Wen-Long Cho

Mosquito Cathepsin B-like Protease Involved in Embryonic Degradation of Vitellin Is Produced as a Latent Extraovarian Precursor

Here we report identification of a novel member of the thiol protease superfamily in the yellow fever mosquito,Aedes aegypti. It is synthesized and secreted as a latent proenzyme in a sex-, stage-, and tissue-specific manner by the fat body, an insect metabolic tissue, of female mosquitoes during vitellogenesis in response to blood feeding. The secreted, hemolymph form of the enzyme is a large molecule, likely a hexamer, consisting of 44-kDa subunits. The deduced amino acid sequence of this 44-kDa precursor shares high similarity with cathepsin B but not with other mammalian cathepsins. We have named this mosquito enzyme vitellogenic cathepsin B (VCB). VCB decreases to 42 kDa after internalization by oocytes. In mature yolk bodies, VCB is located in the matrix surrounding the crystalline yolk protein, vitellin. At the onset of embryogenesis, VCB is further processed to 33 kDa. The embryo extract containing the 33-kDa VCB is active toward benzoyloxycarbonyl-Arg-Arg-para-nitroanilide, a cathepsin B-specific substrate, and degrades vitellogenin, the vitellin precursor. Both of these enzymatic activities are prevented by trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64), a thiol protease inhibitor. Furthermore, addition of the anti-VCB antibody to the embryonic extract prevented cleavage of vitellogenin, strongly indicating that the activated VCB is involved in embryonic degradation of vitellin.

Mosquito ecdysteroid receptor: Analysis of the cDNA and expression during vitellogenesis

An insect steroid hormone, 20-hydroxyecdysone (20E), plays an important role in regulating egg maturation in mosquitoes. To better understand its role, we cloned the cDNA coding for the putative ecdysteroid receptor from the mosquito, Aedes aegypti (AaEcR). The 4158 bp AaEcR cDNA has an open reading frame of 675 amino acids with 10 potential glycosylation sites and a putative phosphorylation polyserine domain. The AaEcR has a DNA binding domain with two zinc fingers and a ligand binding domain characteristic of members of the steroid hormone receptor superfamily. These AaEcR domains share 97 and 87% identities with the respective domains of the Drosophila ecdysteroid receptor (DmEcR). However, the A/B region of the AaEcR shares 35% identity with that of DmEcR-B1 isoform. The F region, located at the carboxyl-terminal of the AaEcR, has only 9% identity with the corresponding region of DmEcR. Potential nuclear targeting and dimerization signals are also present in the AaEcR sequence. There are three AaEcR transcripts of 4.2 kb, 6 kb and 11 kb in adult mosquitoes. 4.2 kb mRNA is predominantly expressed in female mosquitoes during vitellogenesis. In both the fat body and ovaries of the female mosquito, the level of AaEcR mRNA is high at the previtellogenic period and after the onset of vitellogenesis (6 h post blood meal, PBM).

Description of the Transcriptomes of Immune Response-Activated Hemocytes from the Mosquito Vectors Aedes aegypti and Armigeres subalbatus

ABSTRACT Mosquito-borne diseases, including dengue, malaria, and lymphatic filariasis, exact a devastating toll on global health and economics, killing or debilitating millions every year (54). Mosquito innate immune responses are at the forefront of concerted research efforts aimed at defining potential target genes that could be manipulated to engineer pathogen resistance in vector populations. We aimed to describe the pivotal role that circulating blood cells (called hemocytes) play in immunity by generating a total of 11,952 Aedes aegypti and 12,790 Armigeres subalbatus expressed sequence tag (EST) sequences from immune response-activated hemocyte libraries. These ESTs collapsed into 2,686 and 2,107 EST clusters, respectively. The clusters were used to adapt the web-based interface for annotating bacterial genomes called A Systematic Annotation Package for Community Analysis of Genomes (ASAP) for analysis of ESTs. Each cluster was categorically characterized and annotated in ASAP based on sequence similarity to five sequence databases. The sequence data and annotations can be viewed in ASAP at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm . The data presented here represent the results of the first high-throughput in vivo analysis of the transcriptome of immunocytes from an invertebrate. Among the sequences are those for numerous immunity-related genes, many of which parallel those employed in vertebrate innate immunity, that have never been described for these mosquitoes.

Differences in phenotypic and genotypic characteristics among imipenem-non-susceptible Acinetobacter isolates belonging to different genomic species in Taiwan.

In this study, we investigated the distribution of genes encoding various carbapenemases as well as their association with carbapenem resistance in clinical isolates of Acinetobacter genomic species from Taiwan. A total of 129 imipenem-non-susceptible and 79 imipenem-susceptible isolates were examined, of which 185 (88.9%) were Acinetobacter baumannii. Among the 185 A. baumannii isolates, imipenem non-susceptibility was more common in isolates with ISAba1-bla(OXA-51-like) (72/75; 96%), bla(OXA-58-like) (33/33; 100%) or bla(OXA-24-like) (7/7; 100%) than in isolates with only bla(OXA-51-like) (4/72; 5.6%). A metallo-beta-lactamase (MBL) gene was present in two isolates of imipenem-resistant A. baumannii, and bla(OXA-58-like) was also present in these isolates. A total of 18% and 1% of imipenem-non-susceptible isolates of A. baumannii were resistant to tigecycline and colistin, respectively. Among the 23 isolates of non-baumannii Acinetobacter spp., bla(OXA-58-like) and MBL genes were widely disseminated in the imipenem-resistant isolates, and isolates with bla(OXA-58-like) and MBL genes had higher imipenem minimum inhibitory concentrations than those with bla(OXA-58-like) alone. Although the rate of non-susceptibility to colistin was 26.7% among the imipenem-non-susceptible isolates of non-baumanniiAcinetobacter, 93.3% and 100% were susceptible to ciprofloxacin and tigecycline, respectively. In conclusion, different isolates of imipenem-non-susceptible A. baumannii and non-baumanniiAcinetobacter contained different carbapenemases and had different antimicrobial susceptibilities.

Acquisition of a Plasmid-Borne blaOXA-58 Gene with an Upstream IS1008 Insertion Conferring a High Level of Carbapenem Resistance to Acinetobacter baumannii

ABSTRACT The oxacillinase gene was reported to confer limited resistance to carbapenem in Acinetobacter baumannii. In this study, we have demonstrated that an A. baumannii clinical isolate harboring a plasmid, pTVICU53, has 11,037 bp encoding 13 open reading frames. A blaOXA-58 gene with an upstream insertion of truncated ISAba3 (ΔISAba3) and IS1008 was found in this plasmid. ΔISAba3and IS1008 provided two independent promoters for the transcription control of the blaOXA-58 gene. The transformation of pTVICU53 or a shuttle vector bearing IS1008-ΔISAba3-blaOXA-58 to different A. baumannii recipients can increase their MICs of carbapenem 64- to 256-fold. The deletion of promoters provided by IS1008 resulted in dramatic decreases in blaOXA-58 transcription and a 32- to 64-fold reduction in the carbapenem MIC. These findings highlight that A. baumannii might develop carbapenem resistance with a single transformation step, taking up a plasmid containing a genetic construct with a potentially high level of transcription of the blaOXA-58 gene.

Molecular cloning, characterization and tissue expression of prophenoloxidase cDNA from the mosquito Armigeres subalbatus inoculated with Dirofilaria immitis microfilariae.

A cDNA encoding mosquito Armigeres subalbatus pro‐phenol oxidase (As‐pro‐PO) was obtained by rapid amplification of cDNA ends‐polymerase chain reaction (RACE‐PCR) after Dirofilaria immitis inoculation. The 2205 bp As‐pro‐PO cDNA contains a 32 bp 5′‐noncoding region, a 2055 bp open reading frame (685 amino acids), and a 118 bp 3′‐noncoding region. Hydrophobic signal peptide for the endoplasmic reticulum targeting is not found in the NH2‐terminal region. Two potential copper‐binding domains, amino acids 197–245 and 345–412, are highly homologous to those of the other insect pro‐POs. A 2.2 kb As‐pro‐PO transcript was identified by Northern blot analysis using D. immitis microfilariae‐inoculated A. subalbatus. Both in situ hybridization and Northern blot analysis demonstrated that As‐pro‐PO mRNA was synthesized in mosquito haemocytes but not in other tissues, i.e. fat bodies, midguts and ovaries, etc.

Cloning and characterization of cDNAs encoding the antibacterial peptide, defensin A, from the mosquito, Aedes aegypti

Insect defensins are cationic, inducible antibacterial peptides. Four full-length cDNAs encoding defensin A from the mosquito Aedes aegypti were cloned using polymerase chain reaction (PCR) and sequenced. All four cDNAs are 473 base pairs long, bearing an open reading frame of 98 amino acids with a few substitutions in the signal peptide domain. The deduced amino acid sequence of Aedes aegypti defensin (AaDef) contains a signal peptide sequence of 18 amino acids followed by a 40-amino acid putative propeptide domain and a 40-amino acid mature peptide domain. The mature peptide, with a predicted M(r) of 4148, shows 80% identity and 93% similarity to Phormia defensin A, and is identical to the peptide sequencing data for mosquito defensin A of Lowenberger et al. (1995) and B of Chalk et al. (1995). There are three potential phosphorylation sites but no glycosylation sites detected in AaDef. Three putative disulfide linkages between cysteines, characteristic of insect defensins, are conserved in AaDef. Aedes aegypti defensin mRNA is produced in response to a bacterial challenge; it is dramatically enhanced 6 h after bacterial injection, continues to increase through 24 h, and is maintained at high levels until at least 30 h post-bacterial injection.

First identification of blaOXA-51-like in non-baumannii Acinetobacter spp.

Abstract bla OXA-51-like, the intrinsic carbapenemase gene in Acinetobacter baumannii previously found only in this species, was detected in a clinical isolate of Acinetobacter genomic species 13TU. This study aimed to characterize this gene in the isolate. Genomic species identification was confirmed by amplified ribosomal DNA restriction analysis and sequence analysis of 16S-23S ribosomal DNA intergenic spacer, rpoB and recA. The bla OXA-51-like gene, with an upstream ISAba1 insertion, was plasmid-encoded and the surrounding sequences suggested that its origin was from A. baumannii. Transformation of Acinetobacter genomic species 13TU ATCC 17903 with recombinant plas-mid bearing ISAba1-bla OXA-51-like from the isolate increased the minimum inhibitory concentrations (MICs) of meropenem and imipenem 256-fold. This is the first reportof bla OXA-51-like in an organism other than A. baumannii. This plasmid-borne bla OXA-51-like gene with an upstream ISAba1 insertion confers a high level of carbapenem resistance to Acinetobacter genomic species 13TU

Molecular characterization of a defensin gene from the mosquito, Aedes aegypti

Insect immune proteins, defensins, are inducible anti-Gram-positive bacterial peptides. We report here the identification of two defensin genes from the mosquito, Aedes aegypti, which encode a large 541 bp transcript (AaDef Ala) and a small 473 bp transcript (AaDef Asm). The cDNA corresponding to AaDef Ala was cloned, sequenced, and compared with the previously reported AaDef Asm cDNA. The AaDef Ala gene was isolated through genomic library screening and characterized. It putative regulatory region contains a 64 bp intron, a TATA box and a putative arthropod initiator. Two 150 bp long direct and several palindromic repeats are present in this sequence. Similar to other insect immune peptide genes, the AaDef Ala gene contains numerous putative regulatory motifs with impressive similarity to elements of vertebrate acute phase response protein genes.

Contribution of a Plasmid-Borne blaOXA-58 Gene with Its Hybrid Promoter Provided by IS1006 and an ISAba3-Like Element to β-Lactam Resistance in Acinetobacter Genomic Species 13TU

ABSTRACT The contribution of the blaOXA-58 gene and its promoter to β-lactam resistance has not been validated in Acinetobacter spp. other than Acinetobacter baumannii. We identified a multidrug-resistant (including carbapenem resistance) Acinetobacter genomic species 13TU in which blaOXA-58 was the only detected carbapenemase gene. The blaOXA-58 gene was plasmid located, flanked by ISAba3 (downstream) and an ISAba3-like element (upstream). An IS1006 element was inserted into ISAba3-like (IS1006-ΔISAba3-like) to generate a hybrid promoter for blaOXA-58, with a −35 promoter located in IS1006 and a −10 promoter in ISAba3-like. The reference strain of Acinetobacter genomic species 13TU, ATCC 17903, revealed higher MICs of amoxicillin, ticarcillin, and piperacillin and heteroresistance to imipenem and meropenem when it was transformed with a shuttle vector containing a fragment encompassing ΔISAba3-like-blaOXA-58, compared to the same host containing only blaOXA-58. When the fragment was changed from ΔISAba3-like-blaOXA-58 to IS1006-ΔISAba3-like-blaOXA-58, the ATCC 17903 transformant revealed a markedly higher level of blaOXA-58 transcription (12-fold), increased cefuroxime and piperacillin-tazobactam MICs, and homoresistance to imipenem and meropenem. Different roles of the insertion elements preceding the blaOXA-58 gene in Acinetobacter genomic species 13TU are demonstrated. The ISAba3-like--blaOXA-58 construct can mediate resistance to penicillin derivatives but only heteroresistance to carbapenems. The insertion of IS1006 into ISAba3-like, generating a hybrid promoter, could further enhance the transcription of blaOXA-58 and mediate homoresistance to carbapenems and also enhanced resistance to piperacillin-tazobactam.