Abstract

1. Development and validation of insect assays for high-throughput analysis of Francisellaand Burkholderiaspp virulence factors: Yet another way to get rid of cockroaches! Ben Martin1and Nathan Fisher1* 1North Dakota State University, Veterinary and Microbiological Sciences, Fargo, ND, USA Abstract 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 Francisellaand Burkholderiaspecies 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 Francisellaspp and Burkholderiaspp 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. Methods Insect rearing Galleria mellonella larvae were raised in 23 liter clear plastic containers at a constant temperature of 32°C 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 32°C 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 37°C in the dark. The following morning, a single colony was suspended in sterile PBS prior to serial 1:10 dilution in sterile PBS. 20μl 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 37°C 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 tips—an 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. Results & Discussion The 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. ΔFTL_1306 and ΔFTL_1042 are control strains that are not attenuated. • B. thailandensisis virulent toward both B. dubia and G. mellonella. Mutation to T6SS-1is known to attenuate B. pseudomalleiin 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. • Rationale • Simple, 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 by • Inconsistencies in experimental results between shipments, and • Difficulties 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 Burkholderiapseudomallei, B. mallei, and B. thailandensis. • In this study, we sought • To test a smaller, more docile species of roach (Blaptica dubia), • To expand the system for analysis of Francisellatularensis LVS, and • To conduct a side-by-side comparison between the wax moth larvae and roach larvae virulence assays. • 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. tularensisvirulence was indistinguishable in the two assays. • B. thailandensisvirulence 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 Francisellatularensis& Burkholderiapseudomallei. 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 References Contact *corresponding author: nathan.fisher@ndsu.edu Nathan Fisher, PhD, MBA Infectious Disease & Public Health Veterinary and Microbiological Sciences Genomics & Bioinformatics Graduate Program We would like to specifically thank Dr. Jason Huntley at the University of Toledo for providing Francisellatularensis 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 Burkholderiathailandensis two-allele library. 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 Francisellatularensis& Burkholderiapseudomallei . He is continuing some of that molecular pathogenesis work at NDSU using appropriate BSL-1 and BSL-2 surrogate This work was funded by a start-up grant provided to Dr. Fisher by the NDSU Agricultural Research Station