Selective inhibition of penicillin-binding proteins and its effects on growth and architecture of Staphylococcus aureus

We found that the three high molecular weight penicillin-binding proteins (PBP) 1, 2, and 3 of Staphylococcus aureus could be blocked by the β-lactam antibiotics imipenem, cefotaxime, and mecillinam, respectively. The inhibition of any of these PBPs was not sufficient for an antibacterial effect. Even the simultaneous blocking of PBPs 2 and 3, previously supposed to be the lethal targets of β-lactam antibiotics, did not induce bacteriolysis, nor did the combined saturation of PBPs 2, 3, and 4. Instead, PBP 1 seems to play a key role, because on one hand the combined inhibition of PBP 1 with any of the other high molecular weight PBPs led to bacteriolysis, on the other hand, only inhibition of PBP 1 led to a loss of the ‘splitting system’ of the staphylococcal cross wall, similar to that observed in penicillin G-treated cells earlier.     

Interaction of non-lytic beta-lactams with penicillin-binding proteins in Streptococcus pneumoniae

The monobactam aztreonam and the cephalosporin ceftazidime, beta-lactam antibiotics that possess the same side chain R1, showed unusual effects on exponentially growing pneumococci compared to other beta-lactams. Both antibiotics did not induce lysis even at concentrations up to 2 mg ml-1, values well above the respective MICs. However, morphological alterations and growth inhibition of the cells were observed at much lower concentrations. Binding to penicillin-binding proteins (PBPs) in vitro could be monitored directly by using anti-aztreonam antiserum and the Western blot technique. Both antibiotics showed high affinity for PBP 3, but had an extremely low affinity for PBP 2b. It is suggested that the failure to bind to PBP 2b is responsible for the failure to induce lysis in pneumococci.     

Visualization of Penicillin-Binding Proteins during Sporulation of Streptomyces griseus

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.     

A Rapid Latex Agglutination Assay for the Detection of Penicillin-Binding Protein 2′

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2′ (PBP2′) from isolates of staphylococi. PBP2′ present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2′ could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2′ positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2′ negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in β-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of β-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function. Received: 18 March 1998 / Accepted: 26 May 1998     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae

Resistance to penicillin in non-β-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42–581) into an ATG expression vector. When the recombinant PBP 2 molecules were over-expressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2′ (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2′ for [³H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other β-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

Enhanced Calcite Dissolution in the Presence of the Aerobic Methanotroph Methylosinus trichosporium

Microbial aerobic methane oxidation (MOx) is intrinsically coupled to the production of carbon dioxide, favoring carbonate dissolution. Recently, microbial organic polymers were shown to be able to induce carbonate dissolution. To discriminate between different mechanisms causing calcite dissolution, experiments were conducted in the presence of solid calcite with (1) actively growing cells (2) starving cells, and (3) dead cells of the methanotrophic bacterium Methylosinus trichosporium under brackish conditions (salinity 10) near calcite saturation (saturation state (Ω) 1.76 to 2.22). Total alkalinity and the amount of dissolved calcium markedly increased in all experiments containing M. trichosporium cells. After initial system equilibration, similar calcite dissolution rates, ranging between 20.16 (dead cells) and 25.68 μmol L^?1 d^?1 (actively growing cells), were observed. Although concentrations of transparent exopolymer particles declined with time in the presence of actively growing and starving cells, they increased in experiments with dead cells. Scanning electron microscopy images of calcite crystals revealed visible surface corrosion after exposure to live and dead M. trichosporium cells. The results of this study indicate a strong potential for microbial MOx to affect calcite stability negatively, facilitating calcite dissolution. In addition to CO₂ production by methanotrophically active cells, we suggest that the release of acidic or Ca²⁺-chelating organic carbon compounds from dead cells could also enhance calcite dissolution.     

Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis

Penicillin-resistant clinical isolates of Streptococcus pneumoniae contain mosaic penicillin-binding protein (PBP) genes that encode PBPs with decreased affinity for β-lactam antibiotics. The mosaic blocks are believed to be the result of gene transfer of homologous PBP genes from related penicillin-resistant species. We have now identified a gene homologous to the pneumococcal PBP2x gene (pbpX) in a penicillin-sensitive Streptococcus oralis isolate M3 from South Africa that diverged by almost 20% from pbpX of penicillin-sensitive pneumococci, and a central sequence block of a mosaic pbpX gene of Streptococcus mitis strain NCTC 10712. In contrast, it differed by only 2-4% of the 1 to 1.5 kb mosaic block in pbpX genes of three genetically unrelated penicillin-resistant S. pneumoniae isolates, two of them representing clones of serotype 6B and 23F, which are prevalent in Spain and are also already found in other countries. With low concentrations of cefotaxime, transformants of the sensitive S. pneumoniae R6 strain could be selected containing pbpX genes from either S. mitis NCTC 10712 or S. oralis M3, demonstrating that genetic exchange can already occur between β-lactam-sensitive species. These data are in agreement with the assumption that PBPs as penicillin-resistance determinants have evolved by the accumulation of point mutations in genes of sensitive commensal species.     

A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for β-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this β-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N-terminal extracelluiar sensor part, and an intracelluiar C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.     

CRISPR/Cas9‐mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo.In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBPl-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.     

Methicillin-resistant strains of Staphylococcus aureus; presence of identical additional penicillin-binding protein in all strains examined

Abstract The methicillin-resistant strain of Staphylococcus aureus MR-1 previously reported to possess a penicillin-binding protein 3 (PBP 3) with a decreased affinity for β-lactam antibiotics was re-examined and, in common with other resistant strains, found to contain an additional PBP (PBP 2'). Expression of the additional protein, which has a very low affinity for β-lactams, was not influenced by temperature or osmolarity of the medium in contrast with strains examined previously. It was the only PBP still available to bind radioactive β-lactams and therefore still active enzymically when strain MR-1 was grown in the presence of concentrations of β-lactam antibiotics sufficient to kill sensitive strains of S. aureus . Penicillin-peptides derived by partial proteolysis of PBP 2'-penicillin complexes of MR-1 and 3 other methicillin-resistant strains appeared to be identical and different from the penicillin-peptides derived from PBP 1, PBP 2 and PBP 3, each of which gave rise to a unique series of peptides containing covalently-bound penicillin.     

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-d-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.     

Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [³H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [³H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [³H]-benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.     

Simultaneously inhibiting undecaprenyl phosphate production and peptidoglycan synthases promotes rapid lysis in Escherichia coli

Peptidoglycan (PG) is a highly cross‐linked polysaccharide that encases bacteria, resists the effects of turgor and confers cell shape. PG precursors are translocated across the cytoplasmic membrane by the lipid carrier undecaprenyl phosphate (Und‐P) where they are incorporated into the PG superstructure. Previously, we found that one of our Escherichia coli laboratory strains (CS109) harbors a missense mutation in uppS, which encodes an enzymatically defective Und‐P(P) synthase. Here, we show that CS109 cells lacking the bifunctional aPBP PBP1B (penicillin binding protein 1B) lyse during exponential growth at elevated temperature. PBP1B lysis was reversed by: (i) reintroducing wild‐type uppS, (ii) increasing the availability of PG precursors or (iii) overproducing PBP1A, a related bifunctional PG synthase. In addition, inhibiting the catalytic activity of PBP2 or PBP3, two monofunctional bPBPs, caused CS109 cells to lyse. Limiting the precursors required for Und‐P synthesis in MG1655, which harbors a wild‐type allele of uppS, also promoted lysis in mutants lacking PBP1B or bPBP activity. Thus, simultaneous inhibition of Und‐P production and PG synthases provokes a synergistic response that leads to cell lysis. These findings suggest a biological connection that could be exploited in combination therapies.     

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.     

Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin‐binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG‐dependent nascent PG processing in vivo, and bacterial two‐hybrid analysis identified an MltG‐PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild‐type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.     

Pheromone‐binding proteins in the Asian gypsy moth females,Lymantria dispar,recognizing the sex pheromone and plant volatiles

Lepidopterans are known to have different pheromone‐binding proteins with differential expression patterns that facilitate specific signal transduction of semiochemicals. Two PBPs of the Asian gypsy moth, Lymantria dispar, were reported to express in both females and males, but their physiological functions were unknown. Results showed that LdisPBP1 and LdisPBP2 were expressed in the sensilla trichodea of males and the s. trichodea and s. basiconica of females. When LdisPBP1 gene was targeted by RNA interference (RNAi) in males, the expression of LdisPBP1 and LdisPBP2 decreased by 69 and 76%, respectively, and when LdisPBP2 gene was targeted by RNAi, they decreased by 60 and 42%, respectively. In females, after treatment with LdisPBP1 dsRNA, LdisPBP1 and LdisPBP2 levels were reduced by 26 and 69%, respectively, and LdisPBP2 dsRNA reduced the relative expression of them by 4 and 62%, respectively. The expression of LdisPBP1 and LdisPBP2 was interdependent. Electroantennogram (EAG) recordings showed that LdisPBPs participate in the recognition of the sex pheromone in males, and the sex pheromone and plant volatiles in females. The function of LdisPBPs represents the sex‐specific roles.