Reciprocal analysis of Francisella novicida infections of a Drosophila melanogaster model reveal host-pathogen conflicts mediated by reactive oxygen and imd-regulated innate immune response

Abstract

The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response. Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has evolved to counteract immunity reveal a host's immune mechanisms. In this study, we examined the pathogen Francisella novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress. We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two branches of the fly's immunity are important for fighting F. novicida infections in a model host: melanization and an imd-regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be readily explained by its interactions with known immune responses.

Figure 1. Francisella novicida is capable of infecting Drosophila.

Wild-type flies were injected with F. tularensis novicida U112 and survival and growth were monitored over the course of the infection. (A) Survival of wild-type flies following injection of 10³ CFUs of F. novicida. Median-time-to death (MTD) is approximately five days post infection when incubated at 29°C. Log-rank analysis of the Kaplan-Meyer survival curves showed statistical significance with a P value of <0.0001. Figure S1 provides variance data for these and other survival curves. (B) Growth of F. novicida in wild-type flies. Injection of a range of initial doses between 5 and 5*10⁴ CFUs per fly results in bacterial growth to up to approximately 5*10⁷ CFU per fly within 4–5 days post-infection at 29°C. (C) Intracellular and extracellular populations of bacteria within the fly following infection with 5*10³ CFU/fly, as determined by survival of bacteria within cells following injection of the non-cell-permeable antibiotic gentamycin. Horizontal lines indicate mean CFU/fly. (D) GFP expressing F.novicida within a larval hemocyte.

Figure 2. Summary of results of negatively selected mutant phenotypes.

Mutants with confirmed attenuated phenotypes by competitive index are categorized by their sensitivity to oxidative stress and polymyxin and phenotype in imd mutant flies. To be considered attenuated, each mutant listed in this table was determined to have competitive indexes that were statistically significantly less than 1 by one sample t-tests with a maximum p value of <0.05. + indicates increased sensitivity, − indicates decreased sensitivity, and 0 indicates no change. N/A indicates that the assay was not applicable to that mutant, and N/D indicates test not done. In the “imd rescue” column, + indicated that the phenotype is rescued in imd mutants, − indicates no rescue. The “screens in vertebrate models” column indicates which mutants were identified in screens for F.novicida mutants previously.

Figure 3. A negative selection screen of F. novicida mutants identifies bacterial genes important for bacterial growth and survival within the fly.

Transposon Site Hybridization (TraSH) experiments were used to identify bacterial mutants that failed to replicate within Drosophila at a rate similar to wild-type bacteria. (A) Candidate mutants were tested individually using competition assays in which each mutant was injected into flies at a 1∶1 ratio with wild type bacteria. Following 2 days of infection, the bacteria from each fly was plated and a competitive index was determined using the ratio of mutant bacteria to wild type bacteria and comparing that to the input ratio. One sample t-tests showed that all mutants shown had competitive indexes significantly different from 1. The P values for each mutant are mglA<0.0001, pdpB<0.0001, kdpD<0.0001, FTN_0494<0.001, FTN_1657<0.0001, FTN_1654<0.0001, FTN_1099 = 0.007, pilA<0.0001, pilC = 0.0035, FTN_1452<0.0001, fumA = 0.0047, FTN_1719 = 0.0003, FTN_1276<0.0001, FTN_0921 = 0.0001, and talA = 0.0016. Two genes that were identified by TraSH but not confirmed as statistically significant, FTN_0346 and FTN_0392 are shown on the far right. Horizontal lines indicate the geometric mean. (B) Mutants of interest identified in the TraSH analysis include bacteria that are impaired in their ability to resist oxidative stress damage, including the transcriptional regulator oxyR and multiple DNA repair pathway genes. One sample t-tests showed that all mutants shown had competitive indexes significantly different from 1. The P values for each mutant are oxyR<0.0001, uvrA<0.0001, uvrB = 0.0004, recB<0.0001, ssb<0.0001, and ruvC<0.0001. Horizontal lines indicate the geometric mean. (C) Disk diffusion assay comparing oxyR to U112 wild-type bacteria demonstrated increased susceptibility to reactive oxygen produced by hydrogen peroxide (D) DNA damage repair mutants are also sensitive to oxidative stress as measured by disk diffusion assay with hydrogen peroxide. Error bars represent standard error. All of the mutants are statistically different than U112 as measured by two-tailed t-tests with P values of oxyR<0.0001, uvrA = 0.0008, uvrB = 0.0002, recB = 0.0008, mutM = 0.0008, ssb = 0.0006, and ruvC = 0.0005 (E) oxyR and uvrB mutants are rescued in CG3066 mutant flies which are unable to produce a melanization response. Both rescues are statistically significant, with P values in a 2-tailed t-test of 0.0165 and 0.0002 respectively. Horizontal lines indicate mean values.

Figure 4. Negative selection screens in Drosophila immunity mutants identify F. novicida mutants that help the bacteria resist the imd-regulated host innate immune response.

Survival of Toll and imd pathway mutants infected with F. novicida at 29°C. (A) Two null alleles of imd, imd¹⁰¹⁹¹ and imd¹ backcrossed to OR backgrounds were tested, and both are significantly different from OR flies with log-rank test P values of >0.0001. (B)The Toll pathway is represented by loss of function alleles of two Toll pathway members, Dif and MyD88. Neither are statistically different from wild-type with log-rank test P values of 0.0866 and 0.0582 respectively. (C) Confirmation of mutants identified in the TraSH analysis as attenuated in wild-type flies and rescued in imd mutant flies. All rescues are statistically significant as measured by two-tailed t-tests, with P values of pmrA = 0.0006, FTN_0869 = 0.0001, FTN_0889 = 0.0113, udp = 0.0004, glpD<0.0001, nadC = 0.0465, and FTN_0649 = 0.0030. Horizontal lines indicate the geometric means of the samples. (D) Sensitivity of imd rescue mutants to Polymyxin B as measured by disk diffusion assay. Error bars represent standard error. pmrA, FTN_0889, udp, glpD and FTN_0649 are statistically significantly different from U112 with 2-tailed t-test P values of 0.0299, 0.0041, 0.0495, 0.0065 and 0.0447 respectively. FTN_0869 and nadC are not significantly different than wild-type U112, with P values of 0.1404 and 0.8130.

Figure 5. F. novicida deletion mutants of a putative transglutaminase are severely attenuated in virulence and growth.

(A) Survival of wild-type and FTN_0869 mutant bacteria in wild-type flies at 25°C. FTN_0869 mutants demonstrate significantly lower survival compared to wild-type F. novicida with a P value by log-rank analysis of <0.0001. The MTD for U112 is 9 days at 25°C, while the MTD for FTN_0869 is 12 days post infection, with a P value by log-rank analysis of <0.0001. (B) Survival of wild-type and FTN_0869 mutant bacteria in imd mutant flies at 25°C. The FTN_0869 phenotype is now partially rescued as the FTN_0869 mutant and wild-type Francisella die with MTDs of 7 and 8 days respectively at 25°C, a 4-fold decrease in the spread between mutant and wild-type survival. (C) Total wild type and FTN_0869 mutant growth in wild type OR flies, showing a growth defect of FTN_0869 mutants. At each timepoint, U112 and the FTN_0869 mutants are significantly different with P values from a 2-tailed t-test of <0.0001. At 24 hours post-infection, U112 has 8.5-fold more CFU/fly than the FTN_0869 mutant. At 48 hours, U112 has 8.9-fold more bacteria, and at 72 hours, U112 infected flies have a full 45-fold more bacteria than the FTN_0869 mutant, with a difference between mutant and wild-type of 5.9*10⁵ at 24 hours, 1.7*10⁶ at 48 hours, and 1.1*10⁷ at 48 hours. Horizontal lines represent the mean CFU/fly at each timepoint. Error bars represent standard error. (D) Growth of wild-type and FTN_0869 mutant bacteria in imd mutant flies, showing rescue of the growth defect. The difference between the mean number of CFUs of wild-type and FTN_0869 has decreased at every timepoint, with only a 2.9-fold increase in wild-type bacteria compared to FTN_0869 bacteria at 24 hours, 6-fold more bacteria in U112 infected flies at 48 hours, and only 4-fold more U112 bacteria than FTN_0869 bacteria at 72 hours. Horizontal lines represent the mean CFU/fly at each timepoint. Error bars represent standard error.

Figure 6. F. novicida mglA mutants are unable to survive intracellularly, while FTN_0869 mutants are unable to survive extracellularly.

(A) OR flies were treated with gentamycin at 0, 0.5, 1, 2, and 5 hours post infection after incubation at 29°C. Total CFUs per fly and intracellular CFUs as determined following gentamycin treatment for U112 and mglA mutant bacteria. U112 infected flies are represented on the left in red, mglA infection on the right in green. By 30 minutes post infection 1.5% of U122 CFUs are intracellular, at 1 hour post infection this has increased to 2.3%, by 2 hours 9.6% of the bacteria are intracellular, and by 5 hours, 43.4% of U112 is intracellular. In contrast, none of the mglA mutant bacteria is intracellular is intracellular at any timepoint. Horizontal lines represent the mean of all of the data points. (B) Total CFUs in OR wild-type flies and intracellular CFUs and 0, 24, and 48 hours for U112 and FTN_0869 mutant bacteria. U112 infections are graphed on the left in red, FTN_0869 mutant infection on the right in blue. At 24 hours post infection, 8.2% of the U112 CFUs are intracellular, and by 48 hours only 2% is intracellular because the extracellular population is increasing while the intracellular population remains steady. In contrast, at 24 hours post infection 54% of FTN_0869 mutant bacteria is intracellular and 30% is still intracellular at 48 hours, due to the extracellular population failing to increase. Horizontal lines represent the mean. (C) Total CFUs in imd mutant flies and intracellular CFUs and 0, 24, and 48 hours for U112 and FTN_0869 mutant bacteria. U112 infections are graphed on the left in red, FTN_0869 mutant infection on the right in blue. At 24 hours post-infection the percentages of intracellular U112 bacteria in imd flies is similar to those seen in wild-type flies, with 4.6% and 0.4% intracellular at 24 and 48 hours respectively. However, in imd flies the extracellular population of FTN_0868 mutant bacteria is rescued such that only only 15% of the CFUs are intracellular at 24 hours and only 2% is intracellular at 48 hours post-infection. Horizontal lines represent the mean.

Figure 7. The clearance of extracellular FTN_0869 mutant is not due to altered imd pathway activation, antimicrobial peptide induction, or the Drosophila melanization response.

(A) Antimicrobial peptide RNA levels for Cecropin, Diptericin, and Metchnikowan as determined by quantitative RT-PCR. Error bars represent standard error. Antimicrobial peptide induction is not significantly different between wild-type and FTN_0869 mutant F. novicida infections for any of the AMPs tested at any timepoint. (B) Gentamycin chase experiments for early time points, before the induction of antimicrobial peptides. The kinetics of the clearance of extracellular FTN_0869 mutant bacteria are too rapid to be attributed to antimicrobial peptide induction. At one hour post infection, clearance of FTN_0869 mutants has already begun. While only 2.8% of U112 CFUs are intracellular at 1 hour, 29% of FTN_0869 mutant bacteria is already intracellular presumably because the total number of CFUs present in the fly has reduced from 10⁴ per fly to 2*10³ per fly. By two and five hours post-infection at 29°C wild-type bacteria have both extracellular and intracellular populations and have begun to replicate. At 2 hours post infection, the total CFU's of U112 per fly has doubled from 1*10⁴ to 2*10⁴ with 14% of the CFUs intracellular. At 5 hours post-infection, the mean total CFUs per fly is 4*10⁴ with 45% intracellular at 5 hours. In contrast, at 2 hours post infection, 27.6% of the FTN_0869 mutant bacteria are intracellular and the total CFUs per fly remains steady at 10³/fly. At 5 hours post infection the bacterial levels have increased slightly to 3*10³/fly but the intracellular population has increased to 80% of the total CFUs/fly. Horizontal lines indicate the mean. (C) Competitive indexes of FTN_0869 mutants in wild-type w¹¹¹⁸ flies and in non-melanizing CG3066 mutant flies two days post-infection at 29°C. There is no statistically significant difference between the competitive indices in wild-type and non-melanizing flies, with a P value of 0.601 by 2-tailed t-test, showing that the FTN_0869 mutants are not rescued in Drosophila melanization mutants. Horizontal lines represent the geometric mean of each data set.