A novel Erwiniaceae gut symbiont modulates gene expression of the intracellular bacterium Cardinium in the stored product mite Tyrophagus putrescentiae

Abstract

We examined host and bacterial gene expression profiles in the stored product mite Tyrophagus putrescentiae co-infected with Wolbachia (wTPut) and Cardinium (cTPut) while varying the presence of the Erwiniaceae symbiont (SLS). SLS, a novel symbiont in the family Erwiniaceae, with a genome size of 1.7 Mb, is found in 16% of mite species in infected cultures. In addition, SLS was detected in mite feces but not in their eggs. Although Wolbachia expression remained unchanged, the presence or absence of SLS significantly affected Cardinium expression. It indicated that the effect of Wolbachia on SLS was neutral. In SLS-positive samples, Cardinium exhibited 29 upregulated and 48 downregulated genes compared to SLS-negative samples. Furthermore, Cardinium gene expression strongly correlated with mite KEGG gene expression in SLS-positive samples. Positive Spearman's correlations between Cardinium gene expression and mite KEGG immune and regulatory pathways were doubled in SLS-positive compared to SLS-negative samples. The diversity of expressed genes in the mite host decreased in the presence of SLS. Cardinium had more interacting genes to mite host in SLS-positive samples than without SLS. Transposases are the most affected Cardinium genes, showing upregulation in the presence of SLS. Correlation analyses revealed interactions between Cardinium and SLS via mite immune and regulatory pathways, including lysosome, ubiquitin-mediated proteolysis, PIK3_Akt, and cGMP-PKG. The results showed that Cardinium indirectly affects the gut symbionts of mites.IMPORTANCEThis study introduces a new model to analyze interactions between intracellular bacterial symbionts, gut bacterial symbionts, and their mite hosts. Using gene expression correlations, we investigated how the intracellular Cardinium responds to the novel Erwiniaceae gut symbiont in the mold mite Tyrophagus putrescentiae. The data showed that both mite and Cardinium gene expression are different in the samples with and without Erwiniaceae symbionts. In the presence of Erwiniaceae symbionts, Cardinium increased the interaction with the mite host in terms of changes in gene expression. The mite immune and regulatory pathway gene expression is differently correlated to Cardinium genes in relation to Erwiniaceae symbionts. As a well-known producer of allergens, T. putrescentiae physiology and thus its allergen production are influenced by both symbionts, potentially affecting the release of allergens into human environments.

Keywords: Cardinium; Erwiniaceae; Sodalis; Tyrophagus putrescentiae; Wolbachia; allergens; bacterial symbionts; gene expression; stored product mite.

Fig 1

Cardinium (cTPut), a bacterial endosymbiont of the stored food mite, Tyrophagus putrescentiae: (A) Visualization of two Cardinium genomes from mite cultures 5L and 5S using PROKSEE; (B) Venn diagram comparing Cardinium genomes based on KEGG proteins and all predicted proteins (C); (D) dREP comparison of known Cardinium genomes using average nucleotide identity (ANI); (E) scanning electron microscopy (SEM) image of the host, T. putrescentiae; (F) ANI-based comparison of the two Cardinium genomes from cultures 5S and 5L, visualized in PROKSEE.

Fig 2

Phylogenetic affinities and genomic features of Erwiniaceae (SLS) symbiont associated with Tyrophagus putrescentiae: (A) 16S rDNA phylogeny with the TN93 + R111 nucleotide substitution model; (B) maximum likelihood phylogeny based on orthologous protein groups using Mixtamixta tenebrionis as outgroup; (C) comparison of open reading frames (ORFs), with SLS indicated by a red point; (D) GC content comparison, with SLS indicated by a red point; GenBank IDs for genomic and 16S rDNA sequences are listed in Tables S6 and S7; (E) Venn diagram showing predicted protein overlap among select bacterial species; and (F) genomic characteristics of SLS using PROKSEE; phages were identified by VirSorter.

Fig 3

Comparison of Erwiniaceae symbiont (SLS) gene expression in three cultures of Tyrophagus putrescentiae (5L, 5LP, and 5LN): (A) ANOSIM analysis; (B) dbRDA correlation plot illustrating the distribution of samples according to SLS gene expression; (C) heatmap of SLS gene expression among the mite culturesbased on log₂-transformed values; (D) jitter and box plots showing relative numbers of SLS reads among mite cultures (SLS/mite reads), the ANOSIM comparisonshowed variability between samples (between) and inside samples.

Fig 4

Comparison of gene expression of Cardinium in the samples with (SLS+) and without (SLS−) Erwiniaceae symbiont: (A) ANOSIM and (B) heatmap with selected genes. The ANOSIM comparison was calculated for all samples (5L, 5LP, and 5LN versus 5S, 5SN, and 5SP), single-infected cultures (Cardinium infection: 5L versus 5S) and experimental cultures Cardinium/Wolbachia infection: (5LN, 5LP and 5SN, 5SP), the ANOSIM comparison showed variability between samples (between) and inside samples.

Fig 5

Correlational analysis between Cardinium (cTPut) and its mite host Tyrophagus putrescentiae in the samples with (SLS+) and without (SLS−) Erwiniaceae symbiont, based on Spearman correlation coefficients (permutational P < 0.05). (A and B) Positive and negative correlations between cTPut and the mite after Ward clustering: (A) SLS (−) samples, (B) SLS (+) samples. (C and D) Violin plots comparing the number of positive and negative correlations per gene: (C) mite vs cTPut and (D) cTPut vs mite.