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C-type lectin 4 regulates broad-spectrum melanization-based refractoriness to malaria parasites

['Maria L. Simões', 'W. Harry Feinstone Department Of Molecular Microbiology', 'Immunology', 'Bloomberg School Of Public Health', 'Johns Hopkins University', 'Baltimore', 'Maryland', 'United States Of America', 'Yuemei Dong', 'Godfree Mlambo']

Date: 2022-01 

Anopheles gambiae melanization-based refractoriness to the human malaria parasite Plasmodium falciparum has rarely been observed in either laboratory or natural conditions, in contrast to the rodent model malaria parasite Plasmodium berghei that can become completely melanized by a TEP1 complement-like system-dependent mechanism. Multiple studies have shown that the rodent parasite evades this defense by recruiting the C-type lectins CTL4 and CTLMA2, while permissiveness to the human malaria parasite was not affected by partial depletion of these factors by RNAi silencing. Using CRISPR/Cas9-based CTL4 knockout, we show that A. gambiae can mount melanization-based refractoriness to the human malaria parasite, which is independent of the TEP1 complement-like system and the major anti-Plasmodium immune pathway Imd. Our study indicates a hierarchical specificity in the control of Plasmodium melanization and proves CTL4 as an essential host factor for P. falciparum transmission and one of the most potent mosquito-encoded malaria transmission-blocking targets.

Funding: This work was funded by National Institutes of Health grant R01AI12274 (to GD) and National Institutes of Health grant R21AI131574 (to GD), and the Bloomberg Philanthropies (to GD) and the University of California Irvine Malaria Initiative (to GD). The work was also supported by a Johns Hopkins Malaria Research Institute Postdoctoral Fellowship (to MLS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we used CRISPR/Cas9 genome editing to knockout CTL4 and show that this factor is essential for the protection of the clinically relevant human P. falciparum parasite, through a mechanism that does not involve the known complement-like factors, which are important for P. berghei melanization [ 18 – 20 ]. Specifically, we show that the immune factors TEP1, LRIM1, and CLIPA2 do not influence melanization of P. falciparum NF54 ookinetes in CTL4 null mosquitoes while CLIPA14 does. The CTL4 partner, CTLMA2, plays a protective role for P. falciparum even when CTL4 is not present. Intriguingly, the human malaria parasite is not completely blocked by the CTL4-controlled melanization response while the rodent parasite is. Our study points at a significant influence of infection temperature, which is approximately 7 °C higher for P. falciparum, on the kinetics of midgut infection and efficiency of parasite melanization, enabling some human malaria parasites to escape this defense system. We also found that the key anti-P. falciparum innate immune pathway Imd does not influence parasite melanization, while it is known to mediate lysis-based anti-P. falciparum defense [ 21 , 22 , 23 ]. TheCTL4 null mosquitoes are also highly refractory to the fungus Beauvaria bassiana through melanization, but more susceptible to bacterial infections, thereby pointing both agonistic and antagonistic roles of the C-type lectin complex for different pathogens [ 13 , 24 ]. We also show that the mosquito midgut microbiota marginally contributes to the CTL4 null - refractoriness to Plasmodium. Our study proves a major role for CTL4 in the malaria mosquito biology and pathogen transmission and establishes CTL4 as a potent transmission-blocking target for the development of novel malaria control strategies.

We have previously used RNA interference (RNAi)-based gene silencing assays to show that partial depletion of A. gambiae CTL4 results in the melanization of a very small number of P. falciparum ookinetes, but only at unnaturally high infection intensities, and that CTL4 silencing does not affect overall mosquito susceptibility to the human malaria parasite, contrarily to the rodent P. berghei. Hence, previous studies by us and others did not prove a significant P. falciparum host factor role for A. gambiae CTL4, using human malaria laboratory strains and field isolates [ 19 , 20 ].

Together with effector molecule-producing immune signaling pathways, the complement-like system acts as a key effector and regulator of A. gambiae immunity. The core of this system is an ensemble of hemolymph proteins, including the thioester-containing protein TEP1, which is activated by an unknown protease to form TEP1-cut, which is stabilized by the leucine-rich repeat immune proteins LRIM1 and APL1C, which coordinate its binding to the surface of microbes, leading to their elimination through lysis or melanization [ 3 – 9 ]. A complex cascade of serine proteases appears to regulate the TEP1-mediated pathogen killing. The CLIPA2 serine protease inhibits the TEP1-cut deposition on the microbial surface, while the CLIPA14 serine protease also protects P. berghei and other microbes from being melanized, acting more downstream in the process where it appears to regulate phenoloxidase activity [ 10 , 11 ]. Melanization reactions are also key players in A. gambiae immunity to bacteria and fungi and have also been shown to kill pathogens through lysis without the formation of a melanotic capsule [ 9 , 12 – 16 ]. The C-type lectins CTL4 and CTLMA2, which exist mainly as a heterodimer [ 13 , 17 ], have been described as host factors of the rodent parasite P. berghei, protecting the ookinete-stage parasites from the TEP1 complement-like system-regulated melanization, thus enabling them to develop into oocysts and ultimately into sporozoites that can infect the vertebrate host [ 7 , 18 ]. With regard to malaria parasites, most studies on the complement-like defense system have employed the rodent P. berghei model. Whether the same immune factors and mechanisms are involved in eliminating the clinically relevant human P. falciparum has not been clarified and is addressed in the present study.

Plasmodium falciparum is the most prevalent malaria parasite in Africa, accounting for 99.7% of the 213 million malaria cases on that continent in 2018 [ 1 ]. A comprehensive understanding of the biology and transmission of this human-pathogenic parasite through its main mosquito vector, Anopheles gambiae, is paramount for developing new tools to control malaria. Anophelines are not passive vectors: They possess an effective innate immune system that controls infections with diverse microbes, including Plasmodium parasites, bacteria, and fungi, with some degree of specificity. The susceptibility of mosquitoes to Plasmodium and other pathogens, and, hence, vector competence, is an intricate process determined by a fine balance between antagonistic and agonistic immune mechanisms and factors [ 2 ]. Melanization, typically the deposit of a melanin layer on the pathogen surface that results in its encapsulation, is one of the most effective insect defense mechanisms, and extensive studies have shown that A. gambiae can melanize and thereby block infection with the rodent malaria parasite Plasmodium berghei.

Results

CTL4 is a potent transmission-blocking target for malaria First, we compared the susceptibility of all 3 control lines (CTL4-gRNA, the Vasa-Cas9, and the A. gambiae X1 docking line) to P. berghei and P. falciparum infections and found no significant differences among the 3 lines (S2 Fig), suggesting that either one of them can serve as a control. We therefore decided to use the X1 line as a control in subsequent experiments (“control” when used hereafter designates the X1 line). To assess the impact of CTL4 knockout on the susceptibility of A. gambiae to infection with the rodent P. berghei parasite, we fed CTL4null and control (X1) mosquitoes on P. berghei (ANKA 2.34)-infected mice. As documented by several studies, P. berghei achieves unnaturally high infection intensities in A. gambiae, a consequence of A. gambiae not being its natural vector. Therefore, to overcome any possible parasite intensity-related dependence on the outcome of infection, we performed assays with P. berghei at both high and low infection intensities. Contrary to what we and others previously observed for RNAi-mediated silencing of CTL4 in A. gambiae mosquitoes, the CRISPR/Cas9-induced knockout of this gene resulted in a complete (100%) refractoriness to both high and low P. berghei infection intensity (Fig 2A–2C) [7,15,18,20,28–30]. A strain-specific dependence to explain our results can be ruled out, since all the A. gambiae lines used in our study, including CTL4null, are derived from the G3 strain, which was also used in most of the studies on CTL4 mentioned above. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Plasmodium suppression in CTL4null mosquitoes. (A, B) P. berghei infection intensity and prevalence in control and CTL4null A. gambiae females fed on a mouse with low (A) or high (B) gametocytemia and measured at 10 dpi. (C) Images illustrate P. berghei-infected (high gametocytemia) A. gambiae control (upper left) and CTL4null (upper right) midguts showing live parasites and 100% melanized parasites, respectively. Melanized P. falciparum ookinete (lower). (D, E, F) P. falciparum infection intensity and prevalence in control and CTL4null A. gambiae females fed on blood with a low (D, E) or a high (F) gametocytemia and measured in the midgut at 8 dpi (D, F) or in the sg at 14 dpi (E). S5 File displays the underlying data. Dots and inverted triangles indicate the number of parasites in an individual midgut or salivary gland, respectively, and horizontal red bars indicate the median. Two-tailed p-values by Mann–Whitney test were used to compare the live parasites (L); M, melanized parasites (M), and all (L+M) parasites (A). Bars show the percentage of mosquitoes harboring at least one oocyst; the Fisher exact test was used to calculate p-values. Significance of parasite numbers: ****: p < 0.0001; horizontal black lines alone: not significant. dpi, days postinfection; sg, salivary glands. https://doi.org/10.1371/journal.pbio.3001515.g002 We next addressed the permissiveness of CTL4null for the human malaria parasite P. falciparum (NF54). The CTL4-knockout mutants displayed strong P. falciparum suppression at low intensity infection, which mimicked the natural infections observed in the field (Fig 2D and 2E). The median oocyst count was significantly (p < 0.0001) reduced from 1 to 0 parasites/midgut, and the infection prevalence (mosquitoes harboring at least one parasite per total number of mosquitoes) was significantly (p < 0.0001) reduced from 61.3% to 19.7% in CTL4null when compared to the control at 8 d post-blood meal (PBM) (Fig 2D and S2 Table). Interestingly, CRISPR/Cas9-mediated knockout of CTL4 resulted in the melanization of P. falciparum even at the low intensity infection, in contrast to studies based on RNAi-mediated CTL4 silencing, in which P. falciparum melanization was shown to be infection intensity-dependent and hence only observed with a high intensity infection [20]. Accordingly, CTL4 knockout also resulted in a profound decrease in sporozoite loads in the salivary glands of mosquitoes at 14 d PBM (Fig 2E and S2 Table). At a high intensity infection, CTL4-knockout mutants yielded a median of 2 oocysts/midgut, as compared to 32 for the control, and the prevalence of infection also significantly (p < 0.0001) decreased 2.2-fold, from 97.3% to 45.0% at 8 d PBM (Fig 2F and S2 Table). Conversely, at the high infection intensity, the total number of P. falciparum parasites (live and melanin-coated parasites combined) in the CTL4null mosquitoes was significantly (p < 0.0001) lower than the number of total parasites in the control mosquitoes (Fig 2F). This discrepancy was not observed in the low P. falciparum infection intensity assay (Fig 2D) nor in P. berghei infection assays (Fig 2A and 2B), suggesting that CRISPR/Cas9-mediated disruption of CTL4 also results in the killing of P. falciparum without the formation of a melanotic capsule at high infection intensity. In sum, we found that CTL4null mosquitoes were completely refractory to P. berghei as a result of total melanization, and highly refractory to P. falciparum NF54, by melanization, or melanization in addition to another killing mechanism. This contrasts to the A. gambiae L3-5 genetically selected laboratory strain, which melanizes almost all rodent P. berghei, but is unable to melanize sympatric human malaria P. falciparum [31]. The development of P. falciparum was not completely blocked inCTL4null, indicating that the human malaria parasite is capable of partially evading the powerful defense system against which CTL4 protects it. While RNAi-based CTL4 gene silencing resulted in melanization of some parasites but not in a decrease of infection intensity and prevalence, the more extensive melanization and statistically significant reduction in viable human P. falciparum parasites in CTL4nullcorroborates the assertion that the gene silencing will most often yield a hypomorphic phenotype because of an incomplete depletion of target proteins [20]. The observation that the total number of P. falciparum parasites in the CTL4null mosquitoes was significantly lower than the number in the control group suggests that CTL4 protects the human parasite not only from melanization but also from a killing mechanism that is either melanin formation independent or independent of the melanization process and manifests itself at higher levels of infection. Importantly, the level of P. falciparum suppression in CTL4null mosquitoes was significantly higher than that achieved through deleting the FREP1 host factor or by overexpressing the Imd pathway transcription factor REL2 in Anopheles, indicating CTL4 as a powerful transmission-blocking target [25,21].

CTL4 knockout-mediated Plasmodium killing occurs in the midgut epithelium Ookinete-stage Plasmodium invades the mosquito midgut epithelium beginning about 18 to 20 h after ingestion of infected blood, depending on parasite species. To address the question of whether the complete refractoriness of CTL4null to P. berghei (Fig 2A–2C) occurs before or during midgut invasion, or at both stages, we measured the number of P. berghei ookinetes in the blood bolus at 19 h after ingestion, when the majority of parasites have not yet invaded the midgut epithelium. No differences were found in the lumen ookinete loads between CTL4null and control mosquitoes (Fig 3A), indicating that the P. berghei decline in CTL4null mosquitoes occurs when the parasite crosses the midgut epithelium. The peak of P. berghei ookinete invasion of A. gambiae occurs at 24 to 26 h postinfectious blood meal (hpi) [32]. Indeed, we found that all P. berghei ookinetes were melanized in the epithelium of CTL4null mosquitoes at 25 hpi, while the epithelium of control mosquitoes displayed live anti-Pbs28-stained fluorescent parasites (Fig 3C), again showing that the CTL4-dependent Plasmodium protection takes effect exclusively during the invasion of the midgut epithelium. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Spatial–temporal effects of CTL4 knockout on Plasmodium suppression in the A. gambiae midgut. (A, B) P. berghei (A) and P. falciparum (B) ookinete loads in the midgut lumen of A. gambiae females at 19 hpi were not significantly different between the control and the CTL4null groups (two-tailed Mann–Whitney test). S5 File displays the underlying data. (C) Confocal microscopy images illustrate P. falciparum-infected (top panels) and P. berghei-infected (bottom panels) immunostained sections of the A. gambiae CTL4null and control midgut epithelium at 24–25 hpi. Images are representative of 3 independent experiments with at least 10 midguts per replicate. Red indicates parasites probed with α-CTL4 antibody; green indicates live parasites probed with α-Pfs25 (for Pf) or α-Pbs28 (for Pb) antibodies; yellow indicates colocalization of CTL4 and the parasite; and blue indicates DAPI-stained epithelial cells nuclei. Black dots (CTL4null) represent melanized ookinetes. Scale bars 10 μm. hpi, hours postinfection. https://doi.org/10.1371/journal.pbio.3001515.g003 We also wanted to investigate at which invasion stage the observed melanotic capsule-independent killing of P. falciparum occurs during high-intensity infection in CTL4null mosquitoes (Fig 2F). As was true for the rodent parasite, the number of P. falciparum ookinetes in the lumen did not differ between the knockout and control cohorts (Fig 3B), indicating that CTL4 knockout-mediated P. falciparum NF54 lysis, together with melanization, takes place in the midgut epithelium. Melanized P. falciparum ookinetes were visible in the CTL4null midgut epithelium at 24 to 25 hpi, together with live ookinetes (green) (Figs 2D–2F and 3C). Interestingly, while we observed colocalization of CTL4 with the parasite in control mosquitoes, the staining of CTL4 did not perfectly overlap with the contour of the parasite, suggesting that it might not be engaged in a direct interaction (Fig 3C).

CTL4 knockout-mediated Plasmodium melanization is marginally promoted by the mosquito microbiota Studies have shown that the A. gambiae melanization response can be triggered by bacteria in the mosquito hemolymph [12,13]. To exclude the possibility that the extreme phenotype of complete P. berghei melanization in CTL4null mosquitoes is to some degree triggered by bacteria that can drive enzymatic cascades to reach the threshold needed to melanize ookinetes, we treated mosquitoes with an antibiotic cocktail to eliminate the majority of the bacteria from their midgut [22,33–35]. Interestingly, suppression of the bacteria in CTL4null mosquitoes resulted in a few live P. berghei oocysts (median, 0 versus 46.5, respectively; p < 0.0001) (Fig 4A). These results demonstrate that the mosquito microbiota weakly promotes Plasmodium melanization in CTL4null mosquitoes, possibly through the upregulation of immune factors or through bacteria-derived factors that can influence the process. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Effects of microbiota and Imd pathway on Plasmodium suppression in CTL4null mosquitoes. (A, B) P. berghei infection in antibiotics-treated control and CTL4null A. gambiae midguts at 10 dpi, compared to nonantibiotics treated (A) or after injection with dsCaspar or control dsGFP (B). (C, D) P. falciparum infection in control and CTL4null A. gambiae females measured at 8 dpi, after RNAi-mediated silencing of IMD (C) and Caspar (D). S5 File displays the underlying data. Dots indicate the number of parasites in an individual midgut (L, live; M, melanized), and horizontal red bars indicate the median, compared by two-tailed p-values by Mann–Whitney test. Bars show the percentage of mosquitoes harboring at least one oocyst, and the Fisher exact test was used to calculate p-values. Significance of parasite numbers: *: p < 0.05, ****: p < 0.0001; horizontal black lines alone: not significant. dpi, days postinfection; RNAi, RNA interference. https://doi.org/10.1371/journal.pbio.3001515.g004

CTL4-regulated Plasmodium melanization is independent of the Imd pathway The immune deficiency (Imd) pathway is one of the major immune signaling pathways that is activated by, and controls, infections with both bacteria and P. falciparum in A. gambiae. We and others have shown that knockdown of its negative regulator, Caspar, confers a P. falciparum-resistant phenotype in A. gambiae based on parasite lysis but not melanization [22,23]. We have previously shown that the mosquito microbiota augments the Imd pathway [33], and our experiments with aseptic mosquitoes showed a lesser melanization of P. berghei; hence, we hypothesized that perhaps this was due to a modulation of the Imd pathway in the absence of immune-eliciting bacteria in the aseptic mosquitoes [21,33,35,36,37]. In order to investigate whether the Imd pathway could somehow be involved in the CTL4 knockout-induced Plasmodium melanization, we activated the Imd pathway by RNAi-mediated silencing of the pathway’s negative regulator Caspar (gene knockdown (kd) efficiency of 63.25%) in aseptic control and CTL4null mosquitoes (Fig 4B) and compared the melanization phenotypes to mosquitoes treated with a control GFP double-stranded RNA (dsRNA). As shown in Fig 4B, Imd pathway augmentation in aseptic CTL4null mosquitoes did not change the P. berghei melanization phenotype since some live oocysts would still form at the aseptic condition. Hence, the formation of some live P. berghei oocysts in CTL4null mosquitoes at aseptic conditions is not due to a lack of a bacterially mediated modulation of the Imd pathway (Fig 4A and 4B). These data also suggest that the Imd pathway is not involved in CTL4 knockout-mediated melanization of Plasmodium. In A. gambiae, the Imd pathway has emerged as a key defense system against the human malaria parasite P. falciparum through a lytic killing mechanism (not melanization) [22,23]. To further explore a possible implication of the Imd pathway in the CTL4 knockout-induced melanization of P. falciparum, we independently silenced the IMD receptor protein gene (gene kd efficiency of 68.10%) and the negative regulator Caspar to inactivate and activate the pathway, respectively, prior to P. falciparum infection. Silencing IMD did not influence P. falciparum melanization in CTL4null mosquitoes, whereas it did influence their susceptibility to parasite infection with live parasites in both the CTL4null and control groups (Fig 4C), in agreement with our previous studies [22,23]. Silencing Caspar did also not influence the number of melanized P. falciparum ookinetes in the CTL4null mosquitoes, corroborating the independence of the CTL4-regulated melanization from the Imd pathway (Fig 4D).

CTL4-regulated P. falciparum melanization is temperature dependent As described above, P. falciparum infection in CTL4null mosquitoes resulted in a powerful but somewhat leaky phenotype where the infection was not completely abrogated (Fig 1). Optimal infections of A. gambiae with P. berghei and P. falciparum occur at 19 to 20 °C and 27 °C, respectively, and this rather large temperature difference results in a slower rate of development/infection for the rodent parasite than for the human parasite within its vector [38,39]. We hypothesized that it might also influence immune response kinetics and exposure of the parasites to these immune responses, that could explain the complete versus incomplete melanization of P. berghei and P. falciparum, respectively, in CTL4null mosquitoes. To determine whether the leaky P. falciparum infection phenotype of the CTL4null mosquitoes could be attributed to the higher infection temperature, we performed P. falciparum infection experiments at both 19 °C and 27 °C with control and CTL4null mosquitoes fed on the same P. falciparum NF54 gametocyte culture. First, we determined the effect of temperature on P. falciparum development and infection kinetics of the midgut lumen and epithelium in control mosquitoes. Reducing the temperature to 19 °C resulted in a dramatic reduction of ookinetes in both the midgut lumen and epithelium, as well as a significantly reduced number of formed oocysts (Fig 5A and 5B). In addition to the overall reduction of the various parasite stages at 19 °C, we also observed the expected slower midgut invasion and oocyst development kinetics (Fig 5A and 5B). Next, we assessed the effect of the lower temperature on P. falciparum melanization in CTL4null mosquitoes. Interestingly, reducing the temperature to 19 °C did not affect the intensity and prevalence of parasite melanization compared to infection at 27 °C, but it completely abolished the formation of live oocysts in the CTL4null mosquitoes (Fig 5C). These data show that melanization of P. falciparum at 19 °C is more efficient and extensive than that at 27 °C, since the proportion of melanized parasites in relation to the total number of ookinetes in the lumen and midgut epithelium, and live oocysts, is much larger at the lower temperature. These findings show that the observed differences in the proportions of melanized P. berghei and P. falciparum in CTL4null mosquitoes is to a significant extent due to a difference in the temperature required for the sexual development of either parasite, as the more efficient melanization at the lower temperature is likely attributed to a slower invasion kinetics that prolongs the exposure of ookinetes to the mosquito’s melanization-mediated defense. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Influence of temperature on Plasmodium suppression in CTL4null mosquitoes. (A) P. falciparum ookinetes in the lumen of control A. gambiae females at 24 and 40 hpi at 19 °C and 24 hpi at 27 °C, and oocysts at 13 dpi at 19 °C and 8 dpi at 27 °C. (B) P. falciparum ookinetes in the midgut epithelium of control A. gambiae females at 24 and 40 hpi at 19 °C and at 27 °C, and oocysts at 13 dpi at 19 °C and 8 dpi at 27 °C. (C) P. falciparum infection in control and CTL4null A. gambiae females measured at 8 dpi at 19 °C or 27 °C. S5 File displays the underlying data. Dots indicate the number of parasites in an individual midgut (L, live; M, melanized), and horizontal red bars indicate the median, compared by two-tailed p-values by Mann–Whitney test. Bars show the percentage of mosquitoes harboring at least one oocyst, and the Fisher exact test was used to calculate p-values. Significance of parasite numbers: *: p < 0.05, **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; horizontal black lines alone: not significant. dpi, days postinfection; hpi, hours postinfection. https://doi.org/10.1371/journal.pbio.3001515.g005

CTL4 is a selective antagonist of bacterial infection Mosquito immune defense mechanisms have most likely principally evolved to combat infections with pathogens that are more prevalent and virulent than Plasmodium [40], such as bacteria and fungi, which are mainly present in the mosquito’s external environment and intestine. Hence, many of the immune genes and immune signaling pathways that mediate antibacterial and antifungal defenses are also involved in anti-Plasmodium immunity [2]. Knowing that CTL4 is essential for mosquito immunity/tolerance against bacterial systemic infections, we first investigated whether it also contributes to controlling the mosquito’s midgut microbiota. We first compared the number of Luria broth (LB)-culturable bacteria found in the midgut of either sugar-fed control and CTL4null mosquitoes, and the results showed a marginally nonsignificant (p = 0.0612) increased bacterial load in CTL4-deficient mosquitoes (Fig 6A). To also capture contributions of nonculturable bacteria, we compared the total microbiota by qRT-PCR of bacterial 16s ribosomal RNA in both sugar-fed and blood-fed control and CTL4null mosquitoes (Fig 6B). Results again showed that bacterial proliferation in the A. gambiae sugar-fed midgut is not significantly controlled by CTL4 but that CTL4 deletion affects the proliferation of bacteria following a blood meal, which was reduced in the CTL4null mosquitoes, thereby suggesting that CTL4 has an agonistic effect on the midgut microbiota upon blood feeding through an unknown mechanism. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Influences of CTL4 on mosquito interactions with bacteria and fungi. (A, B) Midgut microbial flora of control and CTL4null A. gambiae females was compared using two-tailed p-values by Mann–Whitney (A) and ANOVA followed by a Tukey test (B). (C) Control and CTL4null A. gambiae females were injected with either E. coli (350,000 CFU), S. aureus (420,000 CFU), and B. subtilis (62,000 CFU), or PBS as a control, and longevity was analyzed up to 8 dpi. Kaplan–Meier survival analysis with a log-rank test was used to determine the p-values, and SEs of replicates are indicated. (D) Abdomens were dissected to check bacterial melanization 3 d following E. coli and S. aureus challenge by injection, and the Fisher exact test was used to calculate the difference between control and CTL4null A. gambiae females. (E) Fungi spores were efficiently melanized (white arrows) in the CTL4null abdomens 12 h and 48 h following injection of B. bassiana (2.15 × 106 spores/ml), and the number of live spores (green arrows) was visibly higher in the control abdomens. (F) Melanized and live fungi were measured in midguts tissues from 24 h to 7 d after mosquitoes fed on a sucrose solution containing 2.15 × 108 B. bassiana spores/ml, and the Fisher exact test was used to calculate the difference between control and CTL4null A. gambiae females. (G) Melanized fungi were dectected on the midgut tissue of CTLnull females. (H) Survival of control and CTL4null A. gambiae females after dipping (surface exposure) in B. bassiana spores (1 × 109 spores/ml), compared by the Kaplan–Meier survival analysis with a log-rank test; SE of replicates are indicated. Significance: ****: p < 0.0001. S5 File displays the underlying data. CFU, colony-forming unit; dpi, days postinfection; SE, standard error. https://doi.org/10.1371/journal.pbio.3001515.g006 Next, we investigated the role of CTL4 in controlling systemic bacterial infections by monitoring the survival of CTL4null and control mosquitoes after injection of either control PBS, gram-negative Escherichia coli, or gram-positive Staphylococcus aureus and Bacillus subtilis into the hemolymph. This assay showed a decreased survival of CTL4null mosquitoes upon challenge with gram-negative E. coli, but not with the gram-positive bacteria (Fig 6C), in agreement with previous studies [13,24,32]. To investigate whether the increased CTL4null mortality following E. coli challenge was related to bacteria-induced melanization, which could potentially produce toxic byproducts that are detrimental to the mosquito, we measured the mosquitoes’ abdominal melanization, which is visible in the cuticle 3 d after challenge with E. coli and S. aureus (Fig 6D). No differences were observed in the percentage of melanization between the CTL4null and control cohorts, indicating that the decreased survival of E.coli-challenged CTL4null mosquitoes was not due to a CTL4 knockout-induced increase in bacterial melanization (Fig 6D). Similarly, Schnitger and colleagues [13] did not observe an increase in phenol oxidase enzymatic activity in bacteria-challenged CTL4-silenced mosquitoes. In conclusion, our bacteria challenge assays suggest that CTL4 acts as an antagonist of systemic bacterial infections in a melanization-independent manner. It could be argued that the melanization observed in both CTL4null and control mosquitoes in Fig 6C is a response to wounding provoked by the mechanical injection of bacteria, which equally affected both the CTL4null and control cohorts. However, in contrast to another study in which thoracic melanization of the injection site was observed in CTL4-silenced mosquitoes but not in the dsLacZ control [15], we did not observe any increased abdominal melanization in our CTL4null cohort(Fig 6D).

[END]

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