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Plasmodium falciparum malaria originated when Plasmodium praefalciparum , a gorilla malaria parasite transmitted by African sylvan anopheline mosquitoes, adapted to humans. Pfs47, a protein on the parasite surface mediates P. falciparum evasion of the mosquito immune system by interacting with a midgut receptor and is critical for Plasmodium adaptation to different anopheline species. Genetic analysis of 4,971 Pfs47 gene sequences from different continents revealed that Asia and Papua New Guinea harbor Pfs47 haplotypes more similar to its ortholog in P. praefalciparum at sites that determine vector compatibility, suggesting that ancestral P. falciparum readily adapted to Asian vectors. Consistent with this observation, Pfs47-receptor gene sequences from African sylvan malaria vectors, such as Anopheles moucheti and An. marshallii , were found to share greater similarity with those of Asian vectors than those of vectors of the African An. gambiae complex. Furthermore, experimental infections provide direct evidence that transformed P. falciparum parasites carrying Pfs47 orthologs of P. praefalciparum or P. reichenowi were more effective at evading the immune system of the Asian malaria vector An. dirus than An. gambiae . We propose that high compatibility of ancestral P. falciparum Pfs47 with the receptors of Asian vectors facilitated the early dispersal of human malaria to the Asian continent, without having to first adapt to sub-Saharan vectors of the An. gambiae complex.

The surface protein Pfs47 allows Plasmodium falciparum parasites to survive and be transmitted by making them "undetectable" to the mosquito immune system. P. falciparum parasites express Pfs47 haplotypes compatible with their sympatric vectors, while those with incompatible haplotypes are eliminated by the mosquito. We proposed that Pfs47 serves as a "key" that mediates immune evasion by interacting with a mosquito receptor "the lock," which differs in evolutionarily divergent anopheline mosquitoes. Recombinant Pfs47 (rPfs47) was used to identify the mosquito Pfs47 receptor protein (P47Rec) using far-Western analysis. rPfs47 bound to a single 31-kDa band and the identity of this protein was determined by mass spectrometry. The mosquito P47Rec has two natterin-like domains and binds to Pfs47 with high affinity (17 to 32 nM). P47Rec is a highly conserved protein with submicrovillar localization in midgut cells. It has structural homology to a cytoskeleton-interacting protein and accumulates at the site of ookinete invasion. Silencing P47Rec expression reduced P. falciparum infection, indicating that the interaction of Pfs47 with the receptor is critical for parasite survival. The binding specificity of P47Rec from distant anophelines (Anopheles gambiae, Anopheles dirus, and Anopheles albimanus) with Pfs47-Africa (GB4) and Pfs47-South America (7G8) haplotypes was evaluated, and it is in agreement with the previously documented compatibility between P. falciparum parasites expressing different Pfs47 haplotypes and these three anopheline species. Our findings give further support to the role of Pfs47 in the adaptation of P. falciparum to different vectors.

Significance A previous Plasmodium infection enhances the mosquito immune response to subsequent infections. Priming is mediated by a hemocyte differentiation factor (HDF) consisting of lipoxin A 4 (LXA 4 ) bound to Evokin, a lipid carrier. Insects produce LXA 4 but lack lipoxygenase enzymes. Here we establish that the double peroxidase (DBLOX) enzyme is essential for HDF synthesis and is highly expressed in fat-body oenocytes, a group of specialized cells that increase in number in primed females. The histone acetyltransferase (HAT) AgTip60 is also essential for HDF synthesis, for the persistent increase in oenocyte numbers, and to maintain immune priming. We identified an enzyme essential for lipoxygenase-independent LXA 4 synthesis and show that AgTip60 HAT is also critical to maintain the priming response.

A series of epidemiological explorations has suggested a negative association between national BCG vaccination policy and the prevalence and mortality of COVID-19. However, these comparisons are difficult to validate due to broad differences between countries such as socioeconomic status, demographic structure, rural vs. urban settings, time of arrival of the pandemic, number of diagnostic tests and criteria for testing, and national control strategies to limit the spread of COVID-19. We review evidence for a potential biological basis of BCG cross-protection from severe COVID-19, and refine the epidemiological analysis to mitigate effects of potentially confounding factors (e.g., stage of the COVID-19 epidemic, development, rurality, population density, and age structure). A strong correlation between the BCG index, an estimation of the degree of universal BCG vaccination deployment in a country, and COVID-19 mortality in different socially similar European countries was observed (r2= 0.88;p= 8×10-7), indicating that every 10% increase in the BCG index was associated with a 10.4% reduction in COVID-19 mortality. Results fail to confirm the null hypothesis of no-association between BCG vaccination and COVID-19 mortality, and suggest that BCG could have a protective effect. Nevertheless, the analyses are restricted to coarse-scale signals and should be considered with caution. BCG vaccination clinical trials are required to corroborate the patterns detected here, and to establish causality between BCG vaccination and protection from severe COVID-19. Public health implications of a plausible BCG cross-protection from severe COVID-19 are discussed.Significance StatementThe COVID-19 pandemic is one of the most devastating in recent history. The bacillus Calmette-Guérin (BCG) vaccine against tuberculosis also confers broad protection against other infectious diseases, and it has been proposed that it could reduce the severity of COVID-19. This epidemiological study assessed the global linkage between BCG vaccination and COVID-19 mortality. Signals of BCG vaccination effect on COVID-19 mortality are influenced by social, economic, and demographic differences between countries. After mitigating multiple confounding factors, several significant associations between BCG vaccination and reduced COVID-19 deaths were observed. This study highlights the need for mechanistic studies behind the effect of BCG vaccination on COVID-19, and for clinical evaluation of the effectiveness of BCG vaccination to protect from severe COVID-19.

Significance We have shown in this study that the malaria parasite can rapidly evolve to adapt for loss of an “essential” kinase, PfCDPK1. PfCDPK1 could not be disrupted in the wild-type parasite. However, we were able to disrupt CDPK1 in the transgenic parasites adapted for reduced kinase activity of mutant PfCDPK1. Strategic disruption of PfCDPK1 highlights the importance of understanding the compensatory mechanisms, especially for targets belonging to multigene families. Our study unequivocally demonstrates that PfCDPK1 is critical for mosquito infections and its disruption leads to defective gametogenesis. Our study also suggests involvement of CDPK1 in regulation of sexual stage-specific genes during the asexual proliferation. Targeting PfCDPK1 may be a good strategy for developing transmission-blocking drugs., Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

Significance The introduction of Wolbachia (an intracellular bacterium that does not infect higher organisms) into culicine mosquito populations from endemic areas is a promising strategy to prevent arboviral transmission. Anopheline mosquitoes were thought to be naturally refractory to Wolbachia , but a population of Anopheles gambiae from Burkina Faso infected with Wolbachia was recently reported. We identified a Wolbachia strain in A. gambiae mosquitoes from Mali ( w Anga-Mali). w Anga-Mali infection was associated with reduced prevalence and intensity of sporozoite infection in field-collected females. Experimental infections indicate that w Anga-Mali infection reduces malaria transmission by a mechanism that affects sporozoites and opens the possibility of exploring the introduction of Wolbachia into natural populations of anophelines as a strategy to reduce disease transmission.

In agreement with our published results (1), the epidemiological analysis by Lindestam Arlehamn et al. (2), with COVID-19 data from April 22, 2020, found a significant negative correlation ( P = 0.0006) between mean bacillus Calmette–Guerin vaccination coverage and deaths from COVID-19. However, this correlation did not hold when the authors used updated mortality data from August 1, 2020 (2). Two major factors determine the probability that a person will die from COVID-19: first, whether the person becomes infected; second, for those who become infected, how well they respond to the infection, and this is determined by biological and social variables such as age, gender, preexisting conditions, immunological history (such as bacillus Calmette–Guerin vaccination), and the access and efficacy of medical interventions. The dynamics of the COVID-19 pandemic are complex, and as time goes by, the degree of success of social distancing strategies predominate as the major determinant of the number of people that become infected and those that ultimately die from the infection. For example, COVID-19 mortality in the United States decreased from 6.1% on May 12 to 3.2% on August 12, 2020 (3), due, in part, to the large number of young people infected between June and August (4). We compared the mortality rates in different countries during the first 25 to 30 d of the pandemic, because the infections leading to those early deaths took place before … [↵][1]1To whom correspondence may be addressed. Email: cbarillas{at}niaid.nih.gov. [1]: #xref-corresp-1-1

Plasmodium falciparum malaria originated in Africa and became global as humans migrated to other continents. During this journey, parasites encountered new mosquito species, some of them evolutionarily distant from African vectors. We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. Here, we investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose that Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. A new model, the "lock-and-key theory" of P. falciparum globalization, is proposed, and its implications are discussed.

The malaria parasite, Plasmodium, must survive and develop in the mosquito vector to be successfully transmitted to a new host. The Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. Two alternative approaches were used to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses, and show that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 disrupts JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h postfeeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system.

The mosquito Anopheles gambiae is a primary vector of Plasmodium parasites in Africa. The effect of aging on reproductive output in A. gambiae females from three strains that differ in their ability to melanize Plasmodium and in their systemic levels of hydrogen peroxide (H 2 O 2 ), a reactive oxygen species (ROS), was analyzed. The number of eggs oviposited after the first blood meal decreases with age in all strains; however, this decline was much more pronounced in the G3 (unselected) and R (refractory to Plasmodium infection) strains than in the S (highly susceptible to Plasmodium ) strain. Reduction of ROS levels in G3 and R females by administration of antioxidants reversed this age-related decline in fecundity. The S and G3 strains were fixed for two functionally different catalase alleles that differ at the second amino acid position (Ser2Trp). Biochemical analysis of recombinant proteins revealed that the Trp isoform has lower specific activity and higher K m than the Ser isoform, indicating that the former is a less efficient enzyme. The Trp-for-Ser substitution appears to destabilize the functional tetrameric form of the enzyme. Both alleles are present in the R strain, and Ser/Ser females had significantly higher fecundity than Trp/Trp females. Finally, a systemic reduction in catalase activity by dsRNA-mediated knockdown significantly reduced the reproductive output of mosquito females, indicating that catalase plays a central role in protecting the oocyte and early embryo from ROS damage.

It is remarkable that the two Plasmodium species that infect millions of humans around the world today, Plasmodium falciparum and Plasmodium vivax , both originated in Africa from single common ancestors that infected wild-living apes. Plasmodium species of the subgenus Laverania , which include P. falciparum , exhibit strong host specificity, and no host transfers between humans, gorillas, and chimpanzees have been documented in nature. In contrast, host transfers take place frequently in nature for parasites of the subgenus Plasmodium that includes P. vivax and Plasmodium malariae (1⇓⇓–4). P. falciparum appears to have originated as a result of a single transfer of Plasmodium praefalciparum from gorillas to humans (1). The ability of gorilla parasites to invade the human erythrocytes appears to be a major barrier for interspecies transfer that P. praefalciparum had to overcome (5). However, although P. falciparum does not infect apes in nature, species transfers to bonobos and chimpanzees have been documented in sanctuaries where apes come in close contact with infected humans. Furthermore, experimental infections of chimpanzees with P. falciparum have been achieved multiple times under laboratory conditions (6). This evidence indicates that there is no strong biological barrier for P. falciparum to transfer back from humans to certain ape species. In PNAS, Makanga et al. (7) investigate the potential role of anopheline mosquito vectors as a barrier for interspecies transfer of Plasmodium parasites in an extensive longitudinal survey. Sylvatic anopheline species were collected in two forested wildlife reserves in Gabon (Central Africa) for a period of 15 mo., and 18 different anopheline mosquito species were identified based on morphology and molecular taxonomy. Mosquito whole body and salivary gland were screened for the presence of Plasmodium parasites by PCR amplification and sequencing of a … [↵][1]1To whom correspondence should be addressed. Email: cbarillas{at}niaid.nih.gov. [1]: #xref-corresp-1-1

Plasmodium falciparum lines differ in their ability to infect mosquitoes. The Anopheles gambiae L3-5 refractory (R) line melanizes most Plasmodium species, including the Brazilian P. falciparum 7G8 line, but it is highly susceptible to some African P. falciparum strains such as 3D7, NF54, and GB4. We investigated whether these lines differ in their ability to evade the mosquito immune system. Silencing key components of the mosquito complement-like system [thioester-containing protein 1 (TEP1), leucine-rich repeat protein 1, and Anopheles Plasmodium -responsive leucine-rich repeat protein 1] prevented melanization of 7G8 parasites, reverting the refractory phenotype. In contrast, it had no effect on the intensity of infection with NF54, suggesting that this line is able to evade TEP1-mediated lysis. When R females were coinfected with a line that is melanized (7G8) and a line that survives (3D7), the coinfection resulted in mixed infections with both live and encapsulated parasites on individual midguts. This finding shows that survival of individual parasites is parasite-specific and not systemic in nature, because parasites can evade TEP1-mediated lysis even when other parasites are melanized in the same midgut. When females from an extensive genetic cross between R and susceptible A. gambiae (G3) mosquitoes were infected with P. berghei , encapsulation was strongly correlated with the TEP1-R1 allele. However, P. falciparum 7G8 parasites were no longer encapsulated by females from this cross, indicating that the TEP1-R1 allele is not sufficient to melanize this line. Evasion of the A. gambiae immune system by P. falciparum may be the result of parasite adaptation to sympatric mosquito vectors and may be an important factor driving malaria transmission.