Figure 1.
Anti-lysozyme c-1 antibodies 9122 and 9124 specifically recognized lysozyme c-1.
(A) Western blot showing cross-reaction of lysozyme c-1 antibodies with salivary glands from An. gambiae G3 (lane 1 with 9122; lane 2 with 9124), with salivary glands from An. stephensi (lane 3 with 9122) and no cross-reaction of salivary gland extracts with pre-immune serum (lane 4). (B) Western blot showing cross-reaction of 9122 antibodies (lane 1 and 2) or 9124 (lane 3 and 4) or pre-immune serum (lane 5 and 6) with recombinant lysozyme c-1 purified from E. coli (lane 1, 3 and 5) and from conditioned medium of 4a3B cells (lane 2, 4 and 6). (C) Alignment of the An. gambiae lysozyme c-1 peptide (underlined) used to generate antibodies with other An. gambiae homologues and an orthologue from An. stephensi. (D) Coomassie stained gel showing E. coli expressed recombinant lysozyme c-1 (lane 1); partially purified recombinant lysozyme c-2 (lane 2) and partially purified recombinant lysozyme c-4 (lane 4). (E) Western blot showing cross-reaction of lysozyme c-1 antibodies (9122) with purified recombinant lysozyme c-1 (lane 1) and absence of cross-reactivity with partially purified lysozymes c-2 and c-4 (lanes 2 and 4) and serum proteins from mouse (lane MB).
Figure 2.
Lysozyme c-1 is associated with Plasmodium berghei and Plasmodium falciparum oocysts.
Plasmodium berghei parasites were detected via green fluorescence and anti-lysozyme c-1 antibodies (9122 or 9124 as indicated with white arrows) were detected using Texas-red secondary antibody. All images were collected using confocal microscopy. Representative images of the P. berghei and P. falciparum parasites (indicated with white arrows) are shown. (A) Lysozyme c-1 was not detected on early stage ookinetes of P. berghei (22 h, upper left) but was present on later stages (Days 2 and 5; D2 and D5) of both P. berghei (A, lower left) and P. falciparum oocysts in An. gambiae (B). Pre-immune serum as well as omitting primary antibody showed no staining of parasites (C) indicating a specific labeling by anti-lysozyme antibody 9122 or 9124. Day 5 parasites are shown for control antibodies. (D) Staining of P. berghei oocysts with 9122 as well as 9124 antibodies at day 15 post-infection in An. stephensi. DIC = differential interference contrast; GFP = Green fluorescent protein; Texas red = Goat anti-rabbit secondary antibodies labeled with Texas-red used to visualize 9122 or 9124 primary antibodies for lysozyme c-1.
Figure 3.
Lysozyme c-1 knockdown (KD) significantly reduced prevalence and intensity of P. berghei infections.
The results of three independent replicate experiments for the An. gambiae G3 strain are shown. (A) Shows the significantly decreased prevalence of infection in the LYSC-1 KD mosquitoes. Prevalence was defined as the percentage of mosquitoes that had at least one oocyst per mosquito midgut. (B) Oocyst count data from the three independent replicate experiments including zeros or excluding zeros (mosquitoes that had no infection) is presented. A two-way ANOVA was conducted on individual experiments and p-values are presented along with mean ± SE of oocysts counted in each experiment. The p-value for the totals row indicates the significance of the test when the effect of experiment was included. Parasite numbers were significantly lower in the LYSC-1 KD mosquitoes as compared to control GFP group. ¶Indicates the fold reduction in the expression LYSC-1 as compared to control GFP groups as determined via densitometry analysis (see Materials and Methods).
Figure 4.
Lysozyme c-1 knockdown did not reduce P. berghei oocyst development via increased melanization.
Comparison of midguts infected with a GFP-expressing strain of P. berghei four days after the mosquitoes were injected with either dsGFP or dsLYSC-1. At 3 days post-infection viable parasites were dramatically reduced (GFP) but parasites did not become melanized (DIC).
Figure 5.
Lysozyme c-1 and its muramidase activity were non-detectable in An. gambiae midguts.
(A) A representative Western blot showing an absence of protein cross-reactivity with 9122 antibodies in the midguts as compared to salivary glands. (M = Protein marker). (B) Turbidometric assay showing absorbance values of different samples subtracted from control (which contained only buffer). Muramidase activity was non-detectable in the midgut tissues (n = 10) as compared to positive control (E.coli produced recombinant lysozyme c-1) as well as salivary glands (n = 10 pairs) 24 h after incubation with Micrococcus lysodeikticus cells.
Figure 6.
Silencing of LYSC-1 did not alter the overall number of culturable bacteria in An. gambiae midguts.
Enumeration of culturable bacteria in the midguts of dsGFP and dsLYSC-1 injected mosquitoes. The presented bacterial count data were derived from individual mosquitoes from three biological replicates. Statistical analysis on the count data was performed using software suite R. A two-way ANOVA analysis on the log-transformed data did not reveal any statistical significance in the total number of bacteria in the LYSC-1 knockdown mosquitoes as compared to dsGFP injected mosquitoes (p = 0.11).