dictyNews
Electronic Edition
Volume 44, number 7
March 2, 2018
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Abstracts
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Antimycobacterial drug discovery using Mycobacteria-infected
amoebae identifies anti-infectives and new molecular targets
Valentin Trofimov1, 2, Sébastien Kicka1, Sabrina Mucaria3, Nabil
Hanna1, Fernando Ramon-Olayo4, Laura Vela-Gonzalez Del Peral4,
Joël Lelièvre4, Lluís Ballell4, Leonardo Scapozza3, Gurdyal S.
Besra5, Jonathan A.G. Cox6, * and Thierry Soldati1, *
1Department of Biochemistry, Faculty of Science, University of Geneva,
Geneva, Switzerland
2Present address : Institut Pasteur de Lille, Lille, France
3Pharmaceutical Biochemistry/Chemistry, School of Pharmaceutical
Sciences, University of Geneva, Geneva, Switzerland
4GSK, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
5School of Biosciences, University of Birmingham, Edgbaston,
Birmingham, UK
6School of Life & Health Sciences, Aston University, Birmingham, UK
*Co-corresponding authors:
Scientific Reports, in press
Tuberculosis remains a serious threat to human health world-wide, and
improved efficiency of medical treatment requires a better understanding
of the pathogenesis and the discovery of new drugs. In the present study,
we performed a whole-cell based screen in order to complete the
characterization of 168 compounds from the GlaxoSmithKline TB-set. We
have established and utilized novel previously unexplored host-model
systems to characterize the GSK compounds,i.e. the amoeboid organisms
D. discoideum and A. castellanii, as well as a microglial phagocytic cell line,
BV2. We infected these host cells with Mycobacterium marinum to monitor
and characterize the anti-infective activity of the compounds with quantitative
fluorescence measurements and high-content microscopy. In summary,
88.1% of the compounds were confirmed as antibiotics against M. marinum,
11.3% and 4.8% displayed strong anti-infective activity in, respectively, the
mammalian and protozoan infection models. Additionally, in the two
systems, 13-14% of the compounds displayed pro-infective activity. Our
studies underline the relevance of using evolutionarily distant pathogen
and host models in order to reveal conserved mechanisms of virulence
and defence, respectively, which are potential "universal" targets for
intervention. Subsequent mechanism of action studies based on generation
of over-expresser M. bovis BCG strains, generation of spontaneous
resistant mutants and whole genome sequencing revealed four new
molecular targets, including FbpA, MurC, MmpL3 and GlpK.
submitted by: Thierry Soldati [[log in to unmask]]
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Genetic signatures of microbial altruism and cheating in social amoebas
the wild
Suegene Noh, Katherine S. Geist, Xiangjun Tian, Joan E. Strassmann,
David C. Queller
PNAS, in press
Many microbes engage in social interactions. Some of these have come
to play an important role in the study of cooperation and conflict, largely
because, unlike most animals, they can be genetically manipulated and
experimentally evolved. However, whereas animal social behavior can
be observed and assessed in natural environments, microbes usually
cannot, so we know little about microbial social adaptations in nature.
This has led to some difficult-to-resolve controversies about social
adaptation even for well-studied traits such as bacterial quorum sensing,
siderophore production, and biofilms. Here we use molecular signatures
of population genetics and molecular evolution to address controversies
over the existence of altruism and cheating in social amoebas. First, we
find signatures of rapid adaptive molecular evolution that are consistent
with social conflict being a significant force in nature. Second, we find
population-genetic signatures of purifying selection to support the
hypothesis that the cells that form the sterile stalk evolve primarily through
altruistic kin selection rather than through selfish direct reproduction. Our
results show how molecular signatures can provide insight into social
adaptations that cannot be observed in their natural context and they
support the hypotheses that social amoebas in the wild are both altruists
and cheaters.
submitted by: Dave Queller [[log in to unmask]]
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Title: Repellent and attractant guidance cues initiate cell migration by
distinct rear-driven and front-driven cytoskeletal mechanisms
Authors: Louise P. Cramer1, Robert R. Kay2 and Evgeny Zatulovskiy2,3
1Senior author
1Corresponding author and lead contact: email address: [log in to unmask]
Current Biology, in press
Attractive and repulsive cell guidance is essential for animal life and
important in disease. Cell migration towards attractants dominates
studies [1-8] but migration away from repellents is important in biology,
yet relatively little studied [5, 9, 10]. It is widely held that cells initiate
migration by protrusion of their front [11-15], yet this has not been
explicitly tested for cell guidance because cell margin displacement at
opposite ends of the cell has not been distinguished for any cue. We
argue that protrusion of the front or retraction of the rear or both
together could in principle break cell symmetry and start migration in
response to guidance cues [16]. Here we find in the Dictyostelium
model [6] that an attractant - cAMP - breaks symmetry by causing
protrusion of the front of the cell, whereas its repellent analogue
- 8CPT - breaks symmetry by causing retraction of the rear. Protrusion
of the front of these cells in response to cAMP starts with local actin
filament assembly, whilst the delayed retraction of the rear is
independent of both myosin II polarisation and of motor-based
contractility. On the contrary, myosin II accumulates locally in the rear
of the cell in response to 8CPT, anticipating retraction and required
for it, whilst local actin assembly is delayed and couples to delayed
protrusion at the front. These data reveal an important new concept
in the understanding of cell guidance.
submitted by: Louise Cramer [[log in to unmask]]
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Reclassification of current Dictyostelium mucoroides strain Dm7
based on rRNA sequences and morphological features
Authors:
Kurato Mohri, Shin-ichi Kawakami, Seido Naganoa, Hideko
Urushihara
Mycoscience, in press
Recent classification and phylogenetic analyses of dictyostelids
are based on molecular sequences, but not all strains have been
examined using this approach. Since the original identification in
1869, several strains called Dictyostelium mucoroides have been
isolated; however, molecular taxonomic analyses of these strains
remain insufficient. We therefore attempted to verify classification
of one of these strains (Dm7) using rRNA sequences. As a result,
strain Dm7 was found to be phylogenetically closer to D. clavatum
and D. longosporum than to D. mucoroides. Comparisons of the
morphological features of these strains further supported the
classification based on sequence data, suggesting that Dm7
should be classified as a strain of D. clavatum.
submitted by: Kurato Mohri [[log in to unmask]]
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Xanthomonas citri T6SS mediates resistance to Dictyostelium predation
and is regulated by an ECF-sigma factor and cognate Ser/Thr kinase.
Ethel Bayer-Santos, Lidia dos Passos Lima, Lucas de Moraes Ceseti,
Camila Yuri Ratagami, Eliane Silva de Santana, Aline Maria da Silva,
Chuck Shaker Farah, Cristina Elisa Alvarez-Martinez.
Environmental Microbiology, in press
http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.14085/abstract
Plant-associated bacteria of the genus Xanthomonas cause disease in
a wide range of economically important crops. However, their ability to
persist in the environment is still poorly understood. Predation by
amoebas represent a major selective pressure to bacterial populations
in the environment. In this study, we show that the X. citri type 6
secretion system (T6SS) promotes resistance to predation by the soil
amoeba Dictyostelium discoideum. We found that an extracytoplasmic
function (ECF) sigma factor (EcfK) is required for induction of T6SS
genes during interaction with Dictyostelium. EcfK homologues are found
in several environmental bacteria in association with a gene encoding a
eukaryotic-like Ser/Thr kinase (pknS). Deletion of pknS causes
sensitivity to amoeba predation and abolishes induction of T6SS genes.
Phosphomimetic mutagenesis of EcfK identified a threonine residue
(T51) that renders EcfK constitutively active in standard culture
conditions. Moreover, susceptibility of delta-pknS to Dictyostelium
predation can be overcome by expression of the constitutively active
version EcfKT51E from a multicopy plasmid. Together, these results
describe a new regulatory cascade in which PknS functions through
activation of EcfK to promote T6SS expression. Our work reveals an
important aspect of Xanthomonas physiology that directly affects its
ability to persist in the environment.
submitted by: Ethel Bayer-Santos [[log in to unmask]]
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The saposin-like protein AplD displays pore-forming activity and
participates in defence against bacterial infection during a multicellular
stage of Dictyostelium discoideum
Ranjani Dhakshinamoorthy1+, Moritz Bitzhenner1, Pierre Cosson2,
Thierry Soldati3, and Matthias Leippe1*
1Zoological Institute, Comparative Immunobiology, University of Kiel,
Germany,
2Department of Cell Physiology and Metabolism, Faculty of Medicine,
University of Geneva, Switzerland,
3Department of Biochemistry, Faculty of Science, University of
Geneva, Switzerland
*Correspondence: Prof. Dr. Matthias Leippe
[log in to unmask]
+Present address: Department of Biotechnology, Bhupat and Jyoti Mehta
School of Biosciences, Indian Institute of Technology Madras, India
Front. Cell. Infect. Microbiol, in press
Due to their archaic life style and microbivor behavior, amoebae may
represent a source of antimicrobial peptides and proteins. The amoebic
protozoon Dictyostelium discoideum has been a model organism in cell
biology for decades and has recently also been used for research on host-
pathogen interactions and the evolution of innate immunity. In the genome
of D. discoideum, genes can be identified that potentially allow the synthesis
of a variety of antimicrobial proteins. However, at the protein level only very
few antimicrobial proteins have been characterized that may interact directly
with bacteria and help in fighting infection of D. discoideum with potential
pathogens. Here, we focus on a large group of gene products that
structurally belong to the saposin-like protein (SAPLIP) family and which
members we named provisionally Apls (amoebapore-like peptides) according
to their similarity to a comprehensivelystudied antimicrobial and cytotoxic
pore-forming protein of the protozoan parasite Entamoeba histolytica. We
focused on AplD because it is the only Apl gene that is reported to be
primarily transcribed during the multicellular stages such as the mobile slug
stage. Upon knock-out (KO) of the gene, aplD- slugs became highly
vulnerable to virulent Klebsiella pneumoniae (Kp). AplD- slugs harbored
bacterial clumps in their interior and were unable to slough off the pathogen
in their slime sheath. Re-expression of AplD in aplD- slugs rescued the
susceptibility towards K. pneumoniae. The purified recombinant protein
rAplD formed pores in liposomes and was also capable of permeabilizing
the membrane of live Bacillus megaterium. We propose that the multifarious
Apl family of D. discoideum comprises antimicrobial effector polypeptides
that are instrumental to interact with bacteria and their phospholipid
membranes. The variety of its members would allow a complementary and
synergistic action against a variety of microbes, which the amoeba
encounters in its environment.
submitted by: Matthias Leippe [[log in to unmask]]
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[End dictyNews, volume 44, number 7]
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