Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X

During the intravascular stage of infection, the malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and exits upon rupture of the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also processes itself to generate different isoforms that remain present in terminal schizonts. The function of these different forms is not understood. We have mapped the autoprocessing cleavage sites and constructed parasites with cleavage site mutations. Surprisingly, all the cleavage mutant forms of PM X, including a quadruple mutant that remained full-length, retained in vitro activity, were trafficked normally in the parasites, and supported parasite growth and normal egress and invasion. Further analysis showed that the N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper folding and intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, possibly in a common precursor organelle, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.

Tailoring Pullulanase PulAR from Anoxybacillus sp. AR-29 for Enhanced Catalytic Performance by A Structure-Guided Consensus Approach

Pullulanase is a well-known debranching enzyme that can specially hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four sites (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase PulAR were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Four beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 ℃, pH 5.0 and 6.0, respectively. In addition, its thermostabilities at 60 ℃ and 65 ℃ were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced via rational engineering by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.

New insights into antimalarial chemopreventive activity of antifolates

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, the thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, have yet to be achieved. Using a transgenic P. berghei harboring quadruple mutant dhfr from P. falciparum (Pb::Pfdhfr -4M ) , we demonstrate that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not to a new class of antifolate designed to overcome the resistance such as P218. Detailed investigation to pin-point stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data shows that P218 also inhibited the late hepatic stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg, P218 prevented infection from up to 25,000 pyrimethamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates, as a chemopreventive therapeutic which could lead to a more efficient and cost effective treatment regime.

Listeria monocytogenes 10403S Alternative Sigma-54 Factor σL Has a Negative Role on Survival Ability Under Bile Exposure

Listeria monocytogenes is a Gram-positive bacterium causing listeriosis in animals and humans. To initiate a foodborne infection, L. monocytogenes has to pass through the host gastrointestinal tract (GIT). In this study, we evaluated survival abilities of L. monocytogenes 10403S wild type (WT) and its isogenic mutants in alternative sigma (σ) factor genes (i.e., sigB, sigC, sigH, and sigL) under simulated gastric, duodenal, and bile fluids. Within 10min of exposures, only bile fluid was able to significantly reduce survival ability of L. monocytogenes WT by 2 logs CFU/ml. Loss of sigL showed the greatest bile resistance among 16 strains tested, p<0.0001, (i.e., WT, four single alternative σ factor mutants, six double mutants, four triple mutants, and one quadruple mutant). To further investigate the role of σL in bile response, RNA-seq was conducted to compare the transcriptional profiles among L. monocytogenes 10403S ΔBCH triple mutant (lacking sigB, sigC, and sigH genes; expressing housekeeping σA and σL) and ΔBCHL quadruple mutant (lacking all alternative sigma factor genes; expressing only σA) strains under BHI and 1% bile conditions. A total of 216 and 176 differentially expressed genes (DEGs) were identified in BHI and bile, respectively. We confirmed that mpt operon was shown to be strongly activated by σL. Interestingly, more than 80% of DEGs were found to be negatively regulated in the presence of σL. This includes PrfA regulon and its mediated genes (i.e., hly, hpt, inlB, clpP, clpE, groL, and inlC) which were downregulated in response to bile in the presence of σL. This result suggests the potential negative role of σL on bile survival, and the roles of σL and σB might be in a seesaw model prior to host cell invasion.

Formal proof of the requirement of MESP1 and MESP2 in mesoderm specification and their transcriptional control via specific enhancers in mice

ABSTRACT MESP1 and MESP2 are transcriptional factors involved in mesoderm specification, somite boundary formation and somite polarity regulation. However, Mesp quadruple mutant zebrafish displayed only abnormal somite polarity without mesoderm specification defects. In order to re-evaluate Mesp1/Mesp2 mutants in mice, Mesp1 and Mesp2 single knockouts (KOs), and a Mesp1/Mesp2 double KO were established using genome-editing techniques without introducing selection markers commonly used before. The Mesp1/Mesp2 double KO embryos exhibited markedly severe mesoderm formation defects that were similar to the previously reported Mesp1/Mesp2 double KO embryos, indicating species differences in the function of MESP family proteins. However, the Mesp1 KO did not display any phenotype, including heart formation defects, which have been reported previously. We noted upregulation of Mesp2 in the Mesp1 KO embryos, suggesting that MESP2 rescues the loss of MESP1 in mesoderm specification. We also found that Mesp1 and Mesp2 expression in the early mesoderm is regulated by the cooperation of two independent enhancers containing T-box- and TCF/Lef-binding sites. Deletion of both enhancers caused the downregulation of both genes, resulting in heart formation defects. This study suggests dose-dependent roles of MESP1 and MESP2 in early mesoderm formation.

A Phytochrome B-PIF4-MYC2 Module Tunes Secondary Cell Wall Thickening in Response to Shaded Lighting

Secondary cell walls (SCW) in stem xylem cells provide mechanical strength and structural support for growth. SCW thickening is light- regulated and varies under different light growth conditions. Our previous study revealed that blue light enhances SCW thickening through the activity of MYC2 directed by CRYPTOCHROME1 (CRY1) signaling in stem xylary fiber cells. In this study, we demonstrate that the low ratio of red: far-red light (R:FR) of the shaded light condition inhibits SCW thickening in the inflorescence stem of Arabidopsis. Phytochrome B (PHYB) plays a dominant role in perceiving the R:FR balance. Under white and red-light conditions, phyB mutants display thinner SCWs in xylary fibers, but thicker SCWs are deposited in the PHYTOCHROME INTERACTING FACTORS (PIFs) quadruple mutant pif1pif3pif4pif5 (pifq), suggesting involvement of the PHYB-PIFs signaling module in regulating SCW thickening. Interaction of PIF4 with MYC2 affects MYC2 localization in nuclei and inhibits its transactivation activity on the NST1 promoter. Shade conditions mediate the PIF4 interaction with MYC2 to regulate SCW thickening. Genetic analysis confirms that the regulation of SCW thickening by PIFs is dependent on MYC2 function. Together, these data reveal a molecular mechanism for the effect of shaded light inhibition on SCW thickening in stems of Arabidopsis.

Decreased susceptibility to dihydrofolate reductase inhibitors associated with genetic polymorphisms in Ugandan Plasmodium falciparum isolates

Background The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug susceptibility data for P. falciparum field isolates are limited. Methods We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda from 2016-2020, sequenced 383 isolates, and assessed associations between genotypes and drug susceptibility phenotypes. Results Median IC50’s were 42,100 nM for pyrimethamine, 1,200 nM for cycloguanil, 13,000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, three PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. Conclusion Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

New insights into antimalarial chemopreventive activity of antifolates

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, the thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, have yet to be achieved. Using a transgenic P. berghei harboring quadruple mutant dhfr from P. falciparum (Pb::Pfdhfr-4M), we demonstrate that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not a new class of antifolate designed to overcome the resistance such as P218. Detailed investigation to pin-point stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data shows that P218 also inhibited the late hepatic stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg, P218 prevented infection from up to 25,000 pyrimentamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates, as a chemopreventive therapeutic which could lead to a more efficient and cost effective treatment regime.

Genetic and biochemical characterization of the Na + /H + antiporters of Pseudomonas aeruginosa

Pseudomonas aeruginosa has four Na + /H + antiporters that interconvert and balance Na + and H + gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we have constructed four strains, each of which has only one antiporter: NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na + /H + antiporters. Although the antiporters of P. aeruginosa have previously been studied, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa . The strains expressing only NhaB or Mrp, the two electrogenic antiporters, are able to grow essentially as the wild type across a range of [Na + ] and pH. Strains with only NhaP or NhaP2, which are electroneutral, grow more poorly at increasing [Na + ], especially at high pH, with NhaP the most sensitive. The strain with no Na + /H + antiporters is extremely sensitive to [Na + ] and shows essentially no Na + (Li + )/H + antiporter activity but retains most K + /H + antiporter activity of the wild type at pH 7.5 and approximately half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. RNA-seq analysis of the quadruple deletion strain showed widespread changes, including pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological role of these proteins and their activities in P. aeruginosa . Importance Pseudomonas aeruginosa has four Na + /H + antiporters that connect and interconvert its Na + and H + gradients. We have constructed four deletion mutants, each of which has only one of the four Na + /H + antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH and [Na + ], whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH. We also constructed a quadruple mutant, lacking all four antiporters in which the H + and Na + gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Polymorphism of Antifolate Drug Resistance in Plasmodium vivax From Local Residents and Migrant Workers Returned From the China-Myanmar Border

Drug-resistant Plasmodium vivax malaria impedes efforts to control, eliminate, and ultimately eradicate malaria in Southeast Asia. P. vivax resistance to antifolate drugs derives from point mutations in specific parasite genes, including the dihydropteroate synthase (pvdhps), dihydrofolate reductase (pvdhfr), and GTP cyclohydrolase I (pvgch1) genes. This study aims to investigate the prevalence and spread of drug resistance markers in P. vivax populating the China-Myanmar border. Blood samples were collected from symptomatic patients with acute P. vivax infection. Samples with single-clone P. vivax infections were sequenced for pvdhps and pvdhfr genes and genotyped for 6 flanking microsatellite markers. Copy number variation in the pvgch1 gene was also examined. Polymorphisms were observed in six different codons of the pvdhps gene (382, 383, 512, 549, 553, and 571) and six different codons of the pvdhfr gene (13, 57, 58, 61, 99, 117) in two study sites. The quadruple mutant haplotypes 57I/L/58R/61M/117T of pvdhfr gene were the most common (comprising 76% of cases in Myitsone and 43.7% of case in Laiza). The double mutant haplotype 383G/553G of pvdhps gene was also prevalent at each site (40.8% and 31%). Microsatellites flanking the pvdhfr gene differentiated clinical samples from wild type and quadruple mutant genotypes (FST= 0.259-0.3036), as would be expected for a locus undergoing positive selection. The lack of copy number variation of pvgch1 suggests that SP-resistant P. vivax may harbor alternative mechanisms to secure sufficient folate.