Rapid evolution of decreased host susceptibility drives a stable relationship between ultrasmall parasite TM7x and its bacterial host

Abstract

Around one-quarter of bacterial diversity comprises a single radiation with reduced genomes, known collectively as the Candidate Phyla Radiation. Recently, we coisolated TM7x, an ultrasmall strain of the Candidate Phyla Radiation phylum Saccharibacteria, with its bacterial host Actinomyces odontolyticus strain XH001 from human oral cavity and stably maintained as a coculture. Our current work demonstrates that within the coculture, TM7x cells establish a long-term parasitic association with host cells by infecting only a subset of the population, which stay viable yet exhibit severely inhibited cell division. In contrast, exposure of a naïve A. odontolyticus isolate, XH001n, to TM7x cells leads to high numbers of TM7x cells binding to each host cell, massive host cell death, and a host population crash. However, further passaging reveals that XH001n becomes less susceptible to TM7x over time and enters a long-term stable relationship similar to that of XH001. We show that this reduced susceptibility is driven by rapid host evolution that, in contrast to many forms of phage resistance, offers only partial protection. The result is a stalemate where infected hosts cannot shed their parasites; nevertheless, parasite load is sufficiently low that the host population persists. Finally, we show that TM7x can infect and form stable long-term relationships with other species in a single clade of Actinomyces, displaying a narrow host range. This system serves as a model to understand how parasitic bacteria with reduced genomes such as those of the Candidate Phyla Radiation have persisted with their hosts and ultimately expanded in their diversity.

Keywords: Saccharibacteria; TM7; candidate phyla radiation; interspecies interaction; oral microbiome.

Fig. 1.

TM7x/XH001 coculture contains heterogeneous populations. Phase-contrast image (A) and colony morphology (B–D) of TM7x/XH001 coculture reflect three subpopulations of cells: TM7x-infected XH001 (arrows and irregular colonies), TM7x-uninfected XH001 (arrowheads and regular colonies), and free-floating TM7x (circles). In contrast, XH001 monoculture displayed only TM7x-uninfected XH001 cells (E) and regular colonies (F). (Scale bars: phase contrast, 5 μm; colonies, 1 mm.) (G) Three independent cocultures were passaged for 10 d, and the irregular colony percentage was determined daily. The bar graph is the percent regular (red) or irregular (blue) colonies, whereas the line graph is the total cfu/mL (log scale). The error bars indicate the SD of the triplicate cultures.

Fig. 2.

Single-cell live imaging reveals XH001 growth and cell division. The cell area of individual XH001 cells in monocultures (A) or cocultures (B) were tracked during the live imaging. For the monoculture (A), 26 individual XH001 cells (gray traces) were tracked from six independent experiments, and the average is shown in dark black trace. Each trace was fitted with exponential growth equation to determine growth rate and summarized in C (SI Appendix, Fig. S2A). For coculture (B), TM7x-uninfected (gray lines, n = 22) and TM7x-infected (pink lines, n = 26) hosts were tracked from seven independent experiments. The average growth of uninfected (dark black), infected (dark red), or both (dark blue) are shown. Growth rates were determined and summarized in C (SI Appendix, Fig. S2B). (C) Growth rate summary of single-cell or bulk cell growth measurements (SI Appendix). Each dot reflects growth rate from each trace in A and B or SI Appendix, Fig. S2C. Time-lapse images of monoculture (D) and coculture (E) cells are shown with 40-min interval. (E) TM7x-infected (arrow) and uninfected daughter cells divide differently. (Scale bars: 5 μm.) *P < 0.05, ***P < 0.0001; n.s., not significant.

Fig. 3.

Naïve hosts develop reduced susceptibility to TM7x. Isolated free-floating TM7x (SI Appendix, Fig. S4) was mixed with naïve XH001n strain at 3:1 ratio (TM7x:XH001n). The growth of XH001n was monitored by optical density at 600 nm (A) and the total cfu/mL (blue solid line, circles) (B). The growth of TM7x was monitored by TM7x score (A), irregular colony percentage (red solid line, squire) (B), and qPCR (ratio between TM7x to XH001n) (C). The control experiments with no TM7x is shown in dashed gray lines (triangle, A and B). After mixing the cells, they were passaged every 24 h (x axis). (B) Total cfu/mL and irregular colony percentage are on the right and left y axis, respectively. The XH001n “growth crash points” are indicated by the black arrows (A and B). Error bars indicate the SD of the triplicate measurements. (D) Phase-contrast (Top row) and live/dead (Bottom row) imaging of reinfection experiment at indicated passages. (Scale bars: 5 μm.)

Fig. 4.

Host-reduced susceptibility to TM7x evolves rapidly. The reinfection assay was carried out with regular (blue squares, A and C) or irregular colony (blue squares, B) cells from the recovered reinfection assay. (C) Regular colonies were passaged 5 (∼50 generations) or 10 (∼100 generations) times before they were added to reinfection assay. Gray (circles) or red (triangles) lines are control experiments of colony alone or naïve host cells with TM7x, respectively (A–C). (D) A growth crash is seen in many, but not all, susceptible host Actinomyces species in clade 2 (SI Appendix, green in SI Appendix, Fig. S7A).