Tatiany Patrícia Romão

A second independent resistance mechanism to Bacillus sphaericus binary toxin targets its α‐glucosidase receptor in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the ≈ 60‐kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino‐acid sequence confirmed its identity as an α‐glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19‐nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full‐length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N‐terminal 45‐kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense‐mediated decay pathway of mRNA degradation. In‐gel assays confirmed that a single α‐glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N‐terminus of the receptor.

The orthologue to the Cpm1/Cqm1 receptor in Aedes aegypti is expressed as a midgut GPI-anchored α-glucosidase, which does not bind to the insecticidal binary toxin

Aedes aegypti larvae are refractory to the insecticidal binary (Bin) toxin from Bacillus sphaericus, which is not able to bind to its target tissue in the larval midgut. In contrast, Culex pipiens larvae are highly susceptible to that toxin, which targets its midgut brush border membranes (BBMF) through the binding of the BinB subunit to specific receptors, the Cpm1/Cqm1 membrane-bound α-glucosidases. The identification of an Ae. aegypti gene encoding a Cpm1/Cqm1 orthologue, here named Aam1, led to the major goal of this study which was to investigate its expression. The aam1 transcript was found in larvae and adults from Ae. aegypti and a ≈73-kDa protein was recognized by an anti-Cqm1 antibody in midgut BBMF. The Aam1 protein displayed α-glucosidase activity and localized to the midgut epithelium, bound through a GPI anchor, similarly to Cpm1/Cqm1. However, no binding of native Aam1 was observed to the recombinant BinB subunit. Treatment of both proteins with endoglycosidase led to changes in the molecular weight of Aam1, but not Cqm1, implying that the former was glycosylated. The findings from this work rule out lack of receptors in larval stages, or its expression as soluble proteins, as a reason for Ae. aegypti refractoriness to Bin toxin.

Detection of an Allele Conferring Resistance to Bacillus sphaericus Binary Toxin in Culex quinquefasciatus Populations by Molecular Screening

ABSTRACT The activity of the Bacillus sphaericus binary (Bin) toxin on Culex quinquefasciatus larvae depends on its specific binding to the Cqm1 receptor, a midgut membrane-bound α-glucosidase. A 19-nucleotide deletion in the cqm1 gene (cqm1REC) mediates high-level resistance to Bin toxin. Here, resistance in nontreated and B. sphaericus-treated field populations of C. quinquefasciatus was assessed through bioassays as well as a specific PCR assay designed to detect the cqm1REC allele in individual larvae. Resistance ratios at 90% lethal concentration, gathered through bioassays, were close to 1 and indicate that the selected populations had similar levels of susceptibility to B. sphaericus, comparable to that of a laboratory colony. A diagnostic PCR assay detected the cqm1REC allele in all populations investigated, and its frequency in two nontreated areas was 0.006 and 0.003, while the frequency in the B. sphaericus-treated population was significantly higher. Values of 0.053 and 0.055 were detected for two distinct sets of samples, and homozygote resistant larvae were found. Evaluation of Cqm1 expression in individual larvae through α-glucosidase assays corroborated the allelic frequency revealed by PCR. The data from this study indicate that the cqm1REC allele was present at a detectable frequency in nontreated populations, while the higher frequency in samples from the treated area is, perhaps, correlated with the exposure to B. sphaericus. This is the first report of the molecular detection of a biolarvicide resistance allele in mosquito populations, and it confirms that the PCR-based approach is suitable to track such alleles in target populations.

Novel Mutations Associated with Resistance to Bacillus sphaericus in a Polymorphic Region of the Culex quinquefasciatus cqm1 Gene

ABSTRACT Bin toxin from Bacillus sphaericus acts on Culex quinquefasciatus larvae by binding to Cqm1 midgut-bound receptors, and disruption of the cqm1 gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistance cqm1REC allele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in the cqm1 gene. The cqm1REC allele was detected in the four nontreated populations surveyed at frequencies from 0.001 to 0.017, and sequence analysis from these samples revealed a novel resistant allele (cqm1REC-D16 ) displaying a 16-nucletotide (nt) deletion which is distinct from the 19-nt deletion associated with cqm1REC . Yet a third resistant allele (cqm1REC-D25 ), displaying a 25-nt deletion, was identified in samples from a treated area exposed to B. sphaericus. A comparison of the three deletion events revealed that all are located within the same 208-nt region amplified during the screening procedure. They also introduce equivalent frameshifts in the sequence and generate the same premature stop codon, leading to putative transcripts encoding truncated proteins which are unable to locate to the midgut epithelium. The populations analyzed in this study contained a variety of alleles with mutations disrupting the function of the corresponding Bin toxin receptor. Their locations reveal a hot spot that can be exploited to assess the resistance risk through DNA screening.

The N-terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus.

Heterodimeric binary (Bin) toxin, the major insecticidal protein from Bacillus sphaericus, acts on Culex quinquefasciatus larvae through specific binding to the midgut receptor Cqm1, a role mediated by its 448-amino-acid-long BinB subunit. The molecular basis for receptor recognition is not well understood and this study attempted to identify protein segments and amino acid motifs within BinB that are required for this event. First, N- and C-terminally truncated constructs were evaluated for their capacity to bind to native Cqm1 through pull-down assays. These showed that residues N33 to L158 of the subunit are required for Cqm1 binding. Nine different full-length mutants were then generated in which selected blocks of three amino acids were replaced by alanines. In new pull-down assays, two mutants, in which residues (85) IRF(87) and (147) FQF(149) were targeted, failed to bind the receptor. Competition binding assays confirmed the requirements for the N-terminal 158 residues, and the (147) FQF(149) epitope, for the mutant proteins to compete with native Bin toxin when binding to membrane fractions from the insect midgut. The data from this work rule out the involvement of C-terminal segments in receptor binding, highlighting the need for multiple elements within the proteins N-terminal third for it to occur.

Non conserved residues between Cqm1 and Aam1 mosquito α-glucosidases are critical for the capacity of Cqm1 to bind the Binary toxin from Lysinibacillus sphaericus

The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound α-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native α-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding cqm1 and aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the 159GG160 doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1s structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4s partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

Co‐selection and replacement of resistance alleles to Lysinibacillus sphaericus in a Culex quinquefasciatus colony

The Cqm1 α‐glucosidase, expressed within the midgut of Culex quinquefasciatus mosquito larvae, is the receptor for the Binary toxin (Bin) from the entomopathogen Lysinibacillus sphaericus. Mutations of the Cqm1 α‐glucosidase gene cause high resistance levels to this bacterium in both field and laboratory populations, and a previously described allele, cqm1REC, was found to be associated with a laboratory‐resistant colony (R2362). This study described the identification of a novel resistance allele, cqm1REC‐2, that was co‐selected with cqm1REC within the R2362 colony. The two alleles display distinct mutations but both generate premature stop codons that prevent the expression of midgut‐bound Cqm1 proteins. Using a PCR‐based assay to monitor the frequency of each allele during long‐term maintenance of the resistant colony, cqm1REC was found to predominate early on but later was replaced by cqm1REC‐2 as the most abundant resistance allele. Homozygous larvae for each allele were then generated that displayed similar high‐resistance phenotypes with equivalent low levels of transcript and lack of protein expression for both cqm1REC and cqm1REC‐2. In progeny from a cross of homozygous individuals for each allele at a 1 : 1 ratio, analyzed for ten subsequent generations, cqm1REC showed a higher frequency than cqm1REC‐2. The replacement of cqm1REC by cqm1REC‐2 observed in the R2362 colony, kept for 210 generations, indicates changes in fitness related to traits that are unknown but linked to these two alleles, and constitutes a unique example of evolution of resistance within a controlled laboratory environment.

A new allele conferring resistance to Lysinibacillus sphaericus is detected in low frequency in Culex quinquefasciatus field populations

BackgroundThe Cqm1 α-glucosidase of Culex quinquefasciatus larvae acts as the midgut receptor for the binary toxin of the biolarvicide Lysinibacillus sphaericus. Mutations within the cqm1 gene can code for aberrant polypeptides that can no longer be properly expressed or bind to the toxin, leading to insect resistance. The cqm1REC and cqm1REC-2 alleles were identified in a laboratory selected colony and both displayed mutations that lead to equivalent phenotypes of refractoriness to L. sphaericus. cqm1REC was first identified as the major resistance allele in this colony but it was subsequently replaced by cqm1REC-2, suggesting the better adaptive features of the second allele. The major aim of this study was to evaluate the occurrence of cqm1REC-2 and track its origin in field populations where cqm1REC was previously identified.MethodsThe screening of the cqm1REC-2 allele was based on more than 2000 C. quinquefasciatus larvae from five localities in the city of Recife, Brazil and used a multiplex PCR assay that is also able to identify cqm1REC. Full-length sequencing of the cqm1REC-2 and selected cqm1 samples was performed to identify further polymorphisms between these alleles.ResultsThe cqm1REC-2 allele was found in field samples, specifically in two heterozygous individuals from a single locality with an overall frequency and distribution much lower than that observed for cqm1REC. The full-length sequences from these two cqm1REC-2 copies were almost identical to the cqm1REC-2 derived from the resistant colony but displayed more than 30 SNPs when compared with cqm1 and cqm1REC. The cqm1REC and cqm1REC-2 resistant alleles were found to be associated with two distinct sets of wild-type cqm1 variants found in field populations.C onclusionsThe cqm1REC-2 allele occurs in populations in Recife and was probably already present in the samples used to establish the laboratory resistant colony. The data generated indicates that cqm1REC-2 can be selected in field populations, although its low frequency and distribution in Recife suggest that cqm1REC-2 presents a lower risk of selection compared to cqm1REC.

Phosphorylation and interactions associated with the control of the Leishmania Poly-A Binding Protein 1 (PABP1) function during translation initiation

ABSTRACT The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.