Bacillus ACT 2021 on Bacillus anthracis, cereus and thuringiensis April 26-28, 2021 Online - INRA
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Bacillus ACT 2021
9th International Conference
on Bacillus anthracis, cereus and thuringiensis
April 26-28, 2021
Online
Table of contents
CONTACTS 3
GUIDELINES FOR ATTENDING THE MEETING 4
SPONSORS 5
PROGRAM 7
EPIDEMIOLOGY, ECOLOGY AND ADAPTATION 7
PHYSIOLOGY AND DEVELOPMENT (1) 8
PHYSIOLOGY AND DEVELOPMENT (2) 8
HOST-PATHOGEN INTERACTIONS 9
SPORE PROPERTIES 10
GENOMIC, PHYLOGENY AND MOBILE ELEMENTS 10
TOXINS AND THERAPIES 11
NEXT BACILLUS ACT CONFERENCE 13
ABSTRACTS 15
SPEAKERS 52
2
Contacts
Steering committee
Tjakko Abee (Wageningen University, the Netherlands)
Rakesh Bhatnagar (Jawaharlal Nehru University, India)
Steven Blanke (Illinois University at Urbana-Champaign, USA)
Monika Ehling-Schulz (University of Veterinary Medicine Vienna, Austria)
Arthur M. Friedlander (USAMRIID, USA)
Michel Gohar (INRAE Jouy-en-Josas, France)
Jean-Nicolas Tournier (IRBA, France)
Organizing committee
Véronique Broussolle (INRAE Avignon, France)
Michel Gohar (INRAE Jouy-en-Josas, France)
Leyla Slamti (INRAE, Jouy-en-Josas, France)
Jean-Nicolas Tournier (IRBA, France)
Session chairs
Steve Blanke (University of Illinois at Urbana-Champaign, USA)
Frédéric Carlin (INRAE Avignon, France)
Theodor Chitlaru (Israel Institute for Biological Research, Israel)
Monika Ehling-Schulz (University of Veterinary Medicine Vienna, Austria)
Arthur M. Friedlander (USAMRIID, USA)
Annika Gillis (UCLouvain, Belgium)
Theresa Koehler (UTHealth, USA)
Didier Lereclus (INRAE Jouy-en-Josas, France)
Jacques Mahillon (UCLouvain, Belgium)
Anne Moir (University of Sheffield, UK)
Ole Andreas Økstad (University of Oslo, Norway)
Ben Raymond (University of Exeter, UK)
3
Guidelines for attending the meeting
The BACT 2021 conference will be held on the Zoom webinar platform. There is no need to
create a Zoom account or download any software. You can access the webinar by clicking on
the link that was sent to you a few days prior to the meeting. Please do not share this link and
do not post it online. You can check if the link is operational a few days in advance. Please let
us know if you have any issue with it at bacillusact2020@gmail.com.
For optimal experience, it is best to use a landline connection rather than Wi-Fi.
We have allocated 20 min per presentation: 15 min maximum for data sharing and the
remainder for discussion. For a smooth running of the conference, and out of respect for all
the participants, it is essential that the speakers respect this rule.
Questions should be written down in the chat panel during and after the presentation and will
be directed to the speaker by the session chairs at the end of the presentation.
We ask the participants not to record any presentation, as some data might be unpublished.
There will be 3 types of participants during the meeting:
- Hosts (organizing committee)
- Panelists (speakers and session chairs)
- Attendees (all other participants)
All participants will be Attendees by default when entering the webinar. At the beginning of
each session, the Hosts will change the status of the moderators and the speakers of the
session to Panelists. During the switch, the connection will be interrupted but will resume
within a few seconds. Microphones and cameras are operational for both Hosts and Panelists.
To preserve bandwidth, only the hosts, speakers and chairs of each session will turn their
cameras on.
Using a videoconference platform such as Zoom webinar should hopefully not pose too many
difficulties and many of us have been using these means of communication for a while now.
However, if you feel that you need more information, please visit the Zoom website
(https://zoom.us/).
We thank you for your support and for joining us for the BACT 2021 online conference.
We wish you a pleasant and scientifically rich meeting.
4
Sponsors
We thank our sponsors for their generous support
5We also thank the following people for their precious assistance
Ludovic Bridoux (INRAE, Jouy-en-Josas, France) for the website maintenance
Céline Gautier and the Micalis administrative staff (INRAE, Jouy-en-Josas, France) for the registration
and finance management
6Program
The schedule corresponds to the time in Paris, France.
Monday, April 26
14h00 – 14h10 Welcome address
14h10 – 14h30 Introduction
Arthur M. Friedlander, Senior scientist | United States Army Medical Research
Institute of Infectious Diseases, Frederick, MD and Department of Medicine,
Uniformed Services University of Health Sciences, Bethesda, MD, USA.
Epidemiology, ecology and adaptation
Ben Raymond (University of Exeter, UK) and Jacques Mahillon (UCLouvain,
Belgium)
14h30 – 14h50 Adaptation of Bacillus thuringiensis to plant colonisation affects differentiation
and toxicity #1
Yicen Lin, PhD student | Bacterial Interactions and Evolution Group, DTU
Bioengineering, Technical University of Denmark, Denmark
14h50 – 15h10 The fate of bacteria of the Bacillus cereus group in the amoeba environment #2
Haibo Chen, PhD student | Micalis Institute, INRAE, AgroParisTech, Université
Paris-Saclay, Jouy-en-Josas, France
15h10 – 15h30 Immunological evidence of host variation in exposure and resistance to anthrax
in Kruger and Etosha National Parks #3
Sunday Ochai, PhD student | Department of Veterinary Tropical Diseases, Faculty
of Veterinary Science, University of Pretoria, Onderstepoort, South Africa
15h30 – 15h50 Anthrax: A comprehensive review describing the clinical features of reported
hospitalized cases for all routes of infection published in the english literature,
1880-2018 #4
Katherine Hendricks, Senior scientist | Division of High Consequence Pathogens
and Pathology, Centers for Disease Control and Prevention, Atlanta GA, USA
15h50 – 16h10 Environmental and genetic factors affecting Bacillus anthracis spore
concentrations at anthrax carcass sites #5
Zoë R. Barandongo, PhD student | Department of Forest and Wildlife Ecology,
University of Wisconsin-Madison, Madison WI, USA
Break
7Physiology and development (1)
Monika Ehling-Schulz (University of Veterinary Medicine Vienna, Austria) and
Theresa Koehler (UTHealth, USA)
16h30 – 16h50 Bacillus anthracis’ S-layer is a cell envelope load-bearing component #6
Antonella Fioravanti, Post-doctoral fellow | Structural and Molecular
Microbiology, Structural Biology Research Center, VIB and Structural Biology
Brussels, Vrije Universiteit Brussel, Brussels, Belgium
16h50 – 17h10 A model for the early steps of SCWP assembly in the Bacillus cereus sensu lato
group #7
Anastasia Tomatsidou, Post-doctoral fellow | Howard Taylor Ricketts Laboratory,
Argonne National Laboratory, Lemont IL, USA
17h10 – 17h30 The putative different roles of tasA and calY in the biofilm formation of B. cereus
#8
Ana Álvarez-Mena, PhD student | Institute for Mediterranean and Subtropical
Horticulture "La Mayora", Spanish National Research Council–University of Malaga,
Spain
17h30 – 17h50 Motility and biofilm regulation in the B. cereus group – identification of the
conserved motility genes cdgL and mogR #9
Ole Andreas Økstad, Senior scientist | Section for Pharmacology and
Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo,
Norway
17h50 – 18h10 Role of materials types and positions on the Bacillus cereus [Bc-98/4] fouling and
its resistance to cleaning #10
Piyush Kumar Jha, Post-doctoral fellow | UMET, Univ. Lille, CNRS, INRAE, ENSCL,
Villeneuve d’Ascq, France
18h10 – 18h30 Towards the characterization of an alternative survival mode during infection in
Bacillus thuringiensis #11
Hasna Toukabri, PhD student | Micalis Institute, INRAE, AgroParisTech, Université
Paris-Saclay, Jouy-en-Josas, France
Tuesday, April 27
Physiology and development (2)
Monika Ehling-Schulz (University of Veterinary Medicine Vienna, Austria) and
Theresa Koehler (UTHealth, USA)
14h00 – 14h20 The activation of σP, an ECF σ factor that controls β-lactam resistance in Bacillus
thuringiensis, cereus, and anthracis #12
Kelsie Nauta, PhD student | Department of Microbiology and Immunology,
University of Iowa, Iowa City IA, USA
814h20 – 14h40 Identification of the extracytoplasmic function σ factor σP regulon in Bacillus
thuringiensis #13
Theresa D. Ho, Post-doctoral fellow | Department of Microbiology and
Immunology, Carver College of Medicine, University of Iowa, Iowa City IA, USA
14h40 – 15h00 Transcriptomic response to oxidative stress of Bacillus anthracis htrA-disrupted
and parental wild type strains #14
Galia Zaide, Senior scientist | Department of Biochemistry and Molecular Genetics,
Israel Institute for Biological Research, Ness-Ziona, Israel
Host-pathogen interactions
Didier Lereclus (INRAE Jouy-en-Josas, France) and Steve Blanke (University of
Illinois at Urbana-Champaign, USA)
15h00 – 15h20 Branched-chain amino acid metabolism and virulence in Bacillus anthracis #15
Soumita Dutta, Post-doctoral fellow | Department of Microbiology and Molecular
Genetics, McGovern Medical School, The University of Texas Health Science Center
at Houston, Houston TX, USA
15h20 – 15h40 Distinct contribution of the HtrA protease and PDZ domains to its function in
stress resilience and virulence of Bacillus anthracis #16
Ofer Cohen, Senior scientist | Department of Biochemistry and Molecular
Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
15h40 – 16h00 The sequence of the universal bacterial DNA repair protein Mfd dictates the
pathogenicity of Bacillus cereus strains #17
Delphine Cormontagne, PhD student | Micalis Institute, INRAE, AgroParisTech,
Université Paris-Saclay, Jouy-en-Josas, France
Break
16h20 – 16h40 AtxA-controlled small RNAs of Bacillus anthracis virulence plasmid pXO1 regulate
gene expression in trans #18
Ileana D. Corsi, PhD student | Department of Microbiology and Molecular
Genetics, McGovern Medical School, The University of Texas Health Science Center
at Houston and MD Anderson Cancer Center and UTHealth Graduate School of
Biomedical Sciences, The University of Texas, Houston TX, USA
16h40 – 17h00 Encapsulation with D isomer poly -g-glutamic acid, an especially poor TLR ligand,
inhibits human dendritic cell responses to Bacillus anthracis #19
Tanya M. Jelacic, Investigator | United States Army Medical Research Institute of
Infectious Diseases, Frederick MD, USA
9Spore properties
Anne Moir (University of Sheffield, UK) and Frédéric Carlin (INRAE Avignon,
France)
17h00 – 17h20 Enhanced germination of heat activated Bacillus anthracis spores persists over a
five-week period #20
Andrew Roser, PhD student | Louisiana Tech University, USA
17h20 – 17h40 Characterization of Bacillus anthracis spores lacking dipicolinate synthase #21
Chandler Hassan, PhD student | University of Nevada, Department of Chemistry
and Biochemistry, Las Vegas NV, USA
17h40 – 18h00 A novel pilus superfamily from the endospores of pathogenic Bacilli #22
Pradhan Brajabandhu, PhD student | Structural and Molecular Microbiology, VIB-
VUB Center for Structural Biology, VIB and Department of Bioengineering Sciences,
Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium
18h00 – 18h20 Measurement of interaction force between material and Bacillus spores #23
Anna Ipatova, PhD student | Univ. Lille, CNRS, UMR 8520, Lille, France
18h20 – 18h40 Foam Flow: An eco-efficient strategy for cleaning of contaminated industrial
equipment #24
Heni Dallagi, PhD student | UMET, Univ. Lille, CNRS, INRAE, ENSCL, Villeneuve
d’Ascq, France
Wednesday, April 28
Genomic, phylogeny and mobile elements
Annika Gillis (UCLouvain, Belgium) and Ole Andreas Økstad (University of Oslo,
Norway)
14h00 – 14h20 Dating points along the phylogeny of Bacillus anthracis #25
Gilles Vergnaud, Senior scientist | Université Paris-Saclay, France
14h20 – 14h40 Signatures of selection in core and accessory genomes suggest ecological
diversification between clades of Bacillus cereus sensu lato #26
Hugh White, PhD student | Department of Biosciences, University of Exeter,
Exeter, UK
14h40 – 15h00 A novel Bacillus thuringiensis strain: A highly dynamic plasmid environment #27
Nancy Fayad, Post-doctoral fellow | Laboratory of Biodiversity and Functional
Genomics, UR-EGP, Faculty of Science, Université Saint-Joseph de Beyrouth, Beirut,
Lebanon and Laboratory of Food and Environmental Microbiology, Earth and Life
Institute, UCLouvain, Louvain-la-Neuve, Belgium
1015h00 – 15h20 tip, the intriguing transfer locus of pXO16, a conjugative plasmid from Bacillus
thuringiensis serovar israelensis #28
Pauline Hinnekens, PhD student | Laboratory of Food and Environmental
Microbiology, Earth and Life Institute, UCLouvain, Louvain-la-Neuve, Belgium
15h20 – 15h40 Viral proteins involved in the adsorption process of phage Deep-Purple infecting
members of the Bacillus cereus group #29
Audrey Leprince, PhD student | Laboratory of Food and Environmental
Microbiology, Earth and Life Institute, UCLouvain, Louvain-la-Neuve, Belgium
Toxins and therapies
Arthur M. Friedlander (USAMRIID, USA) and Theodor Chitlaru (Israel Institute for
Biological Research, Israel)
15h40 – 16h00 New insights into the emetic toxin cereulide: Means of distribution, synergistic
actions and prevention strategies #30
Markus Kranzler, Post-doctoral fellow | Institute of Microbiology, Department of
Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria
16h00 – 16h20 Bacillus cereus growth and cereulide formation in different food matrices #31
Katia Rouzeau-Szynalski, Senior scientist | Food Safety Microbiology, Institute of
Food Safety and Analytical Sciences, Food Safety Research Department, Nestlé
Research, Lausanne, Switzerland
Break
16h40 – 17h00 Crystal structure and molecular docking studies of Bacillus cereus Haemolysin BL
lytic component, HblL1 #32
Lainey Williamson, PhD student | School of Biosciences, Cardiff University, Park
Place, Cardiff, UK
17h00 – 17h20 Entry and trafficking of Bacillus anthracis edema factor #33
Emilie Tessier, PhD student | Institut de Recherche Biomédicale des Armées,
Bretigny-sur-Orge, France
17h20 – 17h40 Clindamycin protects nonhuman primates against inhalational anthrax but does
not enhance reduction of circulating toxin levels when combined with
ciprofloxacin #34
Mary E. Wright, Senior scientist | Division of Clinical Research, National Institute
of Allergy and Infectious Diseases, Bethesda MD, USA
17h40 – 18h00 Treatment of experimental anthrax with pegylated circularly permuted capsule
depolymerase #35
Arthur M. Friedlander, Senior scientist | United States Army Medical Research
Institute of Infectious Diseases, Frederick MD and Department of Medicine,
Uniformed Services University of Health Sciences, Bethesda MD, USA
1118h00 – 18h20 The novel diagnostic approaches for B. anthracis in Georgia #36
Nino G. Vepkhvadze, Senior scientist | State Laboratory of Agriculture, Tbilisi,
Georgia
Closing remarks
12Next Bacillus ACT conference
The next Bacillus ACT conference is scheduled to take place in Paris April 24-28,
2022, if the sanitary conditions allow it.
The sessions are planned to be held at the Espace Saint Martin and the gala
dinner will be hosted at the Musée des Arts Forains.
We look forward to seeing you there!
Espace Saint Martin, Paris
13Musée des Arts Forains, Paris
14Abstracts 15
#1
Adaptation of Bacillus thuringiensis to plant colonisation affects differentiation and
toxicity
Yicen Lin1, Monica Alstrup1, Janet Ka Yan Pang1, Gergely Maróti2, Mériem Er-Rafik3,
Nicolas Tourasse4, Ole Andreas Økstad5, Ákos T. Kovács1
1
Bacterial Interactions and Evolution Group, DTU Bioengineering, Technical University of
Denmark, Denmark
2
Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of
Sciences, Hungary
3
National Centre for Nano Fabrication and Characterization, Technical University of
Denmark, Denmark
4
Univ. Bordeaux, CNRS, INSERM, ARNA, France
5
Centre for Integrative Microbial Evolution and Section for Pharmacology and
Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Norway
Although certain isolates from the Bacillus cereus group (Bacillus cereus sensu lato) are used
as plant biologicals, safety concerns remain due to pathogenic traits. For example, toxin
production might shift as an adaptive survival strategy in natural niches (the soil and plant
rhizosphere). Therefore, it is crucial to explore bacterial evolutionary adaptation to the
environment. Herein, we investigated Bacillus thuringiensis (Cry-) adaptation to the
colonisation of Arabidopsis thaliana roots, and monitored changes in cellular differentiation in
experimentally evolved isolates. Isolates from two populations displayed improved iterative
ecesis on roots, reduced biofilm formation on abiotic surfaces, diminished swimming, but
increased swarming, in addition to enhanced haemolysis and toxicity against insect larvae.
Molecular dissection and recreation of a causative mutation revealed the importance of a non-
sense mutation in the rho transcription terminator gene. Finally, transcriptome analysis revealed
how Rho impacts various B. thuringiensis genes involved in carbohydrate metabolism and
virulence. Our work suggests that evolved multicellular aggregates have a fitness advantage
over single cells when colonising plants, creating a trade-off between swimming and
multicellularity in evolved lineages, in addition to unrelated alterations in pathogenicity.
16#2
The fate of bacteria of the Bacillus cereus group in the amoeba environment
Haibo Chen, Emilie Verplaetse, Leyla Slamti, and Didier Lereclus
Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas,
France.
Spores of the Bacillus cereus group bacteria are commonly found in the soil but evidence
suggests that they are unable to grow in such a natural environment in the absence of nutrient
input. Amoebas have been reported to be an amplifier for several species of pathogenic bacteria
and their potential involvement to explain the large amount of B. thuringiensis and B. cereus
spores in soil has been frequently proposed. Here, we studied the fate of Bacillus and amoebas
when cultured together. We show that the virulence factors produced by B. thuringiensis and
B. cereus do not affect the amoeba Acanthamoeba castellanii, which, on the contrary, can
phagocytose and effectively digest vegetative Bacillus cells to grow and prevent the formation
of cysts. Bacterial spores can germinate in the amoeba environment and the vegetative cells can
then form chains or aggregates that appear to be less efficiently phagocyted by the amoeba. The
use of transcriptional fusions between fluorescent reporter genes and stationary phase- and
sporulation-specific promoters showed that the sporulation process occurs more efficiently in
the presence of amoebas than in their absence. Moreover, our results showed the amoeba
environment to promote spore germination and allow the bacteria to complete their
developmental cycle. Overall, this study reports that the amoeba-Bacillus interaction creates a
virtuous circle in which each protagonist helps the other to develop.
17#3
Immunological evidence of host variation in exposure and resistance to anthrax in
Kruger and Etosha National Parks
SO Ochai 1, WC Turner 2, H van Heerden 1
1 Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of
Pretoria, Onderstepoort, South Africa.
2 U.S. Geological Survey, Wisconsin Cooperative Wildlife Research Unit, Department of
Forest and Wildlife Ecology, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.
Anthrax has a global distribution and is enzootic in Kruger National Park (KNP) in South Africa
and Etosha National Park (ENP) in Namibia. These parks have similar savanna ecosystems and
many of the same potential host species, yet the main anthrax host in each park is only a minor
host species in the other. In KNP the main host species is greater kudu (Tragelaphus
strepsiceros) and in ENP the main host species is plains zebra (Equus quagga). We measured
the presence of anti-PA antibodies in zebra and kudu sampled from endemic and non-endemic
areas in each park using Anti-PA ELISA and their ability to neutralise anthrax lethal toxin using
the toxin neutralisation assay (TNA). It was found that kudus in KNP had higher anti-PA
antibody titres than kudus in ENP and a higher proportion of animals that were positive than
ENP (95% versus 40%, respectively). ENP zebras had higher titres and higher proportions of
positive animals than those of KNP (83% versus 63%, respectively). Animals in anthrax
endemic areas in KNP had higher titres than those in non-endemic areas, but this was not so in
ENP. It was seen that ENP kudus and KNP zebras showed better toxin neutralization than their
respective counterparts in each park. In summary, these results indicate that these species differ
in their exposure to and resistance toward B. anthracis between the two parks. These patterns
may be due to environmental differences between these systems and their interplay with host
behavior, leading to differences in exposure frequency and dose, which in turn lead to
immunological trade-offs between resistance versus tolerance to the anthrax lethal toxin.
18#4
Anthrax: A Comprehensive Review Describing the Clinical Features of Reported
Hospitalized Cases for All Routes of Infection Published in the English Literature, 1880-
2018
Katherine Hendricks, MD, MPH&TM1; Marissa K. Person, MSPH1; John S. Bradley, MD2;
Thitipong Mongkolrattanothai, MPH1; Nathaniel Hupert, MD, MPH3; Peter Eichacker, MD4;
Arthur M. Friedlander, MD5; William A. Bower, MD1
1. Division of High Consequence Pathogens and Pathology, Centers for Disease Control and
Prevention; Atlanta, United States of America
2. Division of Infectious Diseases, Rady Children’s Hospital San Diego and the University
of California San Diego School of Medicine; San Diego, United States of America
3. Departments of Population Health Sciences and of Medicine, Weill Cornell Medicine,
Cornell University and New York-Presbyterian Hospital; New York, United States
4. Department of Critical Care Medicine, Clinical Center, National Institutes of Health;
Bethesda, MD, United States of America
5. US Army Medical Research Institute of Infectious Diseases; Frederick, United States of
America
Background: Anthrax, a toxin-mediated zoonotic disease caused by Bacillus anthracis with a
worldwide distribution, has long been considered a potential biowarfare agent. Existing adult
antimicrobial postexposure prophylaxis and treatment recommendations are consensus based.
Methods: To inform an update of clinical guidelines, we completed a systematic review of the
English literature for clinical and demographic characteristics of adults and children
hospitalized with anthrax infection (cutaneous, inhalation, ingestion, injection, primary
meningitis) abstracted from published case reports, case series, and line lists in English during
1880-2018, assessing geographic distribution, exposure source, signs, symptoms, and
complications over the course of hospitalization and outcome by treatment.
Results: Data on 764 adults and 167 children hospitalized with anthrax met completeness
criteria for review; cutaneous anthrax was the most common form for both adults and children.
From 1880 through 1915, reported cases were mostly from Europe; from 1916-1950, most were
from North America; since then, most have come from Western and Central Asia. Since 1960,
adult mortality has ranged from 31% for cutaneous to 90% for primary meningitis. Overall
pediatric mortality was lower – at 22% for the entire time period. For adults, indirect – mostly
occupational – animal contact (e.g., woolmaking, brushmaking, tanning) accounted for half or
more of exposures until the most recent time period, and finished consumer goods accounted
for one quarter to one third of cases through 1950. Almost two-thirds (59%) of pediatric
exposures were direct exposures to ill animals. Most patients with inhalation anthrax developed
pleural effusions, over half with ingestion anthrax developed ascites, and a third with ingestion
anthrax had coagulopathies. Treatment improved survival for those with systemic symptoms,
from ~30% in those untreated to ~70% in those receiving antimicrobials with/without
antiserum/antitoxin.
Conclusions: This review provides an improved evidence base for both clinical care of
individual anthrax cases and public health planning for large-scale anthrax attacks.
19#5
Environmental and genetic factors affecting Bacillus anthracis spore concentrations at
anthrax carcass sites
Zoë R. Barandongo , Yen-Hua Huang , Amelie C. Dolfi , Spencer A. Bruce , and Wendy C.
1* 1 1 2
Turner 3
1
Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, Madison, WI
53706, USA
Department of biological Sciences, State University of New York, Albany, NY 12222, USA
2
U.S. Geological Survey, Wisconsin Cooperative Wildlife Research Unit, Department of
3
Forest and Wildlife Ecology, University of Wisconsin-Madison, Madison, WI 53706, USA
Anthrax is a lethal zoonotic disease posing significantly risk to herbivorous wildlife and
livestock. Upon host death, vegetative cells of Bacillus anthracis are released into the
environment and undergo sporulation. Spores of B. anthracis are highly resistance to adverse
temperatures, drought and ultra-violet radiation enabling them to persist in the soil reservoir for
years. Anthrax transmission requires host exposure to high dose of spores. Therefore,
environmental conditions or infection traits that facilitate higher initial spore concentrations
and higher rates of spore survival will increase the chances that strains present at a particular
carcass site are able to infect future host. This study builds off a long-term study of B. anthracis
spore concentrations in soils at natural plains zebra (Equus quagga) anthrax carcass sites in
Etosha National Park, Namibia. Here we investigate environmental and pathogen genetic
factors affecting initial spore concentrations and spore survival rates over a decade at 40 zebra
anthrax carcass sites. Specifically, we examine how spore concentrations are affected by i) soil
characteristics at carcass sites (e.g., pH, calcium content, percent organic matter or soil
composition); ii) seasonality and weather conditions at the time of host death; and iii) pathogen
genomic diversity. The biggest predictor of variation in spore concentrations was the season in
which the animal died. Zebras dying of anthrax in wet seasons—the peak season for anthrax in
this system—had several orders of magnitude higher spore concentrations in carcass site soils
than did zebras that died of anthrax in dry seasons. Soil characteristics and pathogen genetic
diversity had no significant effects on spore concentrations or survival rates. Future research is
needed to determine if the seasonal patterns in spore concentrations are driven by seasonal
variation in sporulation success or seasonal differences in host health and susceptibility,
affecting terminal cell concentrations.
20#6
Bacillus anthracis’ S-layer is a cell envelope load-bearing component
Antonella Fioravanti1,2, Marion Mathelie-Guinlet3, Yves Dufrêne3, Han Remaut1,2
1) Structural and Molecular Microbiology, Structural Biology Research Center, VIB,
Brussels, Belgium.
2) Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
3) Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-
5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.
As part of its immune evasion strategy Bacillus anthracis presents a dynamic and complex
composition of its cell envelope. In the vegetative bacteria the cell surface is covered by one of
two 2D-protein arrays known as the Sap or EA1 S-layer (surface layer), present during
exponential and stationary growth phase, respectively [1].
S-layers are commonly found as the outermost cell surface components of procaryotic cells [2,
3]. Recently we succeeded in exposing the disruption of S-layer integrity as a mechanism with
therapeutic potential in S-layer carrying pathogens [4]. Using anti-Sap Nanobodies (Nbs [5])
able to destroy Sap S-layer integrity, we could show that acute disruption of B. anthracis S-
layer integrity results in severe morphological cell surface defects and in attenuated growth.
Unlike the genetic knockout of sap, cells with acutely compromised Sap S-layer would wrinkle
and collapse.
In Gram-positive bacteria, the peptidoglycan sacculus is thought to form the major cell
envelope supporting structure, ensuring cell shape and withstanding turgor pressure. To
investigate the unexpected observations that the loss of Sap results in cell collapse, we used a
combination of Atomic Force Microscopy and light microscopy observation to probe the
contribution of B. anthracis S-layer to cell integrity. Our new experiments show that cells
presenting a compromised S-layer by means of Nbs insult or genetic knockout lose their
elasticity and stiffness. Furthermore, our experiments show that S-layer integrity is required to
maintain cell shape in presence of osmotic stresses. These results present compelling
experimental evidence to the hypothesis that the S-layer can serve as prokaryotic exoskeletons
and a major load-bearing component of bacterial cells.
1. Mignot, T., et al., Developmental switch of S-layer protein synthesis in Bacillus anthracis. Mol Microbiol, 2002.
43(6): p. 1615-27.
2. Sara, M. and U.B. Sleytr, S-Layer proteins. J Bacteriol, 2000. 182(4): p. 859-68.
3. Albers, S.V. and B.H. Meyer, The archaeal cell envelope. Nat Rev Microbiol, 2011. 9(6): p. 414-26.
4. Fioravanti, A., et al., Structure of S-layer protein Sap reveals a mechanism for therapeutic intervention in anthrax.
Nat Microbiol, 2019. 4(11): p. 1805-1814.
5. Muyldermans, S., Nanobodies: Natural Single-Domain Antibodies. Annual Review of Biochemistry, 2013. 82(1): p.
775-797.
21#7
A model for the early steps of SCWP assembly in the Bacillus cereus sensu lato group
Anastasia Tomatsidou1,2, Olaf Schneewind(†), Dominique Missiakas1,2
1
Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, IL 60439;
2
Department of Microbiology, University of Chicago, Chicago, IL 60637
†
Deceased 26 May 2019
Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall
polysaccharide (SCWP) that is essential for the retention of Surface (S)-layer and S-layer
homology (SLH) domain proteins. The SCWP is a conserved feature of the Bacillus cereus
sensu lato group of bacteria. In B. anthracis, genetic disruptions of the SCWP biosynthetic
pathway impair growth, cell division and virulence. Growth impairment can be explained by
the irreversible accumulation of biosynthetic intermediates of SCWP assembly. Cell division
and virulence defects result from the loss of S-layer associated proteins including murein
hydrolases that control the separation of daughter cells, and adhesins that promote B. anthracis
invasion of host cells. The SCWP of B. anthracis is comprised of trisaccharide repeat units
composed of one ManNAc and two GlcNAc residues with O3-α-Gal and O4-β-Gal
substitutions. Pyruvylation at O4, O6 of the distal ManNAc and acetylation at O3 of the
penultimate GlcNAc residue account for the retention of S-layer and SLH proteins in the
envelope. Our current model for SCWP assembly purports that trisaccharide repeat units are
assembled onto undecaprenol on the cis side of the plasma membrane, translocated, and
subsequently polymerized by WpaA and WpaB. Ultimately, polymerized units are transferred
onto peptidoglycan by LCP enzymes. Here, we describe the three enzymes that synthesize
repeat units and examine candidate genes for the cis to trans flipping of repeat units across the
plasma membrane.
22#8
The putative different roles of TasA and CalY in the biofilm formation of B. cereus
A. Álvarez-Mena , M. L. Antequera-Gómez , L. Díaz-Martínez , J. Caro-Astorga , O. P.
1 1 1 1
Kuipers , A. de Vicente , D. Romero
2 1 1
Institute for Mediterranean and Subtropical Horticulture "La Mayora", Spanish National
1
Research Council–University of Malaga, Bulevar Louis Pasteur 31 (Campus Universitario de
Teatinos), 29071, Málaga, Spain; Department of Molecular Genetics, Groningen
2
Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University
of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
alvarezmena@uma.es
Bacillus cereus is a human pathogen responsible of many food poisoning due to the ingestion
of contaminated vegetables or processed foods. The biofilm development and spore formation,
are essential stages in the survival and transmission of the bacteria and hence the production of
harmful toxins for the consumer. During the biofilm formation, a subpopulation is differentiated
to extracellular matrix producers, mainly composed by exopolysaccharides, proteins and
extracellular DNA. One of the most fascinating compounds of this matrix are the amyloid
proteins, that shows a high tendency to fibrillate and have a multifunctional role in the bacterial
physiology.
In earlier studies it was identified that B. cereus possess two orthologues genes to the tasA gene
in B. subtilis, initially described as essential in the assembly of amyloid fibers, and they were
named as tasA and calY respectively. Both genes are located in the same genomic region and
the deletion of each one leads to a different phenotype related with a deficient biofilm. In the
case of the tasA mutant the biofilm finally detaches from the well at 72 hours, and the calY
mutant shows a thinner ring phenotype in comparison with the wild type strain. Those
preliminary results could indicate that TasA and CalY have different roles in the biofilm
formation. The level of tasA and calY expression in the biofilm was higher than in planktonic
cells, and it could be differentiated three different subpopulations: both genes are expressed,
only calY or none of them. Amyloid proteins have been described as being involved in adhesion
and host-colonization. A study of the dynamic of tasA gene expression show that is influenced
by the type of vegetable surface.
The results mentioned above indicate that TasA and CalY might complementarily contribute to
biofilm formation.
This work was supported by grants from the National Plan I+D+I of the Ministerio de Ciencia
e Innovación (AGL2016-78662-R and PID2019-107724GB-I00) and by the European
Research Council Starting Grant (BacBio 637971). Ana Álvarez is supported by a PhD
fellowship (BES-2017-081275) from the FPI program of the same Ministry.
23#9
Motility and biofilm regulation in the B. cereus group – identification of the conserved
motility genes cdgL and mogR
Veronika Smith1, Toril Lindbäck2, Ida K. Hegna1, Sarah Finke1, Nicolas J. Tourasse1,
Christina Nielsen-LeRoux , Malin Josefsen1, Ida Kristine Bu Nilssen1, Bjørn Dalhus , Annette
3 4,5
Fagerlund1,6, Ole Andreas Økstad1
1
Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo,
Norway. 2Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life
Sciences, Oslo, Norway. 3INRAE, AgroParisTech, Micalis, Jouy-en-Josas, Paris-Saclay University, France.
4
Department of Medical Biochemistry, Institute of Clinical Medicine, University of Oslo, Postboks 4950
Nydalen, 0424 Oslo, Norway. 5Department of Microbiology, Division of Laboratory Medicine, Oslo University
Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway. 6Nofima, Norwegian Institute of Food, Fisheries and
Aquaculture Research, Ås, Norway.
The second messenger c-di-GMP was previously identified in Gram-negatives as a key
regulator of the switch from planktonic growth to biofilm formation. By searching B. cereus
group genomes, we previously identified ten conserved proteins (CdgA-J), putatively related to
c-di-GMP synthesis (DGCs) or breakdown (PDEs). Furthermore, motility, biofilm formation,
and/or virulence was affected by deletion and/or overexpression of cdg genes. Among the
putative DGCs and PDEs, only two were conserved in Bacillus subtilis, indicating that c-di-
GMP signaling is different in the two groups of bacteria. A novel B. thuringiensis 407 protein,
CdgL, carries a degenerate nucleotide cyclase domain, and cdgL deletion rendered the
bacterium severely reduced in flagellar gene expression, resulting in non-flagellated, non-
motile cells. cdgL deletion also caused a delay in biofilm pellicle formation, which was also
observed by flagellin gene deletion. cdgL was found to reside in a three-gene operon, also
encoding a putative glycosyl transferase and a NupC family transporter, and which is conserved
throughout the B. cereus group with the exception of non-motile B. pseudomycoides. Also, a
newly identified putative transcriptional regulator MogR, is present only in Listeria spp and B.
cereus group bacteria, and was found to affect flagellar gene expression in B. thuringiensis 407.
By global transcriptional profiling B. thuringiensis MogR was also found to affect the
expression of genes related to virulence and biofilm formation, and EMSA analyses suggested
direct repression of flagellar genes. Increased biofilm formation and attenuation of virulence
upon MogR overexpression was confirmed by phenotypic assays including toxicity to Galleria
mellonella larvae, and influence of MogR on biofilm formation and cytotoxicity was found to
be independent of the loss of flagella. LC-MS/MS analyses and microarray experiments
suggested increased total cellular levels of c-di-GMP and higher sinI transcriptional levels,
respectively. Interestingly, mogR was found to be conserved also in non-motile B. cereus group
species such as B. mycoides and B. pseudomycoides, and is expressed in non-motile B.
anthracis. Altogether this provides indications of an expanded set of functions for MogR in B.
cereus group species, beyond motility regulation. Altogether this work further develops the
knowledge of the coordinated regulation between motility, virulence and biofilm formation in
B. cereus group bacteria.
24#10
Role of materials types and positions on the Bacillus cereus [Bc-98/4] fouling and its
resistance to cleaning
Piyush Kumar JHA , Heni DALLAGI*, Elodie Richard**, Thierry BENEZECH*, Christine
*
FAILLE *
*
UMET, Univ. Lille, CNRS, INRAE, ENSCL, 369, Rue Jules Guesde, F-59650 Villeneuve
d’Ascq CEDEX, France
Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, US 41, UMS 2014,
**
PLBS, F-59000, Lille, France
Bacteria can adhere and proliferate on any surface especially in agri-food environments,
producing biofilms that are sometimes very difficult to control. Along with bacterial properties,
environmental factors including material type and surface properties, as well as location within
processing lines, determine the fouling capability of bacteria. However, the role of the vertical
vs horizontal position on fouling and further resistance to cleaning has been the subject of little
research.
In this study, we investigated the role of materials on biofilm properties of Bacillus cereus 98/4,
in vertical and horizontal positions. Four materials with very different topographic and
hydrophilic/hydrophobic properties were tested: stainless steels with 2R (smooth) and 2B
(rough) finishes, glass (hydrophilic) and PP (hydrophobic).
We first analyzed the amount and structure of the 24-h biofilms. For this purpose, biofilms were
produced on coupons vertically or horizontally immersed in a suspension of 10 cells/ml in 1/10
7
TSB (tryptone soy broth). The amount of cultivable cells within biofilms was evaluated by the
plate count method. The covered surface and the organization of the biofilms were analyzed
through microscopic observations (epifluorescence and confocal laser scanning microscopes)
after staining biofilms with orange acridine. The material type only played a minor role in
biofilm formation, while some role of positions was seen on the contamination level. Actually,
vertically immersed PP coupons were contaminated by 1 log more than horizontally immersed
PP coupons, while only a few differences were seen for the other materials. Whatever the
materials and the vertical/horizontal position, biofilms were formed of cell clusters separated
with poorly contaminated areas.
We then investigated the ease of detachment of the different biofilms, using a pilot-scale
cleaning test rig, and a standard rinsing method (5 Pa for 20 min at 30±2 °C). Contrary to what
was observed for biofilm formation, the resistance of biofilms to detachment is clearly affected
by the material, the lowest log reduction being seen for PP surfaces (log reduction for PP
surfaces were under 1 log, while for other materials it was more than 1.5 log). Conversely, no
effect of the vertical/horizontal position could be evidenced in this study.
In conclusion, positions to some extent determined the contamination level, meanwhile,
material type impacted the detachment level.
25#11
Towards the characterization of an alternative survival mode
during infection in Bacillus thuringiensis
Hasna Toukabri, Didier Lereclus and Leyla Slamti
Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350, Jouy-en-Josas,
France
Bacteria of the Bacillus cereus group are able to complete a full developmental cycle in their
host. Using the honeycomb moth, Galleria mellonella (Gm), as an infection model, it was
shown that these bacteria were able to kill their host, to survive in its cadaver via necrotrophism
and to form spores by sequentially activating virulence, necrotrophism (Nec) and sporulation
(Spo) genes. The differentiation course of a Bacillus thuringiensis (Bt) population was
determined by monitoring necrotrophism and sporulation gene expression at the single-cell
level with transcriptional fluorescent reporters. These studies showed that only a part of the
population became Nec+ and among this subpopulation some bacteria differentiated into spores.
A subpopulation composed of cells that did not express any of the fluorescent reporters
remained uncharacterized and did not appear to be in exponential or stationary phase, but the
majority was identified as viable using a cell death marker. All these results suggest that a
subpopulation of cells in an unknown physiological state is able to survive in the host cadaver
without resorting to sporulation which is a costly, complex and irreversible process. To
precisely identify the bacterial cells of interest during infection for subsequent analysis, a
reporter strain harboring optimized and unstable fluorescent reporters was constructed. We
show that the Nec-/Spo- cells accounts for at least 50% of the population in the insect cadaver.
We then examined the metabolic activity of the Nec- cells during infection by assaying their
ability to synthesize proteins using genetic tools. Our data indicate that there is a metabolic
change leading to a potential quiescence in this subpopulation over time. Furthermore,
microscopic observations highlight a strong modification of the bacterial shape from rods
towards coccoid forms. More metabolic activity assays are being performed on this
subpopulation. In order to characterize the factors involved in this physiological state which
has never been described in sporulating Firmicutes during host infection, we started a global
RNA-Seq approach on the subpopulation of cells of interest. This study will help understand
the adaptation, survival and persistence of bacteria in their environment and especially for
B. cereus, that appears today as an important emerging pathogen.
26#12
The activation of σP, an ECF σ factor that controls β-lactam resistance in Bacillus
thuringiensis, cereus, and anthracis
Kelsie Nauta*, Theresa Ho, and Craig Ellermeier
Department of Microbiology and Immunology, University of Iowa, Iowa City, IA; *kelsie-
nauta@uiowa.edu
Antibiotic resistance has reduced the efficacy of antibiotics, resulting in untreatable infections.
Expression of resistance genes can be controlled by extracytoplasmic function (ECF) σ factors;
which are alternative transcription factors found exclusively in bacteria that activate
transcription in response to extracellular stresses. Although ECF σ factors are a diverse and
important group of transcription factors, they remain poorly understood. σP is an ECF σ factor
that controls β-lactamase expression in Bacillus cereus, B. thuringiensis, and some strains of B.
anthracis, resulting in resistance to certain β- lactams. In the absence of these β-lactams, σP is
sequestered by RsiP, the membrane- spanning anti-σ factor that inhibits σP activity. In the
presence of σP-inducing β-lactams, RsiP is proteolytically degraded. This process is generally
accomplished by sequential site-1 and site-2 proteases. We have identified signal peptidase as
the site-1 protease that cleaves RsiP in the extracellular domain. We also showed the conserved
site-2 protease, RasP, is required for cleavage of RsiP at site-2. We identified a penicillin
binding protein (PBP) that is required for site-1 cleavage of RsiP. We find it binds both β-
lactams that activate and β-lactams that don’t activate σP. We hypothesize the PBP binds β-
lactams and this PBP-β-lactam complex is required for a protein-protein interaction that results
in σP activation. The goal of this work is to define a novel mechanism for the control of β-
lactam resistance.
27#13
Identification of the Extracytoplasmic Function σ factor σP regulon in Bacillus
thuringiensis
Theresa D. Ho1, Kelsie Nauta1 & Craig D. Ellermeier 1,2
1
Department of Microbiology and Immunology
Carver College of Medicine
University of Iowa
431 Newton Rd
Iowa City, IA 52242
2
Graduate Program in Genetics,
University of Iowa,
Iowa City, IA 52242, USA
Bacillus thuringiensis and other members of the B. cereus family are resistant to many beta-
lactams. Resistance is dependent upon the Extra Cytoplasmic Function sigma factor SigP. We
used shotgun proteomics to identify proteins whose expression was induced by SigP. We
compared the protein profiles of strains which either lacked SigP or over-expressed SigP and
we identified 8 members of the SigP regulon which included four beta-lactamases as well as
three Penicillin Binding Proteins (PBPs). Using transcriptional reporters, we confirmed that
these genes are induced by beta-lactams in a SigP-dependent manner. Each of these genes were
deleted individually or in various combinations to determine their role in resistance to a subset
of beta-lactams, ampicillin, methicillin, cephalexin and cephalothin. We find that different beta-
lactamases and PBPs are differentially involved in resistance to different beta-lactams. Our data
show that B. thuringiensis utilizes a suite of enzymes to protect itself from beta-lactam
antibiotics.
28#14
Transcriptomic response to oxidative stress of Bacillus anthracis htrA-disrupted and
parental wild type strains
Galia Zaide, Uri Elia, Inbar Cohen-Gihon, Ma'ayan Israeli, Shahar Rotem, Ofir Israeli,
Sharon Ehrlich, Hila Cohen, Shirley Lazar, Adi Beth-Din, Anat Zvi, Ofer Cohen and Theodor
Chitlaru
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research,
Ness-Ziona 74100, Israel
HtrA (High Temperature Requirement A) protease/chaperone is active in the quality control of
protein-synthesis. High throughput genomic/proteomic/serologic surveys of B. anthracis
showed that HtrA is an exposed immunogenic putative-vaccine candidate. B. anthracis HtrA
disruption resulted in a dramatic attenuation in the guinea pig, murine and rabbit models of
anthrax, and therefore an htrA-deleted Sterne-strain served for the development of an
efficacious and safe next-generation live attenuated anthrax spore-vaccine. The phenotype
associated with disruption of the htrA gene established that HtrA is necessary for tolerance to
various stress stimuli and for modulation of several bacterial proteins potentially involved in
the stress response. The htrA-disrupted bacteria exhibited sensitivity to oxidative stress
correlating with a delayed multiplication in macrophages. Here we report a comparative RNA-
seq transcriptomic study generating a database of differentially expressed genes in the B.
anthracis htrA-disrupted and wild-type parental strains under oxidative stress. The study
demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role
influencing expression of more than 1000 genes under stress. Functional analysis of groups or
individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation
in the DhtrA and upregulation in the WT strains of various transcriptional regulators, (ii)
downregulation of translation processes in the WT strain, (iii) downregulation of metal ion
binding functions and upregulation of sporulation-associated functions in the DhtrA strain.
Fifteen genes uniquely upregulated in the wild-type strain were further interrogated for their
modulation in response to other stress regimens.
Reference: Zaide, Elia, Cohen-Gihon, Israeli, Rotem, Israeli, Ehrlich, Cohen, Lazar, Beth-Din,
Shafferman, Zvi, Cohen, Chitlaru. Comparative Analysis of the Global Transcriptomic
Response to Oxidative Stress of Bacillus anthracis htrA-Disrupted and Parental Wild Type
Strains. Microorganisms. 2020, E1896.
29#15
Branched-Chain Amino Acid Metabolism and Virulence in Bacillus anthracis
Soumita Dutta1, Ileana D. Corsi1, 2 and Theresa M. Koehler1, 2
1
Department of Microbiology and Molecular Genetics, McGovern Medical School, The
University of Texas Health Science Center at Houston, Houston, TX, United States
2
MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, The
University of Texas, Houston, TX, United States
Bacillus anthracis exhibits robust proliferation in diverse niches of mammalian hosts. Infection
can result in up to 108 CFU/g of tissue at time of death of the animal. The metabolic attributes
of B. anthracis that permit rapid proliferation to high numbers in multiple mammalian tissues
have not been established. We posit that branched-chain amino acid (BCAA: I, L, and V)
metabolism is key to B. anthracis pathogenesis. B. anthracis carries an unusually large number
of genes predicted to be involved in BCAA biosynthesis and import. Two partially paralogous
operons are associated with BCAA biosynthesis. Six genes are predicted to encode BCAA
transporters (permeases) of the cation symporter family and additional genes are predicted to
encode at least two BCAA-associated ABC transporters. Previous studies have demonstrated
that expression of BCAA-related genes is altered during growth of B. anthracis in bovine blood
in vitro, and the bacterium exhibits valine-auxotrophy during growth in a medium that mimics
blood serum. In addition, our recent RNA-seq data show that transcript levels of many BCAA-
related genes are controlled by AtxA, the transcription regulator that positively affects
expression of the anthrax toxin and capsule genes. We found that B. anthracis growth in defined
media was severely restricted in the absence of exogenously added BCAAs, suggesting even
when BCAA biosynthesis genes are expressed, BCAA transport is required for optimal growth
in vitro. We created mutants deleted for one or multiple BCAA metabolism genes and examined
the null-mutants for (1) growth in defined media, (2) the ability to import BCAAs, and (3)
virulence in a murine model for anthrax. Here we report data indicating that the predicted
BCAA transporter gene brnQ3 is required for optimal isoleucine and valine transport. A brnQ3-
null mutant showed a growth defect in media containing limited concentrations of BCAAs.
Moreover, the mutant was highly attenuated in a murine model for anthrax and tissues of
surviving animals showed no B. anthracis CFU. Interestingly, full B. anthracis virulence also
requires BCAA biosynthesis. An ilvD-null mutant lacking dihydroxy-acid dehydratase, an
enzyme essential for BCAAs synthesis, exhibited unperturbed growth when cultured in media
containing BCAAs, but was highly attenuated for virulence in the murine model. Our findings
suggest that BCAA availability is a critical factor for B. anthracis dissemination and
proliferation in host tissues.
30#16
Distinct Contribution of the HtrA Protease and PDZ Domains to its Function in Stress
Resilience and Virulence of Bacillus anthracis
Ofer Cohen, Ma'ayan Israeli, Uri Elia, Shahar Rotem, Hila Cohen, Avital Tidhar, Adi
Bercovich-Kinori, Theodor Chitlaru
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research,
Ness-Ziona, Israel
The chaperone/protease HtrA (High Temperature Requirement A of B. anthracis) is an
important pathogenicity determinant of B. anthracis. Consequently, disruption of the htrA gene,
results in significant virulence attenuation, despite unaffected ability of DhtrA strains to
synthesize the exotoxins. B. anthracis DhtrA strains exhibited increased sensitivity to stress
regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA)
and down-modulation of the bacterial S-layer. The virulence attenuation associated with
disruption of the htrA gene was suggested to reflect the susceptibility of DhtrA mutated strains
to stress insults encountered in the host. As all HtrA serine proteases, HtrA exhibits a protease
catalytic domain and a PDZ domain. We interrogated the relative impact of the proteolytic
activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the
chaperone activity and/or interaction with substrates) on manifestation of phenotypic
characteristics mediated by HtrA. By inspecting the phenotype exhibited by DhtrA strains
trans-complemented with either a wild-type, truncated (DPDZ), or non-proteolytic form
(mutated in the catalytic serine residue) of HtrA, as well as strains exhibiting modified
chromosomal alleles, it is shown that: (i) the proteolytic activity of HtrA is essential for its N-
terminal autolysis and subsequent release into the extracellular milieu, while the PDZ domain
was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of
the functions related to stress resilience as well as involvement of HtrA in assembly of the
bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to
be dispensable for the role of HtrA in mediating up-regulation of the extracellular protease
NprA under starvation stress, and (iv) in a murine model of anthrax, the HtrA PDZ domain,
was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of
the PDZ domain may represent a unique characteristic of B. anthracis HtrA amongst bacterial
serine proteases of the HtrA family.
31#17
The sequence of the universal bacterial DNA repair protein Mfd dictates the
pathogenicity of Bacillus cereus strains
Delphine Cormontagne1, Seav-Ly Tran1, Samantha Samson2, Solène Albert1,2, Gwenaëlle
André-Leroux2 and Nalini Ramarao1
1
Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-
Josas, France.
2
Maiage, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
The bacterial protein Mfd (mutation frequency decline) is highly conserved among bacteria and
has been shown to be involved in bacterial DNA repair. We have recently shown that this
protein confers bacterial resistance to the host nitrogen response produced during an infection
by the immune system. Indeed, Mfd helps preserving DNA integrity and is involved in the
repair following NO-induced DNA damage. Using a large collection of Bacillus cereus strains
with various pathogenicity, we could further establish a link between Mfd sequences, protein
3D structures and virulence. Protein sequence comparison and 3D modelling were used to
determine specific domains involved in the virulence of B. cereus. Using an in vivo insect model
of infection, we show that the mfd gene of a pathogenic strain could complement the avirulent
phenotype of a non-pathogenic strain. These data strongly suggest that the 3D structure of Mfd
plays an essential role in its function and might be a new and interesting way to discriminate
pathogenic from harmless B. cereus strains.
As Mfd is widely conserved within bacteria, those findings could improve our understanding
on the pathogenicity of potentially a large spectrum of bacteria.
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