Virulence of serotype M3 Group A Streptococcus strains in wax worms (Galleria mellonella larvae)

Abstract

Group A Streptococcus (GAS) causes human infections that range in severity from pharyngitis ("strep-throat") to necrotizing fasciitis ("flesh-eating disease").  To facilitate investigation of the molecular basis of host-pathogen interactions, infection models capable of rapidly screening for differences in GAS strain virulence are needed.  To this end, we developed a Galleria mellonella larvae (wax worm) model of invasive GAS infection and used it to compare the virulence of serotype M3 GAS strains.  We found that GAS causes severe tissue damage and kills wax worms in a dose-dependent manner.  The virulence of genetically distinct GAS strains was compared by Kaplan-Meier survival analysis and determining 50% lethal doses (LD₅₀).  Host-pathogen interactions were further characterized using quantitative culture, histopathology and TaqMan assays.  GAS strains known to be highly pathogenic in mice and monkeys caused significantly lower survival and had significantly lower LD₅₀s in wax worms than GAS strains associated with attenuated virulence or asymptomatic carriage.  Furthermore, isogenic inactivation of proven virulence factors resulted in a significantly increased LD₅₀ and decreased lesion size compared to the wild-type strain, a finding that also strongly correlates with animal studies.  Importantly, survival analysis and LD₅₀ determination in wax worms supported our hypothesis that a newly emerged GAS subclone that is epidemiologically associated with more human necrotizing fasciitis cases than its progenitor lineage has significantly increased virulence.  We conclude that GAS virulence in wax worms strongly correlates with the data obtained in vertebrate models, and thus, the Galleria mellonella larva is a useful host organism to study GAS pathogenesis.

Acknowledgements

This work was supported in part by American Heart Association grant AHA0775045.

Figures and Tables

Figure 1 GAS causes severe disseminated infection and tissue destruction in Galleria mellonella larvae. (A) Wax worm larvae were inoculated with 10⁷ CFU of representative serotype M3 strain MGAS315 by injection through the left hindmost proleg using a 29 G needle. (B and C) Infection with MGAS315 resulted in rapid melanization (dark pigmentation) and high mortality (n = 10 larvae, red bar) by 24 h post-inoculation. No deaths occurred in larvae sham inoculated with sterile saline (PBS, gray bar). (D) Microscopic examination of infected wax worm larvae at 6 h post-inoculation shows an expanding abscess-like lesion at the GAS injection site. An area of extensive soft tissue necrosis and bacterial proliferation (dense blue-violet region in the center of the micrograph, demarcated with a white asterisk) is surrounded by melanized hemocytes (extracellular brown pigment and host immune cells walling off the expanding lesion, demarcated by black arrowheads). Extensive tissue destruction occurred in the thoracic and abdominal body segments (Gram's stain, 4x original magnification). (E) At the site of inoculation, a dense infiltrate of GAS organisms is seen in a background of anucleate agranular host hemocytes (Gram's stain, 40x original magnification). (F) By 6 h post-inoculation, chains of GAS have already disseminated throughout the hemolymph to begin infecting soft tissue near the first true leg (boxed region in A). The Galleria mellonella body cavity contains a variety of soft tissue types, including hemolymph with immune cells, fat, muscle and epithelium, that are similar to those encountered by GAS during invasive human infections such as necrotizing fasciitis (Gram's stain, 40x original magnification).

Figure 2 Virulence of GAS in the Galleria mellonella infection model. (A and B) Wax worm larvae were inoculated with serial dilutions (10⁷, 10⁶, 10⁵ and 10⁴ CFU) of strain MGAS315 (red squares), which is representative of highly virulent serotype M3 GAS strains causing severe invasive infections in humans or strain MGAS12501 (brown triangles), which was recovered from the oropharynx of an asymptomatic carrier. Survival was monitored for 96 h. Kaplan-Meier survival curves were determined for four or more independent experiments and showed a dose-dependent response with both strains (n = 10 larvae per dose per strain per experiment). The highest dose (10⁷ CFU) of either strain resulted in significantly higher mortality than any of the three lower doses tested (p < 0.001 for either MGAS315 or MGAS12501, 10⁷ CFU compared to 10⁶ CFU, logrank test). Similarly, inoculation of 10⁶ CFU resulted in significantly higher mortality than the lowest dose tested (p < 0.001 for MGAS315 and p = 0.033 for MGAS12501, 10⁶ CFU compared to 10⁴ CFU, logrank test). At the 10⁶ CFU dose, invasive strain MGAS315 killed more wax worms than carrier strain MGAS12501 (p = 0.004, logrank test). No deaths occurred in larvae sham infected with sterile saline (PBS, gray circles). (C–D) Survival curves of wax worm larvae inoculated with either invasive strain MGAS315 (red squares) or carrier strain MGAS12501 (brown triangles) were highly reproducible. Results from multiple independent experiments using the highest dose tested (10⁷, solid lines) and lowest dose tested (10⁴ CFU, dotted lines) are shown as individual curves (n = 10 larvae per dose per strain per experiment). (E) Infected larvae demonstrated progressive melanization post-inoculation, indicating a robust host immune response to the invasive GAS infection. Compared to larvae infected with the highly virulent invasive strain MGAS315, wax worms infected with the less virulent carrier strain MGAS12501 developed pigmentation at a much slower rate. (F) In vivo growth of GAS organisms in wax worm larvae inoculated with 10⁵ CFU of strain MGAS315 demonstrates a logarithmic curve. Bacterial burden was quantified from pools of five homogenized larvae at the time points indicated (n = 3 pools per time point).

Figure 3 Virulence of serotype M3 GAS strains in the Galleria mellonella infection model correlates with virulence in mice. Wax worm larvae were infected with serial dilutions (10⁷, 10⁶, 10⁵ and 10⁴ CFU) of representative serotype M3 strain MGAS315 or one of 10 other GAS strains also recovered from human patients, and survival was monitored for 96 h. Results from three or more independent assays are shown (n = 10 larvae per strain per dose per experiment). (A) Kaplan-Meier survival curves were determined for wax worms inoculated with 10⁶ CFU. Compared to all strains tested except MGAS3392 (pink circles), the highly virulent reference strain MGAS315 (red circles) results in a significantly lower survival over time (*indicates p < 0.05 compared to MGAS315, logrank test). (B) The 50% lethal dose (LD₅₀) was determined by Probit analysis. The highly virulent reference strain MGAS315 (red bar) has a significantly lower LD₅₀ (that is, it is more virulent) than other invasive strains known to be less virulent in mice (various blue bars) or strains recovered from the oropharynx of asymptomatic carriers (various brown bars) (* indicates p < 0.05 compared to MGAS315, Mann-Whitney test). (C) Overall, GAS strains such as MGAS315, MGAS3382 and MGAS3392 which are known to be highly virulent in mice and/or monkeys (red checkered bar) have a significantly lower mean LD₅₀ in wax worm larvae (that is, they are more virulent) than GAS strains associated with lower virulence in animals (blue checkered bar) or asymptomatic carriage in humans (brown checkered bar) (* indicates p < 0.01 compared to MGAS315, Mann-Whitney test). (D) The virulence of these 11 serotype M3 GAS strains tested in Galleria mellonella larvae strongly correlates with their virulence in mice (Pearson's correlation r = 0.689 and p = 0.019).

Figure 4 Virulence factors implicated in invasive human disease contribute to GAS virulence in the Galleria mellonella infection model. Wax worm larvae were infected with serial dilutions (10⁷, 10⁶, 10⁵ and 10⁴ CFU) of wild-type strain MGAS315 or one of five different isogenic mutant GAS strains lacking expression of the gene encoding a specific virulence factor, and survival was monitored for 96 hours. Results from three or more independent assays are shown (n = 10 larvae per strain per dose per experiment). (A) Kaplan-Meier survival curves were determined for wax worms inoculated with 10⁶ CFU. Compared to all strains tested except MGAS315ΔslaA (yellow circles), the highly virulent reference strain MGAS315 (red circles) results in a significantly lower survival over time (logrank test). (B) The 50% lethal dose (LD₅₀) was determined by Probit analysis. The highly virulent reference strain MGAS315 (red bar) has a significantly lower LD₅₀ (that is, it is more virulent) than the isogenic mutant strain lacking expression of the gene encoding mtsR (MGAS315ΔmtsR), mga (MGAS315Δ12bpmga), prsA (MGAS315ΔprsA), slaA (MGAS315ΔslaA) or speB (MGAS315ΔspeB) (various green bars, p calculated relative to strain MGAS315, Mann-Whitney test). Gene complementation restored the wild-type virulence phenotype to strains MGAS315ΔmtsRcomp, MGAS315Δ12bpmgacomp and MGAS315ΔprsAcomp (various hatched green bars, p calculated relative to the comparator isogenic mutant strain, Mann-Whitney test). (C) Compared to wild-type reference strain MGAS315, the bacterial burden of the isogenic mutant strains was not significantly different in infected wax worm larvae (student's t-test). Bacterial burden was quantified from pools of five homogenized larvae (n = 4 pools per strain) inoculated with 10⁵ CFU of the indicated strains and harvested at 18 h post-inoculation. (D) Microscopic examination of MGAS315-infected wax worm larvae at three hours post-inoculation shows multiple coalescing abscess-like lesions characterized by extensive melanization (boxed regions). In comparison, the SpeB-deficient strain causes considerably smaller lesions with less melanization (circled regions) (Hematoxylin and eosin stain, 4x original magnification). (E) In vivo expression of mtsR, prsA and speB by wild-type strain MGAS315 was confirmed by TaqMan quantitative real-time PC R analysis.

Figure 5 Subclone 8 GAS strains are significantly more virulent than subclone 5 GAS strains in the Galleria mellonella infection model. Subclone 5 strains of GAS were previously shown to lack necrotizing fasciitis capacity in mice, monkeys and humans.13 Subclone 8 strains of GAS are newly emerged descendents of subclone 5 strains that epidemiologically have regained necrotizing fasciitis capacity in humans.3 To test the hypothesis that subclone 8 strains are more virulent than subclone 5 strains, wax worm larvae were infected with serial dilutions (10⁷, 10⁶, 10⁵ and 10⁴ CFU) of five representative strains from each subclone lineage, and survival was monitored for 96 h. Results from five independent assays are shown (n = 10 larvae per strain per dose per experiment with five strains tested from each subclone lineage). (A) Kaplan-Meier survival curves were determined for wax worms inoculated with 10⁷ CFU. Compared to subclone 5 strains (violet diamonds), subclone 8 strains (pink diamonds) result in significantly lower survival over time (logrank test). (B) The 50% lethal dose (LD₅₀) was determined by Probit analysis. Subclone 8 strains (pink bars) have a significantly lower LD₅₀ (that is, they are more virulent) than subclone 5 strains (violet bars) (Mann-Whitney test).

Table 1 Serotype M3 GAS strains used in this study