Insecticidal genes of Yersinia spp.: taxonomical distribution, contribution to toxicity towards Manduca sexta and Galleria mellonella, and evolution

The toxin complex (Tc) proteins whose insecticidal potential resembles that of the Bacillus thuringiensis Bt-toxin were first purified from Photorhabdus luminescens which lives in symbiosis with nematodes [1]. They have also been identified in other insect-parasitizing bacteria such as Serratia entomophila, Xenorhabdus nematophilus, or Pseudomonas entomophila [2, 3]. Homologous insecticidal toxin genes are present in most genomes of Yersinia strains sequenced so far, including Y. mollaretii, seven Yersinia pestis strains and three Y. pseudotuberculosis strains. They have also been found in Y. frederiksenii and in two Y. enterocolitica strains, T83 and W22703, for which a genome sequence is not yet available [4–6]. However, tc genes are absent in Y. bercovieri and in Y. enterocolitica strain 8081 [7]. Interestingly, Tc proteins of three Serratia species and of Y. frederiksenii are plasmid-encoded, indicating that these sepABC-like genes are part of a horizontally mobile region [4].

Little is known about the biological role of the tc genes in Yersinia spp. The genes of the tc operons have been classified into three types according to their homology, namely tcdA/tcaAB/tccAB (type [A]), tcdB/tcaC (type [B]), and tccC (type [C]) [8]. Tc proteins have recently been shown to be secreted in a type III-dependent manner in Y. pestis [9]. Type [A] and [B] Tc proteins are presumably toxins directed against invertebrate and mammalian gut cells, and the variability in terms of Tc composition and Tc sequences may be due to insect- and tissue-specific activity [8, 10]. A role of the Tc proteins from Y. enterooclitica strain T83, Y. pseudotuberculosis strain IP32953 and Y. pestis KIM in mice gut colonization and in the actin cytoskeleton rearrangement of human gut cells and mouse fibroblast cells, respectively, has been reported [5, 8, 11]. The function of TccC remains unknown, but it has been suggested that TccC homologs could contribute to stable biofilm formation in fleas or combatting yet unknown antibacterial effectors in fleas [12], or that they act as universal activator of, or chaperons for, different toxin proteins [13].

Y. enterocolitica was the first member of the Yersinia genus for which insecticidal activity has been experimentally demonstrated, and tcaA encoding a subunit of the toxin complex was identified to be necessary for this activity [6]. The transcription of tcaA in Y. enterocolitica is completely repressed at 37°C, but strongly induced at lower temperatures with a maximum at approximately 10°C to 15°C. In contrast to Y. enterocolitica W22703, tcaABC expression in Y. pseudotuberculosis strain IP32953 was observed at 15°C and at 37°C [14]. Upregulation of tcaA and tcaB, but not tccC, upon temperature shift from 37°C to 26°C have been shown in two Y. pestis strains [15, 16]. The IP32953 Tc proteins are toxic against M. sexta larvae when expressed heterologously in E. coli [14]. Temperature-independent, but weak oral toxicity of several Y. pseudotuberculosis to this tobacco hornworm has been reported. Y. pseudotuberculosis, unlike Y. pestis, causes acute oral toxicity to fleas [12]. However, when the tcaAB gene pair from Y. pseudotuberculosis was heterologously expressed in E. coli, the lysates did not cause excess mortality in fleas, and a Y. pseudotuberculosis mutant deleted of the tc genes remained toxic toward the arthropod [8]. This is in line with the finding that two Y. enterocolitica strains containing a tcdB-tccC gene pair (strain CS080) or lacking any tc-like genes (strain 8081) were equally toxic to fleas [12].

The insecticidal potential of a variety of Yersinia spp. has not been tested in an insect infection assay, and the correlation of virulence to the presence or absence of tc operons in yersiniae is unknown. The phylogenetic relationship of the insecticidal toxins is not well understood. Here, we report a genome-based comparison of the tc genes in Y. enterocolitica strain W22703, the phylogenetic analysis of tccC genes in Yersinia species, the tc-PAI ^Ye distribution among six biotypes, and the insecticidal activity of Yersinia spp. towards two model organisms, the greater wax moth G. mellonella and the tobacco hornworm M. sexta.

Two basic methods have been used here to determine the insecticidal potential of Yersinia spp., namely the oral application of viable cells and the subcutaneous injection of protein extracts or living bacterial cells. Upon oral application of W22703 and W22703-tcaA::Tn5lux protein extract to M. sexta larvae, we could recently demonstrate the role of TcaA in Y. enterocolitica toxicity towards insects [6]. Further five Yersinia strains were tested here for the first time with respect to their oral toxicity in the M. sexta model (Table 1). The presence of the tc-PAI ^Ye correlates with a higher toxicity of yersiniae towards larvae of the tobacco hornworm, while strains such as Y. ruckeri or Y. aldovae lacking the tc genes are less insecticidal in this assay. The variable insecticidal activity of strains with tc genes might be the result of sequence variations, or the presence of further insecticidal components. Due to a higher toxin concentration, the feeding of protein extracts led to higher lethality rates using strain W22703 [8]. In contrast, subcutaneous infection of G. mellonella does not result in a significantly different toxicity of these strains (Table 2). In comparison to P. luminescens that causes death of G. mellonella larvae within 24 hours following injection of several thousand cells [18], approximately 5 × 10⁵ Y. enterocolitica strain W22730 cells are required to kill G. mellonella within five days. The most surprising result of the injection study performed here was the high variability of the insecticidal potential among Yersinia strains that is probably caused by tc-independent determinants. Examples for factors required for full virulence towards insect larvae are the hemolysin XhlA of X. nematophila or the gene mcf of P. luminescens. [13, 19]. In Y. enterocolitica, XaxAB, an apoptotic AB toxin, and the putative macrophage toxin MT have been identified as candidates with potential insecticidal activity, but their biological role still remains to be uncovered [20]. The overall results of the Galleria bioassay correlate with the finding that among 147 Yersinia isolates from the environment, 15.6% were Y. enterocolitica, but only 0.7% belonged to Y. kristensenii [21].

Although the biological role of Tc proteins has still to be experimentally defined, sequence analysis already revealed several interesting aspects. Regions of significant sequence similarities have been identified in all TcdA-like elements characterized so far [14]. Especially, TcaC is well conserved within the Yersinia genus, but TcaB and TcaA show significant sequence variability [8]. When the TccC sequences derived from the tc-PAI ^Ye of yersiniae were aligned, a high degree of sequence conservation was obtained at amino acids 1–680, followed by a remarkably high sequence diversity [14] as is confirmed by the TccC cladogram (Fig. 2). Some Tc sequences show evidence of undergoing degradation with frameshifts that often result in the splitting of tc genes into two separate ORFs (Fig. 1). Frameshifts in Y. pestis, especially in tcaB of CO92, are discussed as a critical step in the recent evolution of flea-borne transmission in the genus Yersinia due to the loss of one or more of those insect gut toxins [12, 14]. This data indicates that the tc genes of yersiniae may be under diversifying selection [8] which might result in insecticidal proteins with host-specific activity and with varying insecticidal potential.

It has been suggested that the genomes of different strains have taken up different tc genes after strain separation [22]. However, the data presented here point to a common Yersinia ancestor that has aquired the tc-PAI ^Ye . The plasmid-encoded Tc proteins in Y. frederiksenii and a transposon-like element downstream of Y. pestis Angola tcaC hint to putative mechanisms that might have played a role during horizontal transfer of insecticidal toxin genes (Fig. 1). This hypothesis is strongly supported by the common insertion site of the tc-PAI ^Ye that indicates one horizontal gene transfer (HGT) event, by the highly conserved phage-related genes within the tc-PAI ^Ye , and by a similar gene order including tccC1 and tccC2 in all islands investigated. Moreover, the cladogram derived from a comprehensive alignment of TccC protein sequences (Fig. 2) essentially reflects the phylogeny of Yersinia based on 16S rDNA sequences, including the clonal diversity among Y. enterocolitica strains [23]. As an additional insecticidal determinant, tccC genes located outside the tc-PAI ^Ye might have been acquired by a further HGT event following the separation of Y. pseudotuberculosis and Y. enterocolitica, because all available genomes of the Y. pseudotuberculosis and Y. pestis subline share two tccC insertion sites. Thus, reductive evolution by genetic drift might explain the lack of tc-PAI ^Ye in several Yersinia species and strains (Table 2) as examplified by the identification of rudimentary tc genes in biotypes 1A and 1B (Fig. 3).

The prevalence of the tc-PAI ^Ye in many genomes, its proven functionality in Y. enterocolitica and Y. pseudotuberculosis, as well as the common insecticidal potential of Yersinia spp. towards M. sexta and G. mellonella, hints to insects as yet unknown host organisms of yersiniae. This is in line with the hypothesis that environmental predators such as nematodes or insect larvae play a role in the evolution of pathogens [22, 24]. The tc-PAI ^Ye has probably been acquired by an ancestral Yersinia strain before the separation of Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, and others. This ancestor strain could then have evolved the ability to exploit invertebrates by the acquisition of further genetic determinants required for the interaction of yersiniae with those hosts [20]. Distinct sequence variation, and reductive evolution especially within the genomes of Y. pestis serovars, might have allowed yersiniae to occupy specific ecological niches [22]. The role of the tc genes and other insecticidal determinants in proliferation and transmission of the three human pathogenic Yersinia species remains to be elucidated in more detail.