PowerPoint Presentation
Development and validation of insect assays for high-throughput
analysis of Francisella and Burkholderia spp virulence factors: Yet
another way to get rid of cockroaches!
Ben Martin1 and Nathan Fisher1*1North Dakota State University,
Veterinary and Microbiological Sciences, Fargo, ND, USAAbstract
Invading pathogens first interact with the innate immune system
of the host, which presents significant barriers to survival and
growth of microorganisms. Characterizing interactions between the
pathogen and the innate immune system is vital to the ultimate
goals of disease prevention and treatment. Saturating transposon
mutagenesis has recently been achieved in several Francisella and
Burkholderia species and the resulting libraries can be used as a
genetic basis for the evaluation of interactions between these
bacteria and the innate immune system. Unfortunately, small mammal
host model systems do not offer adequate throughput for individual
interrogation of mutant strains. Various attempts have been made to
analyze mutant strains pooled together, but an efficient mechanism
by which to individually test a large number (thousands) of strains
is preferred. Fortunately, this level of throughput is possible
with insect assays. Since the innate immune systems of insects are
strikingly robust and highly similar to those of mammals, their use
to study this interaction has been described for many human
pathogens. Here we describe the development and validation of two
assays for determining resistance to innate immune-mediated killing
of Francisella spp and Burkholderia spp isolates. The first assay
is based on the popular alternative host G. mellonella, while the
second is based on the orange-spotted cockroach, Blaptica dubia.
The new assay based on B. dubia solves significant logistical
difficulties associated with the care and maintenance of G.
mellonella larvae and is also well suited for use by undergraduate
researchers.
RationaleSimple, high-throughput virulence screening assays are
needed to keep pace with modern functional genomic
technologies.Because they contain an intact innate immune system
that is structurally and functionally very similar to that of
mammals, insects have become popular in medium- to high-throughput
virulence assays.In early studies with the commonly used host,
Galleria mellonella (wax moth larvae), we were frustrated
byInconsistencies in experimental results between shipments,
andDifficulties in rearing G. mellonella larvae in the
laboratory.In previous studies (Fisher, et al, 2012), we
demonstrated that the Madagascar Hissing Cockroach could serve as a
virulence assay host for Burkholderia pseudomallei, B. mallei, and
B. thailandensis.
In this study, we soughtTo test a smaller, more docile species
of roach (Blaptica dubia),To expand the system for analysis of
Francisella tularensis LVS, andTo conduct a side-by-side comparison
between the wax moth larvae and roach larvae virulence assays.
Methods
Insect rearing
Galleria mellonella larvae were raised in 23 liter clear plastic
containers at a constant temperature of 32C in the dark. The growth
media was composed of Gerber whole wheat cereal, wheat bran, sugar,
glycerin, water, and amphotecerin B (antifungal). Late instar
larvae were selected for infection based on lack of mellonization
or discoloration to ensure subjects were healthy. Pupae are
harvested and transferred to a mating chamber. As the adult moths
emerged, mating occurs and eggs laid on folded wax paper are
collected for seeding new colonies. Adult female moths produce
around 400-600 eggs, allowing for multiple colonies to be started
from only a few moths, alleviating the need to have a constant
commercial supply.
Blaptica dubia were ordered from BuyDubiaRoaches.com and were
stored at 32C in the dark in 32 liter plastic containers. They were
fed on a diet of fresh vegetables and dry dog food, which was
changed daily. Adult roaches were collected for future rearing,
which is a current focus of the lab. 1 to 1.5 larvae were used in
all studies to date.
Insect infections
Bacteria were grown overnight on either LB (B. thailandensis) or
CHOC (F. tularensis LVS) agar plates incubated at 37C in the dark.
The following morning, a single colony was suspended in sterile PBS
prior to serial 1:10 dilution in sterile PBS. 20l aliquots of each
dilution were injected into groups of 10 G. mellonella or B. dubia
larvae using an 31-gauge insulin syringe loaded into a Dymax
Stepper repetitive dispensing tool. G. mellonella were injected
subcutaneously in the dorsal posterior quadrant, immediately to the
right or left of the midline. B. dubia were injected subcutaneously
on the ventral posterior quadrant, between the 2nd and 3rd segments
on the left side of the midline (red arrow in images above).
Infected larvae were housed in plastic petri dishes and were held
at 37C in the dark. Survival was monitored and recorded daily for
one week. LD50s were determined by the method of Reed & Muench
(ref).
All infections shown here were performed with syringes. However,
subsequent experiments showed no difference in lethality or time to
death for B. dubia infections using sharpened P10XL plastic pipette
tipsan optimization that significantly decreases the time required
to perform these studies, while significantly increasing student
safety. This form of inoculation leads to high numbers of control
group deaths in the G. mellonella assay, presumably due to damage
to the soft cuticle. References
Contact*corresponding author:[email protected]
Fisher, PhD, MBAInfectious Disease & Public HealthVeterinary
and Microbiological SciencesGenomics & Bioinformatics Graduate
Program Results & Discussion
Conclusions
The B. dubia insect virulence screening assay is superior to the
commonly used G. mellonella assay for analysis of the
intracellular, Gram-negative pathogens F. tularensis LVS and B.
thailandensis.B. dubia avoids technical pitfalls inherent to the G.
mellonella assay: inconsistency in larvae health between shipments,
difficulty rearing the insects in the lab, and the requirement for
short-duration studies due to larvae pupation. B. dubia can be
inoculated with a sharpened P10XL plastic pipette tip, increasing
the safety of the operation.F. tularensis virulence was
indistinguishable in the two assays.B. thailandensis virulence
toward B. dubia more closely resembled that seen in mammals.
The B. dubia insect virulence assay can easily be used for
high-throughput screening and functional genomic identification of
virulence factors.
About the Fisher Lab
Dr. Fisher relocated to NDSU in the fall of 2012 after a
successful early career with the US Army and Department of Homeland
Security where he used functional genomics to characterize
pathogenic mechanisms utilized by Francisella tularensis &
Burkholderia pseudomallei. He is continuing some of that molecular
pathogenesis work at NDSU using appropriate BSL-1 and BSL-2
surrogate models, as described here. In addition, the lab has
exciting projects to characterize the virulence of important
nosocomial species of Burkholderia (members of the Bcc complex) and
the emering pathogen Stenotrophomonas maltophilia. (Please see our
other poster on that topic). We are happy to collaborate with any
investigators that may wish to test the virulence of additional
pathogens in the B. dubia assay. Finally, we are actively seeking
enthusiastic graduate students and undergraduate students
interested in summer research experiences. Contact Dr. Fisher
directly, if interested.Acknowledgements
We would like to specifically thank Dr. Jason Huntley at the
University of Toledo for providing Francisella tularensis LVS and
the mutants derived from that strain. We would also like to thank
the Manoil Lab at the University of Washington for making available
the Burkholderia thailandensis two-allele library. This work was
funded by a start-up grant provided to Dr. Fisher by the NDSU
Agricultural Research Station Dr. Fisher relocated to NDSU in the
fall of 2012 after a successful early career with the US Army and
Department of Homeland Security where he used functional genomics
to characterize pathogenic mechanisms utilized by Francisella
tularensis & Burkholderia pseudomallei. He is continuing some
of that molecular pathogenesis work at NDSU using appropriate BSL-1
and BSL-2 surrogate
StrainLD50: B. dubiaLD50: G. mellonellaF. tul LVS1.1 x 104
cfu4.4 x 103 cfuF. tul LVS dsbA>1.7 x 106 cfu2.7 x 106 cfuF. tul
LVS dipA2.6 x 105 cfuNDF. tul LVS FTL_13066.6 x 104 cfuNDF. tul LVS
FTL_10424.3 x 104 cfuNDB. thai E2641.7 x 102 cfu 1.5 cfuB. thai
E264 T6SS-1::TN3.9 x 103 cfu *1.2 cfuB. thai E264 T6SS-2::TN1.6 x
102 cfuNDB. thai E264 clpX::TN4.3 x 102 cfu *NDThe table to the
right gives the observed LD50 for each mutant in the B. dubia and
G. mellonella assays. F. tularensis LVS allelic exchange mutants
were kindly provided by Dr. Jason Huntley at the University of
Toledo. B. thailandensis transposon mutants were provided by the
Manoil lab at the University of Washington. Red boxes contain
parental isolates and mutants known to be attenuated in mice.ND=Not
done. *average of LD50 from two independent TN mutants in the same
locus. Stdev of 663 cfu and 328 cfu, respectively.
F. tularensis LVS is virulent toward B. dubia and the pattern of
attenuation of specific mutants mirrors that observed in mammals.
The dsbA mutant has been shown to be attenuated in mice (ref) and
the dipA mutant has been shown to be defective in intracellular
replication in tissue culture (ref). Both mutants are attenuated in
B. dubia. FTL_1306 and FTL_1042 are control strains that are not
attenuated.
B. thailandensis is virulent toward both B. dubia and G.
mellonella. Mutation to T6SS-1is known to attenuate B. pseudomallei
in hamsters and B. thailandensis in Madagascar hissing cockroaches
(Fisher, et al, 2012). This mutant is attenuated, albeit only
slightly, in the B. dubia assay, but remains fully virulent in the
G. mellonella assay. Moreover, the LD50 differs between the B.
dubia and G. mellonella assays by 2 logs, with the higher virulence
in B. dubia being more reminiscent of the behavior of B.
thailandensis in mammals. T6SS-2::TN is included as a control known
not to be involved in virulence.
