Detection of the Wolbachia-encoded DNA binding protein, HU beta, in mosquito gonads

Abstract

Wolbachia are obligate intracellular bacteria that cause cytoplasmic incompatibility in mosquitoes. In an incompatible cross, eggs of uninfected females fail to hatch when fertilized by sperm from infected males. We used polyacrylamide gel electrophoresis and tandem mass spectrometry to identify Wolbachia proteins in infected mosquito gonads. These included surface proteins with masses of 25 and 18 kDa and the DNA binding protein, HU beta. Using reverse transcriptase polymerase chain reaction, we showed that the HU gene is transcribed in Wolbachia-infected Culex pipiens and Aedes albopictus mosquitoes. We sequenced HU genes from four Wolbachia strains and compared deduced protein sequences with additional homologs from the databases. Among the Rickettsiales, Wolbachia HU has distinct N- and C-terminal basic/acidic amino acid motifs as well as a pair of conserved, cysteine residues.

Figure 1

Electrophoretic analysis of proteins extracted from Cx. pipiens gonads. Panel A. Lanes 1 and 2 are 150 individual testes from Cx. pipiens; lanes 3 and 4 are 30 individual ovaries. Lanes 1 and 3 (+) are from Wolbachia-infected mosquitoes; lanes 2 and 4 are from a tetracycline-cured sister colony. Panel B represents an independent biological replicate from testes of infected (lane 5) and cured (lane 6) Cx. pipiens. Protein bands at 25 kDa and 18 kDa were visible in 8 biological replicates. Lane 5 shows a better replicate of the 18 kDa band as well as smaller bands that were examined by mass spectrometry.

Figure 2

Total peptide coverage detected for the three Wolbachia proteins. A. Wolbachia surface protein, WSP. Coverage varied from 39% – 50% among 5 replicates. B. Wolbachia putative membrane protein. Coverage varied from 14% – 36% among 2 replicates C. Wolbachia DNA binding protein HU beta. Coverage varied from 36% – 67% among 5 replicates. Shaded boxes indicate mass spectrometry identified peptides.

Figure 3

Comparisons among HU beta proteins. A. Amino acid sequence alignment of wPip HU beta and its E. coli orthologp. Asterisks indicate amino acid identities, and the downward-pointing arrow indicates a proline residue important in DNA binding. Under the E. coli sequence, residues that participate in alpha helix (α) and beta sheet structure (β) as defined by Swinger and Rice (2004) are underlined. B. Alignment of HU beta amino acid sequences. Wolbachia genes from strains wPip, wAlbB, wLec, and wStr were experimentally determined in this study; others were from Wolbachia genomes available in the database. C. Amino acid sequence alignment of Wolbachia HU beta from (wPip and wBm) compared with representative orthologs from Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Rickettsia typhi. N and C-terminal charged motifs composed of lysine, arginine, glutamic acid, and aspartic acid in HU beta are shown in black boxes. Cysteine residues are shown in grey. For other residues, dots indicate identities.

Figure 4

Partial linearized genome of Wolbachia pipientis. Genes described in this study are mapped by solid arrows showing the direction of transcription. Flanking the hupB (0612) are primers HUCloneF/R (indicated by small black arrows), which map immediately downstream of rpsI and within pdxJ, respectively, used to clone HU sequences from other Wolbachia strains described in Fig. 3. Amino acid sequences of HU, WSP (0937) and the putative membrane protein (0576) are described in Fig. 2. Genes encoding IHF alpha (0738) and IHF beta (1041), transcribed in opposite directions, are also shown. Locations of ribosomal protein genes potentially transcribed as operons that include hupB (rplM, rpsI) and ihfB (rpsA) are also represented.

Figure 5

RT PCR analysis of the hupB transcript in pooled decapitated mosquitoes and uninfected mosquito cells. M is a DNA marker. Lanes 1 and 11 are RNA extracted from uninfected C7–10 mosquito cells. Lanes 3, 4, and 12 are total RNA extracted from Culex pipiens females. Lanes 5, 6, and 13 are RNA extracted from Culex pipiens males. Lanes 7, 8, and 14 are RNA extracted from Aedes albopictus females. Lanes 9, 10, and 15 are RNA extracted from Aedes albopictus males. Lanes 2, 4, 6, 8, and 10 were assayed without reverse transcriptase as a control for DNA contamination of RNA preparations. Lanes 11–15 are positive controls for RNA quality using primers for the mosquito ribosomal protein RpS3.

Figure 6

IHF amino acid sequence alignments. A. Sequence alignment of wPip IHF alpha with its E. coli ortholog. B. Sequence alignment of known Wolbachia IHF alpha homologs. C. Sequence alignment of wPip IHF beta with its E. coli ortholog. D. Sequence alignment of known Wolbachia IHF beta homologs. Asterisks indicate amino acid identities. Identities relative to the wPip proteins are indicated by dots.