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. 2024 Apr 16;121(16):e2319790121.
doi: 10.1073/pnas.2319790121. Epub 2024 Apr 9.

Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence

Qiu Zhong #  1 , Binyou Liao #  2 , Jiazhen Liu #  1 , Wei Shen  3 , Jing Wang  1 , Leilei Wei  4 , Yansong Ma  5 , Pu-Ting Dong  6 , Batbileg Bor  6   7 , Jeffrey S McLean  8   9 , Yunjie Chang  10   11 , Wenyuan Shi  6 , Lujia Cen  6 , Miaomiao Wu  2 , Jun Liu  12 , Yan Li  2 , Xuesong He  6   7 , Shuai Le  1
Affiliations

Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence

Qiu Zhong et al. Proc Natl Acad Sci U S A. .

Abstract

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Keywords: Saccharibacteria; TM7x; bacteria-phage interaction; episymbiosis; phage resistance.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
TM7x protects host bacteria from phage predation. (A) Plaque assay of LC001 infecting S. odontolytica strain XH001 and TM7x/XH001. (B) The phage LC001 adsorption percentage of surface-grown XH001 and TM7x/XH001. The asterisks mark P-value of <0.05 as calculated by Student’s t test. (C) Microscopic observation of binding between SYBR Green–tagged LC001 and Laurdan-labeled XH001 or TM7x/XH001. The black and red arrows indicate XH001 and TM7x cells, respectively. (D) The surface-grown bacteria of XH001 or TM7x/XH001 were mixed with phage LC001 for 10 min, and the mixture was observed using the cryo-ET. The box with the dashed line indicates the area that was enlarged for closer observation of the interaction between phage LC001 and XH001. The red arrows indicate the phage LC001.
Fig. 2.
Fig. 2.
TM7x-free XH001 derived from TM7x-associated XH001 is sensitive to phage LC001. (A) Schematic showing the isolation of TM7x-free XH001 cells from TM7x/XH001 coculture. (B) TM7x-free XH001 cells were identified by PCR using primers specific for TM7x. (C) The phage LC001 adsorption percentage for surface-grown bacteria of XH001, TM7x/XH001, and TM7x-free XH001. The asterisks mark P-value of <0.05 as calculated by one-way ANOVA. (D) Plaque assay of LC001 infecting XH001, TM7x/XH001, and three TM7x-free XH001 isolates.
Fig. 3.
Fig. 3.
Transcriptomic analysis of surface-growth XH001 and TM7x/XH001. (A) The transcriptomic data were subjected to PCA. The abscissa is the first principal component, and the ordinate is the second principal component. Green and orange dots represent samples of TM7x/XH001 and XH001, respectively, with three biological replicates for each group. (B) The DEGs between TM7x/XH001 and XH001 were grouped and clustered. Genes and samples were represented horizontally and vertically, respectively. Orange indicates highly expressed genes, and green indicates lowly expressed genes. (C) The volcano plot shows the gene expression levels of TM7x/XH001 compared to XH001. The threshold is set for P-value <0.05 and |log2FoldChange| > 1. Orange and green dots indicate significantly up-regulated and down-regulated genes, respectively, and black dots indicate genes with no significant changes. (D) Clusters of orthologous groups (COG). The number of unchanged (black) and significantly differentially expressed (colored) genes in TM7x/XH001 vs. XH001 for each COG is shown. (E) Overall genome arrangement of the putative S. odontolytica CWP gene cluster. The orange and green peaks represent the APY09_00485-540 gene expression levels in the surface-growth XH001 and TM7x/XH001, respectively.
Fig. 4.
Fig. 4.
The impact of the expression of CWP cluster in XH001 on its phage LC001 sensitivity. (A) Schematic map showing generation of XH001::CWP using homologous recombination in XH001. (B) The relative mRNA expression of APY09_00540 was detected by qRT-PCR. The asterisks mark P-value of <0.05 as calculated by Student’s t test. (C) The surface-grown bacteria of XH001, TM7x/XH001, TM7x/XH001::CWP, or XH001::CWP were mixed with the SYBR Green–labeled phage LC001 for 10 min, and the mixture was observed using the fluorescence microscope intermediately. (D) The mean fluorescence intensity of LC001 adsorbed by XH001, TM7x/XH001, TM7x/XH001::CWP, or XH001::CWP was determined by ImageJ software. The asterisks mark P-value of <0.05 as calculated by one-way ANOVA. (E) Plaque assay of LC001 infecting XH001, TM7x/XH001, TM7x/XH001::CWP, or XH001::CWP. (F) The surface-grown bacteria of TM7x/XH001::CWP were mixed with phage LC001 for 10 min, and the mixture was observed using the TEM. The red arrow points to the phage LC001.
Fig. 5.
Fig. 5.
Transcriptomic analysis of surface-grown and planktonic XH001. (A) The volcano plot shows the DEGs under the surface and planktonic growth of XH001. The threshold is set for P-value of <0.05 and |log2FoldChange| > 1. Orange and green dots indicate significantly up-regulated and down-regulated genes, respectively, and black dots indicate genes with no significant changes. (B) Clusters of orthologous groups (COG). The number of unchanged (black) and significantly differentially expressed (colored) genes in planktonic vs. surface-grown XH001 for each COG is shown. (C) Overall genome arrangement of the S. odontolytica CWP gene cluster. The orange and green peaks represent the APY09_00485-540 gene expression levels of XH001 under surface and planktonic growth, respectively. (D) The surface and planktonic growth bacteria of XH001 or XH001::CWP were mixed with the SYBR Green–labeled phage LC001 for 10 min, and the mixture was immediately observed using the fluorescence microscope. (E) The mean fluorescence intensity of LC001 adsorbed by XH001 or XH001::CWP under surface and planktonic growth. The asterisks mark P-value of <0.05 as calculated by two-way ANOVA.
Fig. 6.
Fig. 6.
In vitro bacteria and phage coevolution experiment. (A) Phage LC001 was cocultured with XH001 or TM7x/XH001 in liquid culture, and 1/10 volume of the coculture was transferred to a fresh Brain Heart Infusion (BHI) medium for 10 passages. The bacterial density was calculated by measuring the optical density (OD) at 600 nm, and the supernatant containing LC001 was harvested to detect the PFU of the phage. XH001, TM7x-free XH001, and phage LC001 at transfer 0, 5, and 10 were isolated and purified, and cross-infection experiments were used to test whether the isolated phages were able to infect hosts from the coevolved populations. (B) The OD600 of bacteria showed that the number of hosts in both of the coevolved populations remained stable. (C) Phage titer in each generation was measured by the EOP experiment using the wild-type XH001 as a host, which showed that the phage population was maintained in both of the coevolved populations. (D) The phage infectivity range is based on the ability of each isolated phage to infect 3 XH001 or TM7x-free XH001 clones from the host population of T0, T5, and T10. Infection by phage is marked in orange, and resistance by hosts is marked in green. The phage infectivity range indicates an arms race model between XH001 and LC001, while TM7x delays the coevolution of phage and bacterial populations.
Fig. 7.
Fig. 7.
The model of episymbiont TM7x modulates the phage resistance of its host bacteria and impacts bacterial–phage interaction dynamics. Within the oral cavity, biofilm is the most dominant and relevant growth mode. The coexistence within the biofilm of free-living and TM7x-associated XH001 populations with differential LC001 sensitivity creates source-sink dynamics between the two antagonistic partners to promote the coexistence of bacteria and phage.
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