Supporting InformationWang et al. 10.1073/pnas.1204158109SI TextEthics Statement.All of the animal work was performed accordingto the guidelines of National Institutes of Health and the JohnsHopkins University Animal Care and Use Committee.

Mosquito and Parasite Maintenance. A. gambiae Keele strain and A.stephensi (Dutch strain) were maintained on 5% (wt/vol) sucrosesolution at 27 ± 1 °C and 80 ± 5% relative humidity and wereprovided by the Johns Hopkins Malaria Research Institutemosquito core. P. falciparum strain NF54 gametocyte cultureswere provided by the Johns Hopkins Malaria Research In-stitute parasite core. P. berghei strain ANKA 2.34 was main-tained by passage through 7–8-wk-old Swiss Webster mice usingstandard procedures (1).

Bacterial Strains, Culture Conditions, and Introduction intoMosquitoes via Sugar Meals. P. agglomerans was originally iso-lated from A. stephensi mosquitoes and selected for long-termsurvival in A. stephensi (2). E. coli DH5a (Invitrogen) was usedfor DNA cloning and plasmid amplification. P. agglomerans andE. coli strains were cultured in LB broth or on agar plates at 28 °Cand 37 °C, respectively. Antibiotics ampicillin (100 μg/mL), car-benicillin (60 μg/mL), chloramphenicol (34 μg/mL), kanamycin(100 μg/mL), and apramycin (80 μg/mL) were added to the mediawhen applicable.Cells were harvested by centrifugation (3,000 × g; 10 min),

washed twice in sterile PBS, and resuspended in 5% (wt/vol)sterile sucrose solution to obtain 109 cells/mL. The bacterial sus-pension was added to sterile cotton pads and provided to 2-d-old mosquitoes for 24 h, after which, the bacterial cotton padswere replaced with new sterile cotton pads containing 5% (wt/vol)sterile sucrose solutions.

Plasmid Construction. DNA encoding mPLA2 was synthesized byGenscript with the P. agglomerans preferred coding usage (http://www.kazusa.or.jp/codon/). NcoI and NheI restriction sites wereincorporated into the 5′ end, and SmaI was incorporated intothe 3′ end. An oligonucleotide, GGAATTCCTGATTAACTT-TATAAGGAGGAAAAATCCATGGCTAGC, containing theEcoR I restriction site, the phage T7 gene 10 translational en-hancer (ENH), and the Shine–Dalgarno region (SD) (3) wasinserted upstream of mPLA2 (Fig. S1B). This synthetic DNAsequence was then cloned into the PUC57-simple vector (Gen-script). The resulting plasmid was digested with EcoR I andSmaI, and the expression cassette was cloned into the corre-sponding sites of pEHlyA2SD (4), to generate pmPLA2EHlyA.In this construct, mPLA2 is fused in frame with an epitope tag(E-tag) and the C-terminal 218 aa of the HlyA DNA sequence.The neomycin phosphotransferase promoter (PnptII) was am-plified from pPnptII:gfp (5) by PCR using primers pnptII for-ward (5′-GCTCTAGAGCGTCAGGCTGTAACAGCTCAGA-3′) and pnptII reverse (5′-GGAATTCCATCCTGTCTCTT-GATCTGATCTTG-3′), and XbaI and EcoRI recognition siteswere incorporated at the 5′ and 3′ ends, respectively (restrictionsites are italicized). The PCR product was digested with XbaIand EcoR I and cloned into the XbaI and EcoRI sites ofpmPLA2EHlyA to generate pnptII-mPLA2EHlyA. These con-structs promoted the constitutive expression of anti-Plasmodiumeffector proteins in P. agglomerans under the control of thePnptII promoter. The synthesized scorpine, pbs21scFv-Shiva1,Shiva1, (EPIP)4, Pro, Pro:EPIP, and (SM1)2 (Tables S1 and S2)coding DNAs were then cloned in between the NheI/NcoI and

SmaI sites of pnptII-mPLA2EHlyA. All constructs were verifiedby DNA sequencing.GFP-tagged bacteria were generated by transferring the plasmid

pPnptII:gfp (5) into P. agglomerans. The plasmid pPnptII:gfp ishighly stable and can be maintained for more than 4 d of culturewithout antibiotic selection.

Expression and Detection of Anti-Plasmodium Effector Molecules inP. agglomerans. P. agglomerans bacteria were chemically cotrans-formed with the effector expression plasmids and the low-copy-number plasmid pVDL9.3 (4), which provides the HlyB and HlyDcomponents (Fig. S2). The TolC protein of P. agglomerans isconserved and did not have to be supplied (4). For protein ex-pression, the transformants were grown overnight at 28 °C in LBliquid medium containing the appropriate antibiotics. Cultureswere centrifuged at 3,000 × g for 15 min at 4 °C, and equal vol-umes of the secreted proteins in the culture supernatant wereconcentrated using Amicon Ultra-4 Centrifugal Filter Units ac-cording to the instructions of the manufacturer. These proteinswere separated by 4–20% gradient SDS/PAGE (Bio-Rad) andtransferred to a PVDF membrane (Millipore). Membranes wereblocked in blocking buffer [4% (wt/vol) milk in PBS with 0.1%Tween 20] and probed with the primary rabbit anti-E tag antibody(Novus Biologicals), followed by incubation with an HRP-conju-gated anti-rabbit IgG secondary antibody. Supernatant fromwild-type P. agglomerans was used as a control.

Effect of Human Blood Meal on Bacteria Proliferation in Mosquitoes.To test the effect of human blood meal on the proliferation ofrecombinant P. agglomerans, 2-d-old A. gambiae females werefed with GFP-tagged P. agglomerans in 5% (wt/vol) sucrose so-lution (108 cells/mL). After feeding for 24 h, the mosquitoeswere starved for 8 h and then allowed to feed on a sterile humanblood meal through a glass membrane feeder. Blood-fed mos-quitoes were dissected at 24-h intervals under sterile conditions,and the bacterial midgut population was determined by platingserial dilutions of midgut homogenates on LB agar plates con-taining 100 μg/mL of kanamycin and incubating the plates at30 °C for 2 d. Colonies of fluorescent bacteria were counted.Each time point used at least 15 mosquitoes.

P. falciparum Transmission-Blocking Assay. Recombinant bacteriawere cultured overnight in LB medium containing appropriateantibiotics, washed twice in sterile PBS, and resuspended in 5%(wt/vol) sterile sucrose solution to obtain 1 × 109 cells/mL. Thebacterial suspension was placed on sterile cotton pads and fed to2- to 3-d-old A. gambiae for 24 h, insects were starved for 8 h,followed by an infectious blood meal as follows. Mosquitoeswere deprived of water and sugar for 8 h and then allowed tofeed for 30 min on a P. falciparum NF54 gametocyte–containingblood meal. Feeding was done with a water-jacketed glass feedercovered with a thin Parafilm membrane maintained at 39 °C.Mosquitoes were then placed in an incubator at 26 °C and 80%relative humidity. Mosquitoes that did not feed were removedwithin 24 h. Success of parasite development was evaluated at 8 dpostinfection by counting oocyst numbers on midguts stained with0.1% (wt/vol) mercurochrome. Mean and median oocyst numberper midgut (infection intensity), percentage of infected mosqui-toes (infection prevalence), and percentage of inhibition relativeto control −Bact were calculated for each group of mosquitoes.

P. berghei Transmission-Blocking Assay. Recombinant bacterialsuspensions [1 × 109 cells/mL of 5% (wt/vol) sugar] soaked in

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cotton pads were fed to 2 d-old A. stephensi. Different groups ofmosquitoes were allowed to feed on the same P. berghei–infectedmouse (∼1 exflagellation per field at 400× magnification) for 6min and then maintained at 19 °C at 80% relative humidity. Fullyengorged mosquitoes were separated within 24 h and providedwith a cotton pad soaked with 5% (wt/vol) sterile sucrose solu-tion. Midguts were dissected on day 14 after the blood meal,stained with 0.1% (wt/vol) mercurochrome, and examined forthe presence of oocysts. Mean and median oocyst number permidgut (infection intensity), percentage of infected mosquitoes(infection prevalence), and percentage of inhibition relative tocontrol −Bact were calculated.

Immunofluorescence Assay. Recombinant P. agglomerans bacteriaexpressing SM1 were cultured overnight in LB medium con-taining appropriate antibiotics, and cells were washed twice inPBS, resuspended in 5% (wt/vol) sucrose to obtain 1 × 109 cells/mL, soaked into cotton pads and fed for 2 d to 2-d-old A.gambiae females. The mosquitoes were then allowed to feed ona noninfected mouse for 30 min and then were maintained at26 °C with 5% (wt/vol) sucrose. Midgut sheets were prepared at18 and 24 h after blood feeding and fixed overnight in 4%(wt/vol) freshly prepared paraformaldehyde (Fisher) at 4 °C,washed in PBS, and blocked with 4% (wt/vol) BSA in PBS at4 °C. The gut sheets were then probed with anti-SM1 peptideantibody (1:1,000 dilution) (6) and washed, followed by in-cubation with Alexa Fluor–conjugated goat anti-rabbit IgG(Sigma, 1:1,000). Nuclei were stained with DAPI, and the gut

sheets were mounted in glycerol buffer plus Slowfade gold re-agent (Invitrogen) following the instructions of the manufacturer.

Impact of Recombinant Bacteria on Mosquito Lifespan.Recombinantbacteria were washed with PBS and resuspended in 5% (wt/vol)sterile sucrose solution to obtain 109 cells/mL and fed to 2-d-oldmosquitoes using soaked cotton pads for 24 h. Female mosqui-toes were separated into five groups that were treated as follows:no bacteria (−Bact), recombinant bacterium P. agglomerans ex-pressing E-tagged HlyA alone (HlyA); or an equal mixture ofbacteria expressing anti-plasmodium effectors (EPIP)4 andShiva1 [(EPIP)4 + Shiva1], Pro:EPIP and mPLA2 (Pro:EPIP +mPLA2), or (EPIP)4 and scorpine [(EPIP)4 + scorpine]. Thebacteria-fed mosquitoes were starved for 8 h and then fed ona noninfected mouse. Each treatment was replicated three timeswith 30 mosquitoes/cup per replicate, and the experiments wererepeated twice. The blood-fed mosquitoes were maintained at27 °C with 10% (wt/vol) sucrose. The mortality for each treat-ment was recorded every 12 h, and dead mosquitoes were re-moved until all mosquitoes had died.

Statistical Analyses. Significant difference in oocyst intensitiesbetween two samples was analyzed using the Mann–Whitney test.Multiple-sample comparisons were analyzed using the non-parametric Kruskal–Wallis test, and medians were comparedusing Dunn’s test. Analysis of survival curves used the log-rank(Mantel–Cox) test. All statistics were performed using GraphPadPrism version 5.00 for Windows (GraphPad Software). P < 0.05was considered to be statistically significant.

1. Sinden RE, Butcher GA, Billker O, Fleck SL (1996) Regulation of infectivity ofPlasmodium to the mosquito vector. Adv Parasitol 38:53–117.

2. Riehle MA, Moreira CK, Lampe D, Lauzon C, Jacobs-Lorena M (2007) Using bacteria toexpress and display anti-Plasmodium molecules in the mosquito midgut. Int J Parasitol37:595–603.

3. Stiner L, Halverson LJ (2002) Development and characterization of a green fluorescentprotein-based bacterial biosensor for bioavailable toluene and related compounds.Appl Environ Microbiol 68:1962–1971.

4. Fernández LA, Sola I, Enjuanes L, de Lorenzo V (2000) Specific secretion of active single-chain Fv antibodies into the supernatants of Escherichia coli cultures by use of thehemolysin system. Appl Environ Microbiol 66:5024–5029.

5. Stiner L, Halverson LJ (2002) Development and characterization of a green fluorescentprotein-based bacterial biosensor for bioavailable toluene and related compounds.Appl Environ Microbiol 68:1962–1971.

6. Ghosh AK, Ribolla PEM, Jacobs-Lorena M (2001) Targeting Plasmodium ligands onmosquito salivary glands and midgut with a phage display peptide library. Proc NatlAcad Sci USA 98:13278–13281.

Fig. S1. Visualization of GFP-tagged P. agglomerans in midguts as a function of time after feeding on a sugar/fluorescent bacteria solution. Mosquitoes werefed with cotton pads soaked with 5% (wt/vol) sucrose solution supplemented with either PBS (−Bact) or 109 cells/mL of GFP-tagged P. agglomerans (Pa-GFP).The fluorescent bacteria could be readily visualized by fluorescence microscopy in midguts at 12, 18, and 24 h after feeding. Fluorescent images are shown tothe left and differential interference contrast (DIC) images of the same field are shown to the right.

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Fig. S2. Strategy for protein secretion using the E. coli hemolysin A (HlyA) system in P. agglomerans. (A) This export system consists of three components thatform a membrane pore (inner-membrane components HlyB and HlyD and outer-membrane component TolC) and the HlyA C-terminal secretion signal domain,to guide the fusion protein through the pore and out of the cell. (B) Schematic representation of the effector–HlyA fusion constructs. Synthetic genes encodingeffector molecules were produced with codons harmonized with the P. agglomerans preferred codon usage and cloned between the NcoI/NheI and SmaIcloning sites placing the effector coding sequence in frame with the C-terminal HlyA domain. An “E” epitope tag was added between the effector gene andHlyA to assist in protein detection. E, E-tag epitope; ENH, translational enhancer; pnptII, the constitutive neomycin phosphotransferase promoter; RBS,ribosome-binding site.

Fig. S3. Effector gene constructs. Synthetic DNA encoding anti-Plasmodium effector molecules were synthesized with codons harmonized with the P. agglom-erans preferred codon usage (Table S2) and fused in-frame to the “E” immune tag and the C-terminal HlyA export leader (Fig. S2B). The genes are driven by theconstitutive neomycin phosphotransferase promoter (pnptII). These constructs were separately transformed into P. agglomerans together with the low-copyplasmid pVDL9.3 carrying the HlyB and HlyD genes. E, epitope tag; ENH, translational enhancer; RBS, ribosome-binding site. See Table S1 for description of theeffector genes.

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Fig. S4. Impact of a chitinase propeptide (Pro) on P. falciparum development in Anopheles gambiae. Mosquitoes were fed on sugar solution supplementedwith PBS (−Bact), with a recombinant P. agglomerans expressing an E-tagged HlyA leader peptide alone (HlyA), or with a Pro-HlyA fusion as indicated and thenfed on a P. falciparum gametocytes–infected blood meal. In parallel, the synthetic propeptide (1 mg/mL infected blood; final concentration) was also fed tomosquitoes. Oocyst numbers were determined 8 d after blood meal. Each dot represents the oocyst number of an individual midgut, and the horizontal linesrepresent mean values.

Fig. S5. Impact of recombinant P. agglomerans strains on P. falciparum development in highly infected mosquitoes. A. gambiae mosquitoes were fed on 5%(wt/vol) sugar solution supplemented with either PBS (−Bact) or recombinant P. agglomerans and, 32 h later, fed on a highly infective (>0.3% gametocytemia)P. falciparum blood meal. Oocyst numbers were determined 8 d postinfection. Each dot represents the oocyst number of an individual midgut, and horizontallines indicate mean values. Data were pooled from three biological replicates. P. agglomerans strains were engineered to express the E-tagged HlyA leaderpeptide alone (HlyA) or mPLA2, Pro:EPIP, Shiva1, scorpine, and (EPIP)4 (Table S1) fusion proteins (Fig. S3), as indicated. Inhibition, inhibition of oocyst formationrelative to the −Bact control; Mean, mean oocyst number per midgut; Median, median oocyst number per midgut; N, number of mosquitoes analyzed; Range,range of oocyst numbers per midgut.

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Table S1. Anti-Plasmodium effector molecules used in this study

Effectors and characterization Target parasite Parasite stage(s) Function or mechanism Source

SM1Salivary gland and

midgut peptide 1P. berghei Ookinete and sporozoite Blocks ookinete invasion of the

midgut epithelium or sporozoiteinvasion of the salivary gland epithelium.

Ref. 1

mPLA2Bee venom phospholipase P. falciparum Ookinete Inhibits ookinete midgut invasion,

probably by modifying the propertiesof the midgut epithelial membranes

Ref. 2P. berghei

Pbs21scFv-Shiva1Single-chain immunotoxin P. falciparum Gametocyte to Oocyst A single-chain monoclonal antibody

(scFv) targeting the major ookinetesurface protein pbs21 and linked tothe lytic peptide Shiva1.

Ref. 3P. berghei

ProA chitinase propeptide P. falciparum Ookinete Inhibits the enzyme and blocks ookinete

traversal of the mosquito peritrophic matrixRef. 4

P. bergheiShiva1Cecropin-like synthetic peptide P. berghei Gametocyte to Ookinete Lyses parasites. Ref. 5

P. falciparumScorpineScorpion P. imperator

venomP. falciparum Gametocyte to Ookinete Anti-malarial cecropin and

defensin-like lytic peptide.Ref. 6

P. bergheiEPIPEnolase–plasminogen

interaction peptideP. falciparum Ookinete Inhibits mosquito midgut invasion by

preventing plasminogen bindingto the ookinete surface

Ref. 7P. berghei

Pro:EPIPA fusion peptide

composed of Pro and EPIPP. falciparum Ookinete Blocks ookinete traversal of the

mosquito peritrophic matrix andprevents plasminogen bindingto the ookinete surface

This studyP. berghei

1. Ghosh AK, Ribolla PEM, Jacobs-Lorena M (2001) Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library. Proc Natl Acad Sci USA 98:13278–13281.

2. Moreira LA, et al. (2002) Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes. J Biol Chem 277:40839–40843.3. Yoshida S, Ioka D, Matsuoka H, Endo H, Ishii A (2001) Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes. Mol Biochem Parasitol 113:

89–96.4. Bhatnagar RK, et al. (2003) Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite. Biochem Biophys Res Commun 304:

783–787.5. Jaynes JM, et al. (1988) In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi.. FASEB J 2:2878–2883.6. Conde R, Zamudio FZ, Rodríguez MH, Possani LD (2000) Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom. FEBS Lett 471:165–168.7. Ghosh AK, Coppens I, Gårdsvoll H, Ploug M, Jacobs-Lorena M (2011) Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut. Proc Natl Acad Sci USA 108:

17153–17158.

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Table S2. Amino acid sequences and codon-optimized DNA coding sequences of theanti-plasmodium effector proteins

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Table S2. Cont.

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