food and Drug Administration granted Coronavirus Disease 2019 (COVID-19) emergency use authorizations for the Seraph® 100 Microbind® Affinity Blood Filter. The medical device is aimed to treat critically ill COVID-19 patients with confirmed or imminent respiratory failure. The aim of this life size in vitro pharmacokinetic study was to investigate the in-vitro adsorption of chloroquine and hydroxychloroquine from human plasma using equipment that is also used at the bedside. After start of the hemoperfusion Pre (Cpre ) Seraph® plasma levels were obtained at 5 (C5 ), 10 (C10 ), 15 (C15 ), 30 (C30 ), 60 (C60 ) and 120 (C120 ) minutes into the procedure. At two timepoints (5 min and 120 min) post (Cpost ) Seraph® plasma levels were determined that were used to calculate the plasma clearance. Both drugs were determined using a validated HPLC method Median [IQR] plasma clearance of the Seraph for chloroquine / hydroxychloroquine was 1.71 [0.51-4.38] ml/min / 1.79 [0.21-3.68] ml/min respectively. The lack of elimination was also confirmed by the fact that plasma levels did not change over the 120 min treatment. As neither chloroquine nor hydroxychloroquine were removed by the treatment with the Seraph dose adjustments in COVID-19 patients undergoing this treatment are not necessary. This article is protected by copyright. All rights reserved.
    • In vivo model to study the impact of genetic variation on clinical outcome of mastitis in uniparous dairy cows.

      Rohmeier, L; Petzl, W; Koy, M; Eickhoff, T; Hülsebusch, A; Jander, S; Macias, L; Heimes, A; Engelmann, S; Hoedemaker, M; et al. (BioMed Central (BMC), 2020-01-31)
      BACKGROUND: In dairy herds, mastitis causes detrimental economic losses. Genetic selection offers a sustainable tool to select animals with reduced susceptibility towards postpartum diseases. Studying underlying mechanisms is important to assess the physiological processes that cause differences between selected haplotypes. Therefore, the objective of this study was to establish an in vivo infection model to study the impact of selecting for alternative paternal haplotypes in a particular genomic region on cattle chromosome 18 for mastitis susceptibility under defined conditions in uniparous dairy cows. RESULTS: At the start of pathogen challenge, no significant differences between the favorable (Q) and unfavorable (q) haplotypes were detected. Intramammary infection (IMI) with Staphylococcus aureus 1027 (S. aureus, n = 24, 96 h) or Escherichia coli 1303 (E. coli, n = 12, 24 h) was successfully induced in all uniparous cows. This finding was confirmed by clinical signs of mastitis and repeated recovery of the respective pathogen from milk samples of challenged quarters in each animal. After S. aureus challenge, Q-uniparous cows showed lower somatic cell counts 24 h and 36 h after challenge (P < 0.05), lower bacterial shedding in milk 12 h after challenge (P < 0.01) and a minor decrease in total milk yield 12 h and 24 h after challenge (P < 0.01) compared to q-uniparous cows. CONCLUSION: An in vivo infection model to study the impact of genetic selection for mastitis susceptibility under defined conditions in uniparous dairy cows was successfully established and revealed significant differences between the two genetically selected haplotype groups. This result might explain their differences in susceptibility towards IMI. These clinical findings form the basis for further in-depth molecular analysis to clarify the underlying genetic mechanisms for mastitis resistance.
    • Genetic selection for bovine chromosome 18 haplotypes associated with divergent somatic cell score affects postpartum reproductive and metabolic performance.

      Meyerholz, M M; Rohmeier, L; Eickhoff, T; Hülsebusch, A; Jander, S; Linden, M; Macias, L; Koy, M; Heimes, A; Gorríz-Martín, L; et al. (Elsevier, 2019-11-01)
      The susceptibility of animals to periparturient diseases has a great effect on the economic efficiency of dairy industries, on the frequency of antibiotic treatment, and on animal welfare. The use of selection for breeding cows with reduced susceptibility to diseases offers a sustainable tool to improve dairy cattle farming. Several studies have focused on the association of distinct bovine chromosome 18 genotypes or haplotypes with performance traits. The aim of this study was to test whether selection of Holstein Friesian heifers via SNP genotyping for alternative paternal chromosome 18 haplotypes associated with favorable (Q) or unfavorable (q) somatic cell scores influences postpartum reproductive and metabolic diseases. Thirty-six heifers (18 Q and 18 q) were monitored from 3 wk before calving until necropsy on d 39 (± 4 d) after calving. Health status and rectal temperature were measured daily, and body condition score and body weight were assessed once per week. Blood samples were drawn twice weekly, and levels of insulin, nonesterified fatty acids, insulin-like growth factor-I, growth hormone, and β-hydroxybutyrate were measured. Comparisons between the groups were performed using Fisher's exact test, chi-squared test, and the GLIMMIX procedure in SAS. Results showed that Q-heifers had reduced incidence of metritis compared with q-heifers and were less likely to develop fever. Serum concentrations of β-hydroxybutyrate were lower and insulin-like growth factor-I plasma concentrations were higher in Q- compared with q-heifers. However, the body condition score and withers height were comparable between haplotypes, but weight loss tended to be lower in Q-heifers compared with q-heifers. No differences between the groups were detected concerning retained fetal membranes, uterine involution, or onset of cyclicity. In conclusion, selection of chromosome 18 haplotypes associated with a reduced somatic cell score resulted in a decreased incidence of postpartum reproductive and metabolic diseases in this study. The presented data add to the existing knowledge aimed at avoiding negative consequences of genetic selection strategies in dairy cattle farming. The underlying causal mechanisms modulated by haplotypes in the targeted genomic region and immune competence necessitate further investigation.
    • Characterization of functional traits with focus on udder health in heifers with divergent paternally inherited haplotypes on BTA18.

      Heimes, A; Brodhagen, J; Weikard, R; Hammon, H M; Meyerholz, M M; Petzl, W; Zerbe, H; Engelmann, S; Schmicke, M; Hoedemaker, M; et al. (BioMedCentral, 2019-07-11)
      BACKGROUND: A major challenge in modern medicine and animal husbandry is the issue of antimicrobial resistance. One approach to solving this potential medical hazard is the selection of farm animals with less susceptibility to infectious diseases. Recent advances in functional genome analysis and quantitative genetics have opened the horizon to apply genetic marker information for efficiently identifying animals with preferential predisposition regarding health traits. The current study characterizes functional traits with a focus on udder health in dairy heifers. The animals were selected for having inherited alternative paternal haplotypes for a genomic region on Bos taurus chromosome (BTA) 18 genetically associated with divergent susceptibility to longevity and animal health, particularly mastitis. RESULTS: In the first weeks of lactation, the q heifers which had inherited the unfavorable (q) paternal haplotype displayed a significantly higher number of udder quarters with very low somatic cell count (< 10,000 cells / ml) compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. This might result in impaired mammary gland sentinel function towards invading pathogens. Furthermore, across the course of the first lactation, there was indication that q half-sib heifers showed higher somatic cell counts, a surrogate trait for udder health, in whole milkings compared to their paternal half-sib sisters with the favorable (Q) paternal haplotype. Moreover, heifers with the haplotype Q had a higher feed intake and higher milk yield compared to those with the q haplotype. Results of this study indicate that differences in milk production and calculated energy balance per se are not the main drivers of the genetically determined differences between the BTA18 Q and q groups of heifers. CONCLUSIONS: The paternally inherited haplotype from a targeted BTA18 genomic region affect somatic cell count in udder quarters during the early postpartum period and might also contribute to further aspects of animal's health and performance traits due to indirect effects on feed intake and metabolism.
    • Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.

      Brodhagen, Johanna; Weikard, Rosemarie; Thom, Ulrike; Heimes, Annika; Günther, Juliane; Hadlich, Frieder; Zerbe, Holm; Petzl, Wolfgang; Meyerholz, Marie M; Hoedemaker, Martina; et al. (BioMed Central (BMC), 2019-05-22)
      Background: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. Results: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challengeassociated biological signaling pathway patterns. Conclusions: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.
    • Human antibody responses against non-covalently cell wall-bound Staphylococcus aureus proteins.

      Romero Pastrana, Francisco; Neef, Jolanda; Koedijk, Dennis G A M; de Graaf, Douwe; Duipmans, José; Jonkman, Marcel F; Engelmann, Susanne; van Dijl, Jan Maarten; Buist, Girbe; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-02-19)
      Human antibody responses to pathogens, like Staphylococcus aureus, are important indicators for in vivo expression and immunogenicity of particular bacterial components. Accordingly, comparing the antibody responses to S. aureus components may serve to predict their potential applicability as antigens for vaccination. The present study was aimed at assessing immunoglobulin G (IgG) responses elicited by non-covalently cell surface-bound proteins of S. aureus, which thus far received relatively little attention. To this end, we applied plasma samples from patients with the genetic blistering disease epidermolysis bullosa (EB) and healthy S. aureus carriers. Of note, wounds of EB patients are highly colonized with S. aureus and accordingly these patients are more seriously exposed to staphylococcal antigens than healthy individuals. Ten non-covalently cell surface-bound proteins of S. aureus, namely Atl, Eap, Efb, EMP, IsaA, LukG, LukH, SA0710, Sle1 and SsaA2, were selected by bioinformatics and biochemical approaches. These antigens were recombinantly expressed, purified and tested for specific IgG responses using human plasma. We show that high exposure of EB patients to S. aureus is mirrored by elevated IgG levels against all tested non-covalently cell wall-bound staphylococcal antigens. This implies that these S. aureus cell surface proteins are prime targets for the human immune system.
    • Staphylococcal serine protease-like proteins are pacemakers of allergic airway reactions to Staphylococcus aureus.

      Stentzel, Sebastian; Teufelberger, Andrea; Nordengrün, Maria; Kolata, Julia; Schmidt, Frank; van Crombruggen, Koen; Michalik, Stephan; Kumpfmüller, Jana; Tischer, Sebastian; Schweder, Thomas; et al. (2017-02)
      A substantial subgroup of asthmatic patients have "nonallergic" or idiopathic asthma, which often takes a severe course and is difficult to treat. The cause might be allergic reactions to the gram-positive pathogen Staphylococcus aureus, a frequent colonizer of the upper airways. However, the driving allergens of S aureus have remained elusive.
    • Determining the bacterial cell biology of Planctomycetes.

      Boedeker, Christian; Schüler, Margarete; Reintjes, Greta; Jeske, Olga; van Teeseling, Muriel C F; Jogler, Mareike; Rast, Patrick; Borchert, Daniela; Devos, Damien P; Kucklick, Martin; et al. (2017-04-10)
      Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.
    • Costs of life - Dynamics of the protein inventory of Staphylococcus aureus during anaerobiosis.

      Zühlke, Daniela; Dörries, Kirsten; Bernhardt, Jörg; Maaß, Sandra; Muntel, Jan; Liebscher, Volkmar; Pané-Farré, Jan; Riedel, Katharina; Lalk, Michael; Völker, Uwe; et al. (2016)
      Absolute protein quantification was applied to follow the dynamics of the cytoplasmic proteome of Staphylococcus aureus in response to long-term oxygen starvation. For 1,168 proteins, the majority of all expressed proteins, molecule numbers per cell have been determined to monitor the cellular investments in single branches of bacterial life for the first time. In the presence of glucose the anaerobic protein pattern is characterized by increased amounts of glycolytic and fermentative enzymes such as Eno, GapA1, Ldh1, and PflB. Interestingly, the ferritin-like protein FtnA belongs to the most abundant proteins during anaerobic growth. Depletion of glucose finally leads to an accumulation of different enzymes such as ArcB1, ArcB2, and ArcC2 involved in arginine deiminase pathway. Concentrations of 29 exo- and 78 endometabolites were comparatively assessed and have been integrated to the metabolic networks. Here we provide an almost complete picture on the response to oxygen starvation, from signal transduction pathways to gene expression pattern, from metabolic reorganization after oxygen depletion to beginning cell death and lysis after glucose exhaustion. This experimental approach can be considered as a proof of principle how to combine cell physiology with quantitative proteomics for a new dimension in understanding simple life processes as an entity.
    • Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β.

      Seeger, Bettina; Klawonn, Frank; Nguema Bekale, Boris; Steinberg, Pablo; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Consumers of fruits and vegetables are frequently exposed to small amounts of hormonally active pesticides, some of them sharing a common mode of action such as the activation of the human estrogen receptor α (hERα) or β (hERβ). Therefore, it is of particular importance to evaluate risks emanating from chemical mixtures, in which the individual pesticides are present at human-relevant concentrations, below their corresponding maximum residue levels. Binary and ternary iso-effective mixtures of estrogenic pesticides at effect concentrations eliciting a 1 or 10% effect in the presence or absence of 17β-estradiol were tested experimentally at the hERα in the yeast-based estrogen screen (YES) assay as well as in the human U2-OS cell-based ERα chemical-activated luciferase gene expression (ERα CALUX) assay and at the hERβ in the ERβ CALUX assay. The outcome was then compared to predictions calculated by means of concentration addition. In most cases, additive effects were observed with the tested combinations in all three test systems, an observation that supports the need to expand the risk assessment of pesticides and consider cumulative risk assessment. An additional testing of mixture effects at the hERβ showed that most test substances being active at the hERα could also elicit additive effects at the hERβ, but the hERβ was less sensitive. In conclusion, effects of the same ligands at the hERα and the hERβ could influence the estrogenic outcome under physiological conditions.
    • Global antibody response to Staphylococcus aureus live-cell vaccination.

      Selle, Martina; Hertlein, Tobias; Oesterreich, Babett; Klemm, Theresa; Kloppot, Peggy; Müller, Elke; Ehricht, Ralf; Stentzel, Sebastian; Bröker, Barbara M; Engelmann, Susanne; et al. (2016)
      The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration.
    • Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells.

      Bauer, Isabel; Günther, Juliane; Wheeler, Thomas T; Engelmann, Susanne; Seyfert, Hans-Martin; Helmholtz Center for Infection Research (2015)
      Considerably divergent data have been published from attempts to model the E. coli vs. S. aureus specific immune reaction of the udder using primary cultures of bovine mammary epithelial cells from cows (pbMEC). Some groups reported a swift, strong and transient inflammatory response against challenges with E. coli and only a weak and retarded response against S. aureus, in agreement with the respective reaction of the udder. Others found almost the reverse. Presence or absence of fetal calf serum distinguished the experimental setting between both groups. We examined here if this causes the divergent reaction of the pbMEC towards both pathogen species. We challenged pbMEC with proteins from heat killed E. coli or S. aureus pathogens or purified TLR2 and TLR4 ligands. The stimuli were applied in normal growth medium with (SM10) or without (SM0) 10% fetal calf serum, or in the basal medium supplemented with 10 mg/ml milk proteins (SM Milk).
    • Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion is correlated with disease progression.

      Stentzel, Sebastian; Sundaramoorthy, Nandakumar; Michalik, Stephan; Nordengrün, Maria; Schulz, Sarah; Kolata, Julia; Kloppot, Peggy; Engelmann, Susanne; Steil, Leif; Hecker, Michael; et al. (2015-07-05)
      Although Staphylococcus aureus is a prominent cause of infections, no vaccine is currently available. Active vaccination relies on immune memory, a core competence of the adaptive immune system. To elucidate whether adaptive immunity can provide protection from serious complications of S. aureus infection, a prospective observational study of 44 patients with S. aureus infection complicated by bacteremia was conducted. At diagnosis, serum IgG binding to S. aureus extracellular proteins was quantified on immunoblots and with Luminex-based FLEXMAP 3D™ assays comprising 64 recombinant S. aureus proteins. Results were correlated with the course of the infection with sepsis as the main outcome variable. S. aureus-specific serum IgG levels at diagnosis of S. aureus infection were lower in patients developing sepsis than in patients without sepsis (P<0.05). The pattern of IgG binding to eight selected S. aureus proteins correctly predicted the disease course in 75% of patients. Robust immune memory of S. aureus was associated with protection from serious complications of bacterial invasion. Serum IgG binding to eight conserved S. aureus proteins enabled stratification of patients with high and low risk of sepsis early in the course of S. aureus infections complicated by bacteremia.
    • A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems.

      Halbedel, Sven; Reiss, Swantje; Hahn, Birgit; Albrecht, Dirk; Mannala, Gopala Krishna; Chakraborty, Trinad; Hain, Torsten; Engelmann, Susanne; Flieger, Antje (2014-11)
      Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.
    • Metaproteomics to unravel major microbial players in leaf litter and soil environments: challenges and perspectives.

      Becher, Dörte; Bernhardt, Jörg; Fuchs, Stephan; Riedel, Katharina; Ernst-Moritz-Arndt-University of Greifswald, Institute of Microbiology, Greifswald, Germany. (2013-10)
      Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, changes in environmental parameters but also human interferences may alter species composition and elicit alterations in microbial activities. Soil and litter metaproteomics, implying the assignment of soil and litter proteins to specific phylogenetic and functional groups, has a great potential to provide essential new insights into the impact of microbial diversity on soil ecosystem functioning. This article will illuminate challenges and perspectives of current soil and litter metaproteomics research, starting with an introduction to an appropriate experimental design and state-of-the-art proteomics methodologies. This will be followed by a summary of important studies aimed at (i) the discovery of the major biotic drivers of leaf litter decomposition, (ii) metaproteomics analyses of rhizosphere-inhabiting microbes, and (iii) global approaches to study bioremediation processes. The review will be closed by a brief outlook on future developments and some concluding remarks, which should assist the reader to develop successful concepts for soil and litter metaproteomics studies.