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A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase

Abstract

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.

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Fig. 1: Annual P. vivax cases and A. sinensis susceptibility to P. vivax infection.
Fig. 2: Effect of gut symbiotic Serratia bacteria of wild mosquitoes on Plasmodium development.
Fig. 3: Identification of the secreted antimalarial lipase AmLip in S. ureilytica Su_YN1.
Fig. 4: AmLip disrupts and kills parasites.
Fig. 5: S. ureilytica Su_YN1 bacteria spread efficiently through mosquito populations.

Data availability

The entire 16S rRNA gene sequence dataset reported in this paper has been deposited in the National Center for Biotechnology Information Sequence Read Archive (accession no. PRJNA642229). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers JACAAS000000000, JACAAT000000000, JACAAU000000000 and JACAAV000000000. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (grants nos. 31830086, 32021001 and 31472044) to S.W.; the National Key R&D Program of China (grant no. 2019YFC1200800) to S.W.; the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB11010500) to S.W.; the Key Research Program of the Chinese Academy of Sciences (grant no. KFZD-SW-219) to S.W.; the National Institutes of Health (grant no. R01AI031478) to M.J.-L.; the Johns Hopkins Malaria Research Institute Insectary, Parasite Core Facilities; the Bloomberg Philanthropies and the Jiangsu Provincial Department of Science and Technology (grant no. BM2018020) to J.C. We thank F. Li for rearing mosquitoes.

Author information

Affiliations

Authors

Contributions

S.W. conceived the project. S.W., H.G. and L.B. designed the study. L.B., X.L., S.L., G.Z. and J.C. collected wild mosquitoes. L.B. conducted the gut symbiotic bacteria isolation, gut colonization, RNA interference and effects of isolated bacteria on P. berghei infection and mosquito biology assays. L.B. and X.L. performed the A. sinensis susceptibility to P. vivax infection assays. H.G. conducted the in vitro anti-Plasmodium activity, mass spectrometry, gene disruption, western blot, AmLip-mediated anti-Plasmodium activity and immunofluorescence assays. Y.J. conducted the bacterial transmission and cage experiments. W.H. conducted assays to determine the effect of Serratia bacteria on P. falciparum infection. Z.H. and L.B. investigated the effect of Serratia culture supernatant on P. falciparum gametocyte development. D.W. provided P. vivax epidemic data. S.Z. performed phylogenomic analysis. L.J. and M.J.-L. provided materials. H.G., L.B. and S.W. analysed the data. H.G., L.W. and S.W. wrote the manuscript. M.J.-L. and S.W. edited the manuscript.

Corresponding author

Correspondence to Sibao Wang.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Microbiology thanks Hitotaka Kanuka and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Persistence of non-symbiotic bacteria in mosquito midguts.

GFP-tagged E. coli and S. aureus were administered to newly emerged female mosquitoes via sugar feeding. Bacteria midgut colonization was determined by plating serially diluted midgut homogenates on LB agar plates containing 100 μg/ml kanamycin. a,b, E. coli and S. aureus bacteria numbers in mosquitoes maintained with sugar. c,d, E. coli and S. aureus bacteria numbers in mosquitoes post a blood meal. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 2). Each replicate contains 10 mosquito midguts.

Source data

Extended Data Fig. 2 Serratia strains stably colonize the mosquito midgut and do not impact mosquito longevity.

a, b, Effect of Serratia strains on An. sinensis (a) and An. stephensi (b) survival post blood meal. Bacteria were administered to two-day-old female mosquitoes via sugar meal and then fed blood. Survival was monitored daily. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 2). Each replicate contains 10 mosquito midguts. c,d, Serratia bacteria numbers in the midgut of female An. sinensis (c) and An. stephensi (d) post blood meal. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 2). Each replicate contains 10 mosquito midguts. e, Visualization of GFP-tagged Su_YN1 and Sm_YN3 bacteria in the midgut of An. stephensi at 24 h after a blood meal. Bright-field images (left) are paired with the corresponding fluorescent images (right). The experiments were repeated twice with similar results.

Source data

Extended Data Fig. 3 Effect of bacteria on P. berghei oocyst formation in An. stephensi.

a, P. berghei oocyst load in An. stephensi mosquitoes carrying Asaia, Acinetobacter or Pantoea bacteria from YN wild caught mosquitoes. b, P. berghei oocyst load in An. stephensi mosquitoes fed with different Serratia strains from different wild-caught mosquito populations. Circles represent the number of oocysts in individual midguts, and horizontal lines indicate the median number of oocysts per midgut. The sample size (n Number) of each group is listed in the table of the lower panel. The statistical significance of the oocyst intensity between the bacteria-fed mosquitoes and PBS-fed mosquitoes (Ctrl) was analysed using the two-tailed Mann-Whitney test. ****P < 0.0001, P > 0.05, not significant (ns). The exact P values in (a) were as follows: Asaia, 0.4508; Acinetobacter, 0.8728; Pantoea, 0.4265. The exact P values in (b) were as follows: Su_YN1, < 0.0001; Sm_YN3, < 0.0001; Sf_JS1, 0.0271; Sf_JS2, 0.0135; Su_JS3, 0.0117; Sm_LN1, 0.0220.

Source data

Extended Data Fig. 4 The effect of Serratia Su_YN1 and Sm_YN3 on An. stephensi blood feeding, fecundity and oviposition rate.

Serratia Su_YN1 and Sm_YN3 do not impact An. stephensi mosquito blood feeding behaviour (n = 100 mosquitoes each group) (a), egg production (Ctrl, n = 46, Su_YN1, n = 39, Sm_YN3, n = 44) (b) or oviposition rate (n = 100 mosquitoes each group) c, Two-day-old An. stephensi mosquitoes were fed on a sugar meal containing bacteria or 5% sugar alone (Ctrl). Three days later, female mosquitoes were fed on a mouse and three days later eggs were collected from individual females. The experiments were repeated three times with similar results. No significant differences were detected among the groups (one-way ANOVA or two-tailed Mann-Whitney test).

Source data

Extended Data Fig. 5 Effect of Rel1 and Rel2 silencing on Su_YN1- or Sm_YN3-mediated anti-Plasmodium activity.

Rel1 and Rel2 were silenced in An. stephensi by systemic injection of double-stranded RNA dsRel1, dsRel2 or dsGFP. The injected mosquitoes were fed on a sugar meal containing Su_YN1 or Sm_YN3. Three days later, the mosquitoes were allowed to feed on the same P. berghei infected mouse. The injected double-stranded RNA (ds) and presence of bacteria are indicated below each column. Each dot represents the oocyst number of an individual midgut, and the horizontal lines indicate the median number of oocysts per midgut. Data are from n = 25 to 28 mosquitoes per group. The sample size (n Number) of each group is listed in the table of the lower panel. The statistical significance of oocyst intensity differences between the dsRel1- or dsRel2-injected and dsGFP-injected mosquitoes carrying the same bacteria (Su_YN1 or Sm_YN3) was analysed using the two-tailed Mann-Whitney test. ****P < 0.0001, P > 0.05, not significant (ns). The exact values were as follows: dsRel1+Su_YN1, 0.8519; dsRel2+Su_YN1, 0.5605; dsRel1+Sm_YN3, < 0.0001; sRel2+Sm_YN3, 0.4851.

Source data

Extended Data Fig. 6 Su_YN1 culture supernatant inhibited P. berghei ookinete formation.

a, Inhibition by Serratia culture supernatants of P. yoelii ookinete formation in vitro. Ookinete formation was quantified by luminescence measurements using the Py.17XNL reporter strain. RLU, relative light units. The experiments were repeated three times with similar results. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 2). b, Effect of Su_YN1 culture supernatant on P. berghei ookinete formation in vitro assay using Giemsa staining. Transformation rate was quantified by comparison with the PBS control. The experiments were repeated three times with similar results. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 3). Statistical significance of the ookinete transformation rate was compared with the PBS control using two-tailed Student’s t-test, ****P < 0.0001. The exact P value was, < 0.0001.

Source data

Extended Data Fig. 7 Haemolytic activity assay of Su_YN1 and Sm_YN3.

a, Bacteria culture supernatant was added (10% V/V) and the mixture incubated with erythrocytes for 12 h. b, The supernatants were collected and the absorbance at 540 nm was measured to evaluate haemoglobin release. Saponin was used as a haemolytic positive control. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 3). Statistical significance of haemolytic activity was compared with the PBS control using two-tailed Student’s t-test. P > 0.05, not significant (ns). The exact P values were as follows: Su_YN1, 0.1672; Sm_YN3, 0.3754.

Source data

Extended Data Fig. 8 Antimalarial activity of different Su_YN1 culture supernatant fractions.

a, Antimalarial activity of organic solvent extracts. Su_YN1 culture supernatant was extracted with solvents of various polarities. The extracted fractions were dried and dissolved in DMSO and the remaining aqueous phase was vacuum treated to remove residual organic reagents. All fractions were tested for antimalarial activity. b, Antimalarial activity assay of Su_YN1 culture supernatant separated using a 3 kDa cut-off centrifugal filter. The retentate and the filtrate were tested. c, Trypsin digestion of Su_YN1 culture supernatant abolishes antimalarial activity. Coomassie Brilliant Blue staining in the lower panel shows the protein patterns before and after treatment. Data points in (b) and (c) are mean ± s.d. The dots represent biologically independent replicates (n = 3). Statistical significance of the ookinete inhibition rate was compared with the PBS control using two-tailed Student’s t-test, ****P < 0.0001, P > 0.05, not significant (ns). The exact P values in (b) were: Trypsin, < 0.0001; Trypsin-inactivated, 0.0552. The exact P values in (c) were: Retentate (compared with PBS), < 0.0001; Filtrate (compared with Retentate), < 0.0001.

Source data

Extended Data Fig. 9 AmLip gene expression in different Serratia strains.

Detection of AmLip transcript abundance in different Serratia strains by qRT-PCR using Serratia 16 s rRNA as an internal reference. Data points are mean ± s.d. The dots represent biologically independent replicates (n = 3). The experiments were repeated twice with similar results.

Source data

Extended Data Fig. 10 Synthesis and purification of Serratia AmLip protein.

a, Western blot detection using HA antibody, of 3HA-tagged AmLip protein in bacterial extracts of Su_YN1 wild type, knock-out mutant AmLip-KO and a mutant AmLip-KO complemented with the AmLip:HA gene. The experiments were repeated twice with similar results. b, Knockout of AmLip was verified by Western blot assay using an AmLip mouse antiserum. The experiments were repeated twice with similar results. c, Expression and purification of AmLip protein expressed E. coli BL21 (DE3). Coomassie blue staining showing the AmLip protein before and after purification on a nickel column. The experiments were repeated twice with similar results. d, Lipase activity test of the purified AmLip protein using the egg yolk lipoprotein plate degradation assay. e, Lipase activity assay of the purified AmLip protein using the trioleoylglycerol- rhodamine B plate degradation assay.

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Supplementary information

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Gao, H., Bai, L., Jiang, Y. et al. A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase. Nat Microbiol 6, 806–817 (2021). https://doi.org/10.1038/s41564-021-00899-8

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