Abstract
Dactylopius species, known as cochineal insects, are the source of the carminic acid dye used worldwide. The presence of two Wolbachia strains in Dactylopius coccus from Mexico was revealed by PCR amplification of wsp and sequencing of 16S rRNA genes. A metagenome analysis recovered the genome sequences of Candidatus Wolbachia bourtzisii wDacA (supergroup A) and Candidatus Wolbachia pipientis wDacB (supergroup B). Genome read coverage, as well as 16S rRNA clone sequencing, revealed that wDacB was more abundant than wDacA. The strains shared similar predicted metabolic capabilities that are common to Wolbachia, including riboflavin, ubiquinone, and heme biosynthesis, but lacked other vitamin and cofactor biosynthesis as well as glycolysis, the oxidative pentose phosphate pathway, and sugar uptake systems. A complete tricarboxylic acid cycle and gluconeogenesis were predicted as well as limited amino acid biosynthesis. Uptake and catabolism of proline were evidenced in Dactylopius Wolbachia strains. Both strains possessed WO-like phage regions and type I and type IV secretion systems. Several efflux systems found suggested the existence of metal toxicity within their host. Besides already described putative virulence factors like ankyrin domain proteins, VlrC homologs, and patatin-like proteins, putative novel virulence factors related to those found in intracellular pathogens like Legionella and Mycobacterium are highlighted for the first time in Wolbachia. Candidate genes identified in other Wolbachia that are likely involved in cytoplasmic incompatibility were found in wDacB but not in wDacA.
Many insects possess vertically-transmitted bacterial symbionts that provide them with amino acids and vitamins (Moran 2006). While most insect endosymbionts belong to the Gammaproteobacteria there are others in many other phyla (Moran et al. 2008). A remarkable case is the Wolbachia endosymbiont that infects between 40% (Zug and Hammerstein 2012) to 66% (Hilgenboecker et al. 2008) of arthropod species. Wolbachia are phylogenetically affiliated to the Alphaproteobacteria, not distantly related to Rickettsia, Ehrlichia, and Anaplasma (Williams et al. 2007). There are 16 phylogenetic supergroups of Wolbachia identified, and 10 of them are associated with arthropods (Augustinos et al. 2011). Based on phylogenomic analysis, six Wolbachia supergroups have been separated in eight species (Ramírez-Puebla et al. 2015).
Wolbachia are nematode as well as arthropod symbionts (Hilgenboecker et al. 2008; Sommer and Streit 2011), and have different effects in their hosts ranging from parasitism to mutualism with spatial and temporal spread of infections in some insect populations (Vavre and Charlat 2012). In nematodes, Wolbachia provide vitamins, energy, help in embryo development, and are capable of evading the host immune response (Darby et al. 2012; Landmann et al. 2014). In arthropods, Wolbachia have been found infecting many tissues inside the insect body including reproductive tracts and somatic cells as bacteriocytes (Dobson et al. 1999; Clark et al. 2005; Hosokawa et al. 2010; Sacchi et al. 2010; Saha et al. 2012). They alter the host reproduction by induction of parthenogenesis (Stouthamer et al. 1999), male-killing (Duplouy et al. 2013), feminization (Stouthamer et al. 1999), and strain incompatibility (Rousset et al. 1992). However, it is also known that Wolbachia may confer benefits to insects by playing an important role in insect development and survival (Dedeine et al. 2001). For example, removal of Wolbachia with antibiotics in Asobara tabida wasps inhibits maturation of oocytes (Dedeine et al. 2001). In Drosophila, Wolbachia may confer protection against virus infections (Teixeira et al. 2008; Chrostek et al. 2013) and provide a fecundity benefit to females when subjected to low or high iron diets (Brownlie et al. 2009). Thus, Wolbachia inside insects may not be only facultative symbionts, but can also be obligate endosymbionts necessary for survival (Dedeine et al. 2001).
There are 12 Dactylopius species (Ben-Dov 2006; Van Dam and May 2012). Six of them are present in Mexico, including the smallest and most distantly related Dactylopius tomentosus (Portillo and Vigueras 2006; Chávez-Moreno et al. 2009). Dactylopius insects feed exclusively on the sap of cactus plants of the genera Opuntia and Nopalea (Pérez-Guerra and Kosztarab 1992). Females of these scale insects spend all their lives on the host plant surface, whereas males are winged and short lived. These insects feed on a poor nutritional and low-calorie diet since cactus sap consists mainly of water (88–95%) and is low in nitrogen (0–0.5%) (Stintzing and Carle 2005). The red pigment carmine is obtained from cochineal insects of the genus Dactylopius, especially from D. coccus, which is a domesticated species. Carmine has been used as a natural dye to color food, medicines, cosmetics, textiles, and artworks, is considered safe for human consumption (Dapson 2005), and has antimicrobial and insecticidal properties (Eisner et al. 1980; Pankewitz et al. 2007).
Previously, we described a betaproteobacterium, Candidatus Dactylopiibacterium carminicum, and other diverse bacterial species associated with Dactylopius species present in Mexico (Ramírez-Puebla et al. 2010). Here, we extend the knowledge of Dactylopius endosymbionts by reporting the presence and genome sequences of two strains of Wolbachia, Candidatus Wolbachia bourtzisii wDacA (supergroup A) and Candidatus Wolbachia pipientis wDacB (supergroup B) obtained from Mexican D. coccus.
Materials and Methods
Sample collection
D. coccus insects were provided by Campo Carmín Greenhouse (Morelos, Mexico) and were maintained on cactus plants (Opuntia ficus indica var. Campo Carmín) in a growth room with controlled photoperiod (12L:12D), temperature (25°), and humidity (40–60%). Other Dactylopius species were collected from different states in Mexico: D. confusus from Tlaxcala, D. ceylonicus from Estado de México, D. opuntiae from Querétaro and Mexico City, and D. tomentosus from Hidalgo.
DNA extraction for detection of Wolbachia in Dactylopius individuals
DNA from the whole bodies of adult females of Dactylopius species collected in Mexico were extracted and purified with DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer’s instructions. PCRs were performed using primer pairs wsp81F/wsp691R (Braig et al. 1998) and 27F/1492R (Lane 1991) directed to the wsp and 16S rRNA genes, respectively.
Recovery of Wolbachia genomes
Sequence and assembly of metagenomic DNA from samples of pooled D. coccus individuals, as well as the recovery of the Wolbachia genomes from the metagenome, were previously reported (Ramírez-Puebla et al. 2016). For 454 sequencing, 2 g (20 individuals) of adult females were superficially disinfected with 70% ethanol, rinsed with sterile distilled water, and dissected with sterile forceps to remove the exoskeleton and guts. Cells in the hemolymph and debris were separated by centrifugation in a Percoll gradient (adapted from Charles and Ishikawa 1999), phases were observed under a microscope, and those with cells were selected for DNA extraction. For PacBio sequencing, eight individuals were superficially disinfected as previously described. Guts and exoskeleton were removed with sterile forceps. Hemolymph from all individuals was pooled for DNA extraction. For Illumina sequencing, guts, ovaries, and Malpighian tubules from 40 females were dissected using sterile forceps under a stereoscopic microscope. These organs were pooled, suspended in PBS, and macerated using a sterile plastic pestle. In all cases, DNA was extracted with DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer´s instructions. Sequencing was performed at Macrogen Inc. (Korea) for Illumina and 454 and at Duke University Genome Sequencing Core Facility (USA) for PacBio.
Genome analysis
The RAST server was used for gene prediction and annotation (Aziz et al. 2008). Manual curation of relevant genes was performed after comparisons with sequences deposited in the following databases: nr and Refseq via BLASTX (Benson et al. 2013), the Conserved Domain Database at GenBank (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), the Protein families (PFAM) database (Finn et al. 2014), and the Transport Classification Database (Saier et al. 2014). Genome completeness was assessed by the presence of single-copy widespread orthologs with BUSCO (Simão et al. 2015). Four out of the forty genes evaluated by BUSCO are absent in all the sequenced Wolbachia genomes and this was taken into consideration for the calculations.
Data availability
The genome sequences of Wolbachia strains wDacA and wDacB have been deposited in the GenBank database under accession numbers LSYX00000000 and LSYY00000000, respectively.
Results
Wolbachia in Mexican Dactylopius spp.
Previously, no Wolbachia sequences were found by PCR amplification of the 16S rRNA gene with primer pair fD1/rD1 (Weisburg et al. 1991) from several Dactylopius samples collected in Mexico and Brazil (Ramírez-Puebla et al. 2010). We reassessed the presence of these endosymbionts by PCR amplification of the Wolbachia-specific wsp gene (Braig et al. 1998). Amplicons of the expected size were obtained from D. ceylonicus, D. coccus, D. confusus, D. opuntiae, and D. tomentosus, although not all individuals of each species gave a positive reaction. To identify the Wolbachia inhabiting D. coccus in Mexico, 16S rRNA gene amplification with primer pair 27F/1492R and sequencing was performed. Two divergent sequences were found; one affiliated with Wolbachia supergroup A and the other with supergroup B. Sequences of the latter supergroup were more abundant in all surveyed D. coccus individuals (Table 1).
Divergence between Wolbachia from Dactylopius from different countries
We have recently reported the recovery of two contig bins matching Wolbachia from a metagenome of D. coccus (Ramírez-Puebla et al. 2015, 2016). A phylogenomic analysis of those bins (Ramírez-Puebla et al. 2015), confirmed that they corresponded to the genomes of two different Wolbachia strains belonging to Candidatus Wolbachia bourtzisii (supergroup A) and Candidatus Wolbachia pipientis (supergroup B), which will be referred to here as wDacA and wDacB, respectively.
Wolbachia from supergroups A and B were previously reported in Dactylopius sp. collected in Lanzarote, Canary Islands, Spain (Pankewitz et al. 2007). The Canarian and Mexican Wolbachia from supergroups A and B showed, respectively, 99.8% and 98.3% identity at the ftsZ gene, and 100% and 98.3% identity at the wsp gene. Thus, Wolbachia infecting Dactylopius sp. populations in the Canary Islands are closely related but distinct to the Mexican Wolbachia, the divergence being more pronounced among supergroup B representatives. Recently, a Wolbachia genome was recovered during a genome sequencing of D. coccus (Campana et al. 2015). The reported wCoc1 genome was found to belong to supergroup B by ftsZ gene sequence analysis, but no analysis of the genome was provided. wCoc1 showed 92.4% and 98.2% ANI values with wDacA and wDacB, respectively, indicating that wCoc1 and wDacB belong to the same species. No further comparison against our strains was performed because the wCoc1 genome assembly was highly fragmented (1064 contigs, N50 size = 1387 bp), and also because that genome may represent a chimera as it is the product of sequences originating from two different and geographically distant D. coccus populations, one from Oaxaca in Mexico and the other from Peru.
Genomes sequences of Wolbachia strains wDacA and wDacB
The number of contigs and N50 sizes of genome assemblies of wDacA and wDacB were 157 and 13.7 kb and 198 and 14.5 kb, respectively (Table 2), values that were average in comparison to released WGS genomes of Wolbachia. Genome completeness assessed with BUSCO (Simão et al. 2015) indicated that the recovered genomes of wDacA and wDacB represented 92% and 94%, respectively, of their whole genomes. It should be pointed out that closed Wolbachia genomes are reported as 92–94% complete by BUSCO because from one to three of the evaluated genes are either missing or fragmented in any given genome. Read coverage was widely different between both genomes, 2700 × in wDacB vs. 174 × in wDacA, indicating that the first Wolbachia strain is predominant in the tissues of D. coccus used in this study. Detection of each Wolbachia strain by 16S rRNA gene PCR amplification and sequencing in isolated individuals of D. coccus seemed to corroborate that wDacB is more abundant than wDacA (Table 1). As in other Wolbachia strains, wDacA and wDacB strains showed reduced genomes and low G + C contents (Table 2). Hypothetical genes represented 35% and 23% of the CDS genes in wDacA and wDacB, respectively. Wolbachia strains show high genome plasticity compared with other insect endosymbionts. The presence of a high proportion of mobile DNA and insertion sequences (Bordenstein and Reznikoff 2005; Cordaux et al. 2008) may promote this plasticity. The two Wolbachia strains of D. coccus were not exceptions, although it is worth mentioning that the genome of wDacB has a higher number of genes annotated as coding for mobile genetic elements and transposases (404, 24% of the CDS genes) in comparison to wDacA (120, 9% of the CDS genes).
Vitamin, coenzymes, cofactors, and nucleotide synthesis
Both Wolbachia strains from D. coccus seemed able to synthesize riboflavin and ubiquinone (coenzyme Q). They also had genes required for purine and pyrimidine nucleotide biosynthesis. They lacked complete biosynthesis genes for biotin, thiamine, coenzyme A, NAD, and folic acid. Nevertheless, an uptake system for biotin and a gene for folate salvage were found encoded in each genome. Both strains also possessed a bacterioferritin gene and heme biosynthesis genes.
Metabolism
The set of genes for the tricarboxylic cycle was complete in both genomes. There were genes for the pentose phosphate pathway but not the oxidative reactions. The phosphofructokinase gene is absent, suggesting that there may be gluconeogenesis but not glycolysis. Cytochrome c oxidase, as well as components of the respiratory complex, were found in both strains.
As has been observed in other Wolbachia and other Rickettsiales, most amino acid biosynthesis pathways were incomplete. However, catabolic genes for proline, aspartate, glutamate, and possibly cysteine were identified in both strains. Genes for glutamate dehydrogenase, glutamine synthetase (GS), and glutamate synthase (GOGAT) required for ammonia assimilation were also present. NifU was identified but no other nitrogen fixation genes. Nif proteins involved in the formation of FeS clusters or other metallo clusters can be found in organisms that do not fix nitrogen.
A complete set of genes for fatty acid biosynthesis were present in both genomes as well as for the synthesis of the phospholipids phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine. No genes for lipopolysaccharide biosynthesis were found in either genome. wDacA and wDacB had genes for peptidoglycan synthesis but no transpeptidase genes for chain cross-linking were found.
Transport
Both genomes encoded genes for ATP-binding cassette (ABC) transporters for uptake of phosphate (pstABCS genes), ferric iron, zinc, and possibly lipids; one for export of heme; and one gene for a Mg+2 (or Co+2) transporter-E (MgtE) family importer. Several genes for putative amino acid symporters were shared by both genomes including five of the major facilitator superfamily (MFS), three of the alanine/glycine:cation symporter (AGCS) family, and one of the dicarboxylate/amino acid:cation symporter (DAACS) family. Strain wDacA but not wDacB had genes coding for an ABC uptake transporter for glutamine/glutamate. On the other hand, strain wDacB possessed three uptake systems of the drug/metabolite transporter (DMT) superfamily, and two genes for organophosphate:phosphate MFS antiporters that were not present in wDacA. The former DMT transporters were > 75% similar to the S-adenosylmethionine (SAM) uptake transporter of Rickettsia prowazekii (Tucker et al. 2003), and the highest similarities (∼49%) of the latter MFS antiporters were to proteins of R. prowazekii which have been implicated in triose phosphate uptake used for phospholipid biosynthesis (Frohlich and Audia 2013). No hexose transporter genes were found, supporting the theory that there is no glycolysis in both strains.
Few export transporters were found in both genomes. Besides the heme exporter, both genomes encoded an ABC transporter putatively involved in organic solvent resistance, a CorC-family transporter for magnesium or cobalt efflux, and a cation diffusion-facilitator (CDF) family exporter for zinc or cadmium. In addition, the wDacA genome encoded two ABC superfamily transporters, one of the heavy metal transporter (HMT) family related to exporters for phytochelatins-Cd complexes and the other of the multidrug resistance (MDR) family.
Secretion systems
Of the two systems for protein export into the periplasm, only the general secretion sec system was found encoded in wDacA and wDacB genomes. Protein secretion into the extracellular environment is accomplished by several types of secretion systems, of which only two were found in the Wolbachia strains of Dactylopius. Both genomes coded for the inner membrane component and the membrane fusion protein of a type I secretion system (T1SS) whose products were 95% and 83% identical, respectively, between the strains. The outer membrane TolC, a channel that acts in conjunction with the other T1SS components, was coded elsewhere in the genomes.
Both strains possessed one type IV secretion system (T4SS). The gene organization was similar to that found in other Wolbachia with two separated clusters, one including virB3, virB4, and four copies of virB6, and another cluster with virB8, virB9, virB10, virB11, and virD4. As it is also observed in other Wolbachia, there was one paralogue of each of virB4, virB8, and virB9 coded elsewhere in the genomes. Genes virB1, virB2, virB5, and virB7 have been reported as being absent in Wolbachia and in Rickettsiales in general (Pichon et al. 2009). However, we found four and three homologs of the pilin virB2 gene in wDacA and wDacB, respectively. The virB2 homologs were not clustered with each other or with other vir genes. BLAST searches recovered virB2 homologs in many Wolbachia genomes (data not shown) that are annotated mostly as hypothetical or membrane proteins.
In the symbiotic wBm strain of the nematode Brugia malayi, the transcriptional regulators wBmxR1 and wBmxR2 bind to the promoter regions of some vir genes (Li and Carlow 2012). wBmxR1 seems to regulate the virB8 operon (which includes the upstream riboflavin biosynthesis gene ribA) and the second copy of virB9, while wBmxR2 controls the expression of the second copy of virB4 (Li and Carlow 2012). Homologs coding for proteins > 74% identical to the wBmxR1 product were found in both our Wolbachia strains, while a homolog to wBmxR2 was found only in wDacA (78% identity).
Stress response
Although living in a relatively protected environment inside their host cells, endosymbionts still retain genes required to cope with stressful conditions. Potassium homeostasis is important to react to changes in osmotic pressure and pH changes. One TrkG potassium uptake protein was found encoded in wDacA, while there were two in wDacB. Both strains had a glutathione-regulated potassium-efflux system KefKL. An HtrA protease/chaperone for degradation of misfolded or mislocalized cell-envelope proteins was encoded in each genome. Genes to contend with oxidative stress, like those for a Fe superoxide dismutase, an alkyl hydroperoxide reductase, three glutaredoxins, and glutathione biosynthesis, were also found. A single gene for a bacterial flavohemoglobin in each genome may be used to contend with nitrosative stress. Common proteins used for temperature stress, such as DnaK-DnaJ-GrpE composing the DnaK chaperone system and GroEL-GroES composing the GroE chaperonin machinery, were found encoded in both genomes as well as a CspA-family cold shock protein. A single sigma factor RpoH protein was encoded in each genome and may be used for stress response.
Virulence factors
Ankyrin domains are involved in protein–protein interactions and, by interacting with specific regions of the host chromatin, can modulate host gene transcription in other bacteria (Iturbe-Ormaetxe et al. 2005; Siozios et al. 2013). Genes coding proteins with ankyrin domains were found in both strain genomes, although wDacB had double the number of genes in comparison to wDacA (34 vs. 17 genes). Neither strain possessed genes for chemotaxis or motility via flagella or type IV pilus.
Two of the five MFS transporters of wDacA and wDacB belonged to the phagosomal nutrient transporter (Pht) family (Chen et al. 2008). Amino acid and pyrimidine transporters of this family are required for intracellular survival of Legionella pneumophila in macrophages (Sauer et al. 2005; Fonseca et al. 2014). Homologs to Pht transporters were found encoded in many Wolbachia genomes and they were distributed in two major phylogenetic clusters which suggests functional divergence (Supplemental Material, Figure S1).
One gene in each Wolbachia strain coded for a protein bearing the mammalian cell entry (MCE) domain. Proteins in this family have been identified as necessary for intracellular colonization and survival by Mycobacterium tuberculosis and M. bovis (Arruda et al. 1993; Flesselles et al. 1999). Several genomes of Wolbachia (Table S1) as well as other Rickettsiales encoded homologs for MCE proteins. Although not restricted to intracellular bacteria, MCE homologs are present in other facultative or obligate endosymbiotic and parasitic bacteria (Table S2).
Finally, genes coding for proteins that have been highlighted as candidates to induce cytoplasmic incompatibility were also found. Both genomes possessed two copies of the DNA-binding protein HU beta (Beckmann et al. 2013). wDacB but not wDacA had genes coding for proteins that are homologs to WPIP0282, which seems to be present only in Wolbachia strains inducing cytoplasmic incompatibility (Beckmann and Fallon 2013). wDacB possessed two homologs of the transcriptional regulator wtrM gene whose product is able to upregulate the expression of a host gene implicated in cytoplasmic incompatibility (Pinto et al. 2013). The wtrM gene of wDacA was split into two halves by a frameshift mutation.
Phages
Several genes of gene clusters encoding phage proteins were found in both Wolbachia genomes. A complete cluster including phage head-baseplate or head-baseplate-tail genes was not recovered; clusters included either head, baseplate, or tail genes. Paralogous genes located in different head or baseplate clusters in each genome suggested that each strain possesses more than one phage, although it was not possible to determine if any of these phages is complete. The tail clusters, one in each genome, were associated with putative virulence genes: two homologs of the VlrC protein in wDacA, and an ankyrin domain protein and a patatin-like protein in wDacB.
Discussion
The presence of Wolbachia in D. coccus (Campana et al. 2015) and Dactylopius sp. (Pankewitz et al. 2007) has been reported. In this study, Wolbachia PCR products were obtained from DNA extracted from Mexican samples of D. ceylonicus, D. coccus, D. confusus, D. opuntiae, and D. tomentosus. These data show that Wolbachia might have started its endosymbiotic state with cochineal insects before the genus Dactylopius had diverged.
We report the presence of two Wolbachia strains, wDacA and wDacB, in Mexican individuals of D. coccus. The large difference in read coverage between the genomes of wDacA and wDacB indicates that the latter strain is prevalent in D. coccus, at least in the tissue samples used here. Interestingly, wDacB but not wDacA possessed homologs coding for proteins that are likely involved in causing cytoplasmic incompatibility, a mechanism promoting persistence and dissemination of Wolbachia in their hosts. In wasps, double infections of supergroup A and group B strains have been found to influence reproductive and ecological isolation among sibling Nasonia species; therefore, Wolbachia has been implicated in wasp speciation (Bordenstein et al. 2001).
Dactylopius insects feed on low-nutrient cactus sap and therefore have to develop strategies to acquire nutrients lacking in their diet from their symbiotic relationships with bacteria. In other insects, riboflavin is produced by their endosymbionts such as the gammaproteobacterium Wigglesworthia for Tsetse flies (Akman et al. 2002) and Buchnera for aphids (Nakabachi and Ishikawa 1999). Recently, it was demonstrated that Wolbachia contributes to the growth, survival, and reproduction of bedbugs by riboflavin provisioning (Moriyama et al. 2015). Furthermore, it has been postulated that Wolbachia strains can also act as heme providers and/or helpers in maintaining iron homeostasis in the host (Brownlie et al. 2009; Kremer et al. 2009). Both Wolbachia strains from D. coccus possessed genes for the biosynthesis of riboflavin, heme, and the iron-storage protein bacterioferritin. All these genes are common in Wolbachia from insects, even in the sex-manipulator strains that negatively affect the host fitness. For riboflavin, Moriyama et al. (2015) have found evidence that its provisioning can counteract the negative effects caused by Wolbachia in their hosts.
As has been observed in other Wolbachia, wDacA and wDacB do not have genes to produce most amino acids that their insect hosts require, which may be provided by other bacteria present in D. coccus (Ramírez-Puebla et al. 2010) that could act as symbionts. The lack of most amino acid biosynthetic capabilities suggests the dependence of Wolbachia on its host or on other endosymbionts. Retention of amino acid biosynthesis defines primary insect symbionts and its absence seems to be a characteristic of secondary symbionts (Darby et al. 2012). Lack of a functional glycolysis pathway and the presence of several amino acid uptake systems indicate that Wolbachia utilizes amino acids instead of sugars as nutrients. Many of the MFS transporters may be proline uptake systems that, together with the presence of PutA for proline catabolism, suggest that this amino acid could be a major nutrient for Wolbachia. In fact, high-level expression of PutA has been demonstrated by proteomic analysis of Wolbachia (Baldridge et al. 2014). Interestingly, proline is an excellent precursor for riboflavin production in the legume endosymbiont Sinorhizobium (Phillips et al. 1999).
Symbiotic and pathogenic bacteria can use effector proteins delivered to their host via the T4SS to promote intracellular colonization and persistence (Juhas et al. 2008). T4SS is widely found in Wolbachia strains (Pichon et al. 2009) and was also found in wDacA and wDacB. It was surprising to note that virB2, coding for the major T-pilus component, was reported as being absent in Wolbachia and other Rickettsiales (Rancès et al. 2008; Pichon et al. 2009). We found several virB2 homologs in wDacA and wDacB, as well as in many Wolbachia genomes. This is in agreement with recent data obtained in other Rickettsiales, like Anaplasma phagocytophilum (Dugat et al. 2014) and Neorickettsia risticii (Lin et al. 2009) which do possess several virB2 paralogues. In N. risticii, VirB2 is located on the cell surface in agreement with its function as the major T4SS pilus protein (Lin et al. 2009). In A. phagocytophilum, the AnkA protein is exported via a T4SS (Lin et al. 2007). Although T4SS are known to transport proteins and/or DNA, an intriguing possibility is that they can act as nutrient transporters in Wolbachia given the scarcity of nutrient export systems in the genomes of these bacteria.
Several efflux systems for heavy metals were found in both genomes suggesting that Wolbachia from D. coccus have to cope with metal toxicity, perhaps contributed by their host diet. In relation to this, the mucilage of Opuntia cacti acts as a good water-soluble chelating polymer (polyelectrolyte) able to remove heavy metals from water (Barka et al. 2013), and metal-bound phytochelatin can be found in Opuntia shoots (Landero Figueroa et al. 2007). Besides heavy metals, other harmful conditions are likely acting on wDacA and wDacB as both their genomes carried several genes to contend with abiotic stresses. Proteomic profiling of a mosquito Wolbachia strain has revealed a profile dominated by chaperones and stress proteins (Baldridge et al. 2014).
Another secretion system used by bacteria to interact with eukaryotes is the T1SS. In pathogenic bacteria, virulence factors such as hemolysins are secreted via this system. Secretion of some ankyrin domain proteins by T1SS has been reported in Rickettsia (Kaur et al. 2012) and Ehrlichia (Wakeel et al. 2011). Proteins bearing typical T1SS-secretion motifs could not be found in either of our Wolbachia genomes, but it is worth noting that several ankyrin domain proteins were coded near the gene for the T1SS inner membrane component in wDacB.
In both Wolbachia strains, we found transporters of the Pht family, which have been described as virulence factors in L. pneumophila (Sauer et al. 2005; Fonseca et al. 2014). These transporters are present in other Legionellales, in Chlamydiales, as well as in other Rickettsiales besides Wolbachia, all having intracellular lifestyles. A protein encoded by wDacA and wDacB showed homology to the virulence factor Mce of Mycobacterium which, when expressed in nonpathogenic Escherichia coli, confers the ability to invade and survive within macrophages (Haile et al. 2002). The presence of all these putative virulence factors has not been previously pointed out in Wolbachia.
Acknowledgments
The authors wish to thank Michael Dunn and Julio Martínez for technical support and for reading the manuscript, and Campo Carmín Greenhouse for providing D. coccus insects. Consejo Nacional de Ciencia y Tecnología (CONACyT) grant 154453 provided financial support. S.T.R.-P. and A.V.-P.L. were in the Programa de Doctorado en Ciencias Biomedicas, Universidad Nacional Autónoma de México and received a scholarship from CONACyT. All the authors declare no conflict of interest, financial or otherwise, that might potentially bias this work.
Footnotes
Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.116.031237/-/DC1
Communicating editor: B. Oliver
- Received May 16, 2016.
- Accepted August 18, 2016.
- Copyright © 2016 Ramírez-Puebla et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
