X. Fang1, I. Ahmad2, A. Blanka2, M. Schottkowski2, A. Cimdins2, M. Galperin3,
U. Römling2, M. Gomelsky1
1Department of Molecular Biology, University of Wyoming, Laramie, USA
2Department of Microbiology Tumor and Cell Biology, Karolinska Institute, Stockholm,
Sweden
3National Center for Biotechnology Information National Library of Medicine,
National Institutes of Health, Bethesda, USA
Background
Numerous enzymes are involved in c-di-GMP synthesis and degradation in
enterobacteria. In contrast, only a handful of c-di-GMP receptors/effectors have been identified.
Objectives
The aim of this study was to identify new c-di-GMP receptors and to further characterize them.
Methods
In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE.
Conclusions
The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E.
coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of
unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding.
We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain
containing glycosyltransferase BcsA.
FEMS-0777
Host manipulation and bacterial survival Offered by Pathogens and Disease Salmonella's intracellular toolkit
D. Holden1
1MRC Centre for Molecular Bacteriology and Infection, Imperial College London,
London, United Kingdom
Following entry of Salmonella into host cells, this pathogen resides in a membrane- bound compartment called the Salmonella-containing vacuole (SCV). Here, the bacteria sense the acidic pH and poor nutritional status of the vacuole lumen and either enter a viable non-replicating state or begin to divide. Replication is dependent on activation of the SPI-2 type III secretion system (T3SS). The T3SS comprises an envelope-spanning secretion system and associated translocon pore in the vacuolar membrane. Bacteria then sense the near-neutral pH of the host cell cytoplasm; this results in dissociation and degradation of a bacterial membrane-bound regulatory complex, which activates translocation of approximately 30 bacterial effector proteins into the host cell.
We are currently studying the biochemical and physiological functions of various effectors, which have been implicated in several activities, including lysosomal detoxification, SCV localisation and membrane dynamics, interference with immune signalling and the induction of cytotoxicity. In this talk I will discuss our recent progress in these areas.
FEMS-2853
Host manipulation and bacterial survival Offered by Pathogens and Disease Manipulation of host membrane transport by Type IV effector proteins
C. Roy1
1Microbial Pathogenesis, Yale University School of Medicine, New Haven, USA
Effector proteins delivered into the cytosol of host cells by Dot/Icm type IV secretion systems modulate processes important for creating a vacuole that supports bacterial intracellular replication. Here we describe the biochemical function of
Legionella pneumophila and Coxiella burnetii effector proteins that play specific roles
in controlling host membrane transport. These studies reveal new enzymatic activities and protein structures that demonstrate these pathogens encodes novel
effector proteins that can manipulate evolutionarily conserved host proteins
that control membrane transport processes, which provide insight into how bacterial pathogens are able to construct a unique vacuole inside phagocytic host cells.
FEMS-1844
Host manipulation and bacterial survival Offered by Pathogens and Disease IDENTIFICATION OF LEGIONELLA PNEUMOPHILA VIRULENCE FACTORS REQUIRED TO SUBVERT HOST AUTOPHAGY
V. Lelogeais1, M. Faure2, F. Vavre3, P. Doublet1 1Legionella pathogenesis,
International Center for Infectiology Research INSERM U1111 CNRS UMR 5308 EN S Lyon UCBL, Villeurbanne, France
2Autophagy Infections Immunity,
International Center for Infectiology Research INSERM U1111 CNRS UMR 5308 EN S Lyon UCBL, Lyon, France
3Evolutionary genetic and genomic,
Biometry and Evolutionary Biology UMR CNRS 5558, Villeurbanne, France
Background
Legionella pneumophila is the causative agent of legionnaire’s disease, a public
health problem leading to at least 10% mortality rate. Via effectors secreted by its type IV secretion system, this intravacuolar bacterium interacts with many intracellular pathways of host cells including autophagy. This highly conserved pathway allows eukaryotic cells to recycle end-life cytosolic components in order to regulate cell homeostasis. Autophagy is also a degradative pathway essential to fight intracellular pathogen infections, but numerous microorganisms have evolved strategies in order to subvert this mechanism. The interaction between L. pneumophila and autophagy has been reported but remains still unclear.
Objectives
Our objective is to better decipher the L. pneumophila/autophagy interplay to
determine whether this pathogen is controlled by autophagy or whether it gets benefit from this cellular process to survive/proliferate.
Methods
We used HeLa cells as a model to follow autophagy modulation upon L. pneumophila infection. Cells were infected with the northern Europe endemic strain L. pneumophila Paris.
Conclusions
Our experiments show that L. pneumophila infection induces autophagy in a type IV secretion system dependent manner, early post infection within infected cells but also in neighboring non-infected cells. Otherwise, preliminary data suggest that inhibition of autophagy is linked to a decreased bacterial replication, therefore autophagy could be benificial for L. pneumophila. Thus, the autophagy stimulation could allow the acquirement of nutrients by the bacterium, provide membrane source for the
expanding of L. pneumophila vacuole and/or lead to the delay of the fusion between
FEMS-1274
Host manipulation and bacterial survival Offered by Pathogens and Disease PASTEURELLA MULTOCIDA TOXIN MANIPULATES T CELL DIFFERENTIATION
K. Kubatzky1, D. Hildebrand1, K. Heeg1
1Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg,
Germany
Background
Pasteurella multocida are gram-negative bacilli, which cause respiratory diseases in a
number of wild and domestic animals. Toxigenic serotype A and D strains produce the Pasteurella multocida toxin (PMT), a classical AB toxin containing a deamidase activity in its catalytical domain. The toxin constitutively activates heterotrimeric G proteins and induces downstream signalling cascades involved in cytoskeleton rearrangement, proliferation, differentiation or survival of the host cell. Pathologically, PMT causes porcine atrophic rhinitis characterized by an increased number of osteoclasts and bone resorption at the nasal turbinate bones. However, PMT does not only act on cells of the bone system but we could demonstrate that it also modulates the signalling of many other hematopoietic cells such as B cells, macrophages or monocytes.
Objectives
We investigated how PMT treatment affects the differentiation of primary human T lymphocytes.
Methods
Proliferation studies showed that the toxin amplifies CD3/28 activated proliferation of T cells through the induction of cell cycle progression. Characterisation of
transcription factor activation and the release of cytokines showed that PMT
manipulates lineage determination towards TH17 and Treg cells as PMT-treated T cells
are double positive for the lineage specific transcription factors FOXp3 (Treg) and
RORγT (TH17) but predominantly behave like inflammatory IL-17-releasing TH17
cells.
Conclusions
These results indicate that a bacterial toxin is able to manipulate the direction of TH-
cell differentiation and thereby presumably adjusts the sensitive balance of the immune response towards a condition that benefits the pathogen.
FEMS-0663
Host manipulation and bacterial survival Offered by Pathogens and Disease R-SPONDIN-2 MEDIATES SUSCEPTIBILITY TO INTESTINAL INFECTION WITH CITROBACTER RODENTIUM
S. Gruenheid1, O. Papapietro1, S. Teatero1, A. Thanabalasuriar1, K. Yuki2, E. Diez1,
L. Zhu1, E. Kang1, Y. Durocher3, M. Marcinkiewicz4, D. Malo2
1Microbiology and Immunology, McGill University, Montreal, Canada 2Human Genetics, McGill University, Montreal, Canada
3Biotechnology Research Institute, National Research Council of Canada, Montreal,
Canada
4Cytochem, Cytochem Inc, Montreal, Canada
Background
Citrobacter rodentium is a mouse pathogen widely used as a model for
Enteropathogenic and Enterohemorrhagic Escherichia coli infections in humans. While C. rodentium causes self-limiting colitis in most inbred mouse strains, it induces fatal diarrhea in hyper-susceptible strains.
Objectives
Here we sought to identify the gene and molecular mechanisms underlying this differential response to intestinal infection.
Methods
We used a forward genetics approach to unambiguously localize the locus conferring this differential outcome to a 4 Mb interval. Systematic evaluation of the genes within this region pinpointed R-spondin -2 as the gene underlying infection susceptibility. Conclusions
Robust induction of R-spondin -2 expression during infection in susceptible mouse strains leads to pathological activation of WNT signaling, subsequent loss of intestinal differentiation, and animal death. Conversely, mouse strains that do not induce R-
spondin -2 expression following infection undergo milder, self-limiting disease with no
mortality. Our data demonstrate a previously unknown role of R-spondins and WNT signaling in susceptibility to infectious diarrhea and identify R-spondin -2 as a key molecular link between enteric infection and control of intestinal homoeostasis.
FEMS-2272
Host manipulation and bacterial survival Offered by Pathogens and Disease THE ROLE OF GLYCOGEN DEGRADATION AND METABOLISM IN BACTERIAL VAGINOSIS
R. Hertzberger1, A. Lewis1, W. Lewis1
1School of Medicine, Washington University, St. Louis, USA
Background
Bacterial vaginosis (BV) is a common condition of the human vagina in which the lactobacilli that normally dominate this niche are absent and replaced by a plethora of fastidious anaerobes. BV is associated with an increased risk of acquiring sexually transmitted disease and adverse health outcomes during pregnancy. Factors that drive the microbial dynamics in BV are poorly defined.
Glycogen is deposited in high levels in human vaginal epithelial cells. It has been postulated to be a major carbon source for vaginal bacteria.
Objectives
Here we test the hypothesis that BV-associated bacteria digest and metabolize glycogen.
Methods
We evaluated glycogen degrading enzyme activity in vaginal specimens of women with and without BV. Growth assays were conducted to examine the ability of
lactobacilli and BV-associated bacteria to utilize glycogen. We examined evidence for glycogen-degrading enzymes among vaginal bacteria.
Conclusions
These experiments provide strong evidence that BV associated bacteria degrade glycogen and metabolize the released sugar residues, whereas most vaginal
lactobacilli do not utilize glycogen. These results provide an explanation of the recent observation by Mirmonsef et al. (1) that glycogen levels in BV are reduced.
1. Mirmonsef P, Hotton AL, Gilbert D, Burgad D, Landay A, Weber KM, Cohen M, Ravel J, Spear GT. 2014. Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH. PLoS One. 9:e102467.
FEMS-2874
Fungal plant pathogens
Transcriptional networks controlling pathogenicity and polarized growth in U. maydis
B. Faist1, J. Ulrich1, K. Heimel2, J. Kämper1
1Institute for Apllied Biosciences, Karlsruhe Institute of Technology, Karlsruhe,
Germany
2Institute for Microbiology and Genetics, University of Göttingen, Göttingen, Germany
The transition from yeast-like saprophytic growth to the formation of pathogenic filamentous hyphae is a critical step in the life cycle of the basidiomycete Ustilago
maydis. The switch is controlled by a pheromone/receptor system and the
heterodimeric bE/bW transcription factor complex, encoded by the a and b-mating type loci, respectively. As both pathways control polar growth and cell cycle, they have to be closely interconnected and cross-controlled. The transcription factor Rbf1, a central node for gene regulation during pathogenic development, responses to inputs from both a and b pathways. Coordination is achieved via the interaction of Rbf1 and bW with the Clp1 protein that renders the bE/bW complex inactive and converts Rbf1 to a repressor of the pheromone pathway, releasing both the a and bE/bW mediated cell cycle arrest. We are currently investigating the molecular mechanisms by which the factors independently, cooperatively, and after interaction with Clp1, regulate gene expression during the dimorphic switch, using a combination of reporter gene assays, in vitro DNA binding studies and the genome-wide
identification and occupation of promoter binding sites.
∆clp1 strains are capable to infect maize plants, but, as the cell cycle block is not
released, development is stalled after formation of the appressorium, before the first cell division. In both wildtype and ∆clp1 hyphae, nuclear envelopes start do dissolve before appressorium formation, which is typical for nuclei entering mitosis. In wildtype cells, nuclei migrate into the invading hyphae and divide. However, nuclei of ∆clp1 cells remain in the appressorium, suggesting a novel function for Clp1 in the coordination of nuclear division and nuclear migration.
FEMS-2833
Fungal plant pathogens
Pathogenomics of Verticillium wilt diseases
B. Thomma1, M. Seidl1, X. Shi1, J. Boshoven1, M. van Damme1, J. Li1, E. Rojas Padilla1, Y. Song1, G. van den Berg1, L. Faino1
1Laboratory of Phytopathology, Wageningen University, Wageningen, Netherlands
Fungi cause severe crop losses and threaten food security worldwide. The soil-borne fungal pathogen Verticillium dahliae causes vascular wilt disease on hundreds of plant species, and disease control is challenging because resistance in plants is relatively rare. Moreover, V. dahliae has a flexible genome allowing it to escape host immunity and maintain aggressiveness. So far, knowledge on mechanisms governing this genomic flexibility remains limited.
Through comparative population genomics we have started to unravel mechanisms to establish the genomic diversity that is essential for adaptive genome co-evolution during the continued arms race with host plants. To this end, two V. dahliae genomes were assembled from telomere-to-telomere using long-read sequencing technology and optical mapping, and compared these to the genomes of other Verticillium spp., revealing a pre-speciation genome duplication event. Comparative genomics using the two finished V. dahliae genomes furthermore revealed recent segmental
duplications that established lineage-specific regions. Interestingly, these regions are enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization, and thus contribute to the evolution of virulence. Our evidence suggests that error-prone homology-dependent DNA repair has caused genomic rearrangements, leading to extensive structural variations. Re-sequencing of additional strains showed that independent losses of genetic material favored the escape of host recognition and, likely, host specificity. We propose that evolution of V. dahliae is linked to segmental genome duplications mediated by improperly repaired DNA breaks.
In addition to genome evolution, we also study the role of epigenetic modifications on virulence of V. dahliae and the biological functions of effector proteins. Collectively, these research lines provide insight in mechanisms that make this fungus such a successful broad host range pathogen.
FEMS-1897
Fungal plant pathogens
FGSSP6, A CERATO-PLATANIN PROTEIN WHICH CONTRIBUTES TO