Conservation of Ancient Genetic Pathways for Intracellular Persistence Among Animal Pathogenic Bordetellae - PubMed (original) (raw)

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

Israel Rivera et al. Front Microbiol. 2019.

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 animal-pathogenic 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. Knock-out 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.

Keywords: Bordetella; evolution; intracellular survival; macrophages; stress response and adaptation; transcriptomics.

Copyright © 2019 Rivera, Linz, Dewan, Ma, Rice, Kyle and Harvill.

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Figures

FIGURE 1

FIGURE 1

Intracellular survival of B. bronchiseptica in murine-derived macrophages. (A) Recovery of viable B. bronchiseptica RB50 and K. aerogenes post internalization by RAW 264.7 macrophages. (B) Z-stack fluorescence microscopy localizing B. bronchiseptica inside RAW 246.7 cells at 2 h post internalization (p.i.). Purple - F-actin; blue - nucleus; green - B. bronchiseptica. (C) Transmission electron microscopy of a RAW 264.7 macrophage containing B. bronchiseptica RB50 at 2 h p.i. Red arrows depict bacteria in the cell phagosomes. Scale bar: 1 μm.

FIGURE 2

FIGURE 2

Comparative analysis of genes upregulated during intracellular survival and their presence/absence in non-classical Bordetella species. Analysis of protein similarity of (A) 318 B. bronchiseptica genes upregulated in macrophages and (B) 4,981 genes in the entire genome of B. bronchiseptica strain RB50 in comparison to the non-classical Bordetella species. From outside to inside: Circle 1: Virtual genome of B. bronchiseptica strain RB50. Circles 2–8: Visual representation of protein similarity between B. bronchiseptica RB50 and classical (circles 2–3) and the non-classical species (circles 4–8) represented as color shades with darker shades indicating higher protein similarity. (C) 77–81% of the genes upregulated in intracellular B. bronchiseptica were conserved among the non-classical species, (D) in contrast to only 46–55% of the 4,981 B. bronchiseptica genes in the entire genome. (E) Line plot showing the frequency of protein similarities.

FIGURE 3

FIGURE 3

Intracellular survival of non-classical bordetellae within macrophages. The non-classical bordetellae were recovered from macrophages in numbers similar to B. bronchiseptica. The only exception, B. avium, failed to survive internalization by macrophages.

FIGURE 4

FIGURE 4

Assessment of B. bronchiseptica deletion mutants for intracellular survival. Deletion of malate synthase regulator gene BB0096 or tripartite tricarboxylate transporter gene BB1908 resulted in significantly reduced bacterial recovery. Plasmid-borne complementation of the gene deletion restored the wildtype phenotype. ∗∗p < 0.01; ∗∗∗p < 0.001.

FIGURE 5

FIGURE 5

Schematic representation of Bordetella exposure to eukaryotic phagocytes and its transcriptional response. Hypothetical scenario depicting Bordetella exposure to interconnected lifecycles and adaptation from environmental phagocytes to animal phagocytes.

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