Conservation of Ancient Genetic Pathways for Intracellular Persistence Among Animal Pathogenic Bordetellae

Abstract

Animal and human pathogens of the genus Bordetella are not commonly considered to be intracellular pathogens, although members of the closely related classical bordetellae are known to enter and persist within macrophages in vitro and have anecdotally been reported to be intracellular in clinical samples. B. bronchiseptica, the species closest to the ancestral lineage of the classical bordetellae, infects a wide range of mammals but is known to have an alternate life cycle, persisting, replicating and disseminating with amoeba. These observations give rise to the hypothesis that the ability for intracellular survival has an ancestral origin and is common among animalpathogenic and environmental Bordetella species. Here we analyzed the survival of B. bronchiseptica and defined its transcriptional response to internalization by murine macrophage-like cell line RAW 264.7. Although the majority of the bacteria were killed and digested by the macrophages, a consistent fraction survived and persisted inside the phagocytes. Internalization prompted the activation of a prominent stress response characterized by upregulation of genes involved in DNA repair, oxidative stress response, pH homeostasis, chaperone functions, and activation of specific metabolic pathways. Cross species genome comparisons revealed that most of these upregulated genes are highly conserved among both the classical and non-classical Bordetella species. The diverse Bordetella species also shared the ability to survive inside RAW 264.7 cells, with the single exception being the bird pathogen B. avium, which has lost several of those genes. Knockout mutations in genes expressed intracellularly resulted in decreased persistence inside the phagocytic cells, emphasizing the importance of these genes in this environment. These data show that the ability to persist inside macrophage-like RAW 264.7 cells is shared among nearly all Bordetella species, suggesting that resisting phagocytes may be an ancient mechanism that precedes speciation in the genus and may have facilitated the adaptation of Bordetella species from environmental bacteria to mammalian respiratory pathogens.

Evolution and Conservation of Bordetella Intracellular Survival in Eukaryotic Host Cells

“…Recent transcription analyses of RisA mutants under Bvg + and Bvg − conditions showed that RisA very likely plays a major role during intracellular survival in mammalian phagocytes. The RisA-dependent in vitro transcription profiles (Coutte et al, 2016) were largely congruent with the transcriptional response of B. bronchiseptica and B. pertussis inside macrophages (Rivera et al, 2019;Petrackova et al, 2020). Under Bvg + conditions, BvgA is phosphorylated and induces transcription of virulence-activated genes as well as of the transcriptional repressor BvgR.…”

“…By contrast, the expression of genes involved in bacterial virulence, which is a hallmark of Bvg-based modulation of gene expression in the mammalian host, is suppressed. None of the known virulence factors, including toxins, T6SS and T3SS, and the adhesins PRN and FHA, were transcriptionally active (Rivera et al, 2019), and neither were any of the 205 genes that were previously identified to encode proteins that are secreted under Bvg + conditions (Luu et al, 2017). A similar expression pattern was recently reported for B. pertussis inside macrophages, where expression of genes encoding virulence factors such as the T3SS, ACT, PTX, PRN, fimbriae 2 (FIM2), tracheal colonization factor (TCF), and the serum resistance protein BrkA was also suppressed (Petrackova et al, 2020).…”

“…This seems to be a contradiction. However, all tested classical and non-classical Bordetella species with the sole exception of B. avium were able to persist in murine macrophages (Rivera et al, 2019); yet, the presence of virulence factors in Bordetella species was shown to be species-specific (Linz et al, 2016) due to multiple events of gene acquisition and gene loss in the genus. For example, the well-known virulence factors of the classical bordetellae, including PTX, are not present in the non-classical species (Linz et al, 2016).…”

“…The ability to survive inside macrophage spans beyond the classical bordetellae (Rivera et al, 2019). Comparative genome analyses between B. bronchiseptica and non-classical Bordetella species revealed the conservation of genes involved in intracellular persistence; approximately 80% of the 318 transcriptionally upregulated B. bronchiseptica genes during intracellular persistence were present in the genomes of non-classical Bordetella spp., in contrast to ~50% of the total of 4,981 evaluated B. bronchiseptica genes.…”

“…Phenotypic analyses validated the significance of this observation by demonstrating that both the classical and the non-classical Bordetella spp. can persist inside RAW 264.7 macrophages (Rivera et al, 2019). The shared ability for intracellular persistence, and evolutionary conservation of the genes suspected to be involved in it, strongly suggests that intracellular survival represents an ancestral trait, the origin of which precedes speciation in the genus.…”