The saccharibacterium TM7x elicits differential responses across its host range

TM7x has restricted host range

In this study we expanded the number of Actinomyces host species/strains that were previously tested on TM7x infection [8] and conducted thorough phenotypic and comparative genomic analyses. TM7x cells were isolated apart from their original co-cultivated bacterial host XH001 (Actinomyces odontolyticus strain) and added back to cultures of diverse Actinomyces strains (n = 27) that span the Actinomyces lineage, as well as other common oral bacterial strains (n = 10) in an established re-infection assay (see methods, Table S1). By 16S phylogeny, Actinomyces lineages are divided into two major clades (clade-1 and −2), with XH001 in clade-2, agreeing with previous study [8] (Fig. 1). TM7x did not grow on any clade-1 Actinomyces strains after multiple passages, nor the common oral bacteria; while all tested strains (12 in addition to XH001) in clade-2 were infected with TM7x over multiple passages (Fig. 1) based on imaging techniques and PCR. These results suggest that the tested Actinomyces species fall into two major groups: resistant or susceptible to TM7x infection (Fig. S1a).

Different phenotypic responses of bacterial hosts to TM7x infection

Infection of naïve XH001 cells by TM7x induces a “growth-crash”, in which host cell density drops precipitously, followed by recovery in their bacterial hosts (Fig. S1b) [8]. This is analogous to a previously hypothesized cyclically-recurring population crash during parasite-host dynamics [30, 31], but interestingly in our case only a single crash was observed followed by stable growth. Recovered XH001 were found to have single-nucleotide variants relative to their naïve ancestors, presumably imparting the observed regain of fitness [7, 8]. The host growth is measured by cell density (OD600) whereas TM7x abundance is scored visually by phase-contrast imaging (see methods; [8]. These methods assess the host and TM7x abundances qualitatively but rapidly and accurately [8].

To further investigate the initial response to TM7x infection, the re-infection assay was conducted by adding TM7x to the 12 susceptible Actinomyces strains with a three-to-one TM7x-to-host cell ratio, and their growth was monitored by OD600 and TM7x scores (Fig. 2). Nine of these hosts displayed varying growth/crash/recovery patterns, and all of these included a clear crash phase and thus are referred to as “permissive” hosts (Fig. 2a–i). However, the remaining three hosts (F0311, ICM47, ICM58) lacked a discernable crash phase, hereafter referred to as “nonpermissive” hosts (Figs. 2j–l, S1c). Furthermore, three of the nine permissive hosts (ATCC17982, F0543, W712) had extended, 4–5 passage-long growth-crash phases before recovery while the rest of the hosts had only one passage-long growth-crashes (Fig. 2a–c). TM7x scoring was consistent with the observed host growth-crashes. When initial increase of the TM7x score was plotted for all hosts (Figs. 2, S1d), the three nonpermissive hosts (F0311, ICM47, ICM58) had a late increase in TM7x score compared to the rest of the hosts. F0310 was the only permissive host to have very late TM7x increase and growth-crash at passage twelve (Fig. 2i).

Previously, during the growth-crash phase, both attached and free-floating TM7x cells were observed, with individual XH001 cells often infected with multiple TM7x cells [8]. This induced host cell swelling and elongation, both common morphological stress responses, with XH001 cell length increasing from ~1.7 µm in monoculture to ~3.7 µm in cocultures, and eventually led to cell death [17]. Phase-contrast imaging illustrated similar results, with increased numbers of attached and free-floating TM7x observed for all nine permissive hosts and one nonpermissive host (Fig. S2a–i, j). However, two of the nonpermissive hosts (ICM47, IMC58) did not display an increased level of TM7x bacteria on their surfaces, nor increased cell length (Fig. S2k, l). To assess cell length quantitatively, we measured the cell length for all 12 bacterial hosts after infection. All hosts had significantly increased cell length (Figs. S1e, S3a, b, d–j) except two nonpermissive (ICM47, ICM58) and one permissive (W712) strains maintained or even slightly decreased their cell length after TM7x infection (Fig. S3c, k, l). The decrease in W712 cell length could be a result of W712 having the longest cells before TM7x infection or an inherent limitation in the image analysis of long cells (see methods). Nevertheless, W712 cells were swollen when they were infected with TM7x (Fig. S2c). Furthermore, although F0311 is a nonpermissive host, it did show many TM7x bacteria on its surface during the infection (Fig. S2j), which could be contributing to its increased cell length. Our findings suggest that TM7x-susceptible hosts divide into two broad categories (Fig. S1a): permissive and nonpermissive, though the permissive strains do present a spectrum of crash intensity and duration (Fig. 2).

Host sensitivity to TM7x infection

Our data showed that even though similar TM7x-to-host ratios were used in re-infection experiments, different hosts displayed drastically different crash/recovery dynamics (Fig. 2), suggesting these hosts have differential sensitivity to TM7x. Notably, a rapid increase of TM7x abundance within the first two passages was observed for three strains: A. odontolyticus ATCC17982 and two A. meyeri strains (W712 and ATCC35568) (Fig. 2a, c, f). To investigate this differential sensitivity further, dose-dependent TM7x infection of naïve XH001 cells was carried out. Results showed that the passage at which XH001 crashed, referred to as the ‘crash point’, was TM7x concentration dependent —with increasing TM7x, we observed earlier crash points (Figs. 3a, S4). Total colony forming units and irregular colony numbers, reflecting the number of total viable hosts and the TM7x infected hosts, respectively [8], were also determined during all passages. By these measurements, the crash points were dependent on the number of TM7x added to the assay. TM7x was able to infect at extremely low concentrations (three TM7x per 4.5 × 10⁶ XH001 cells), and able to completely inhibit XH001 at higher concentrations (2.7 × 10⁸ TM7x per 4.5 × 10⁶ XH001 cells). A similar pattern of TM7x and XH001 growth dynamics were observed at each TM7x concentration (Fig. S4). During the XH001 crash phase (by OD600 or total cfu), the amount of TM7x (by TM7x score or irregular colony) always increased to a maximum and then decreased during XH001 recovery. The crash points determined by total colony forming unit always occurred ~1–1.5 passages before the OD600 crash point, which was consistent with our previous study [8]. This passage difference may be explained by the fact that dead cells can contribute to the cell density measurements.

The sensitivity of A. meyeri strain W712 to TM7x was similarly tested. Remarkably, while dose-dependent growth-crash was also observed (Figs. 3b, S5), it took close to tenfold fewer TM7x cells (3.5 × 10⁷ TM7x per 4.5 × 10⁶ W712 cells) to completely inhibit the initial growth of W712 compared to XH001 (Fig. 3b), suggesting that the sensitivity of W712 to TM7x allows faster TM7x growth at the expense of W712. This was reflected by both the OD600/TM7x score and total/irregular colony measurements (Fig. S5). Again, similar to what was observed in the initial coculture experiment (Fig. 2c), all growth-crashes in W712 had prolonged growth-crashes (Fig. S5). In contrast, the nonpermissive strain ICM47 was completely resistant to growth-crash even at the TM7x-to-ICM47 ratio of 4.9 × 10⁸:4.5 × 10⁶ (Figs. 3c, S6). Despite TM7x infection and growth on ICM47, no growth-crash was observed by cell density measurement and total colony forming units. ICM47 strains also did not form obvious irregular colony morphology, suggesting TM7x does not stress or damage host growth as with the other strains.

TM7x has unique cell localization on the nonpermissive ICM58

TM7x and XH001 have various morphological cell shapes depending on growth conditions and nutrient availability (Fig. S7a) [17]. For all permissive and nonpermissive strains, we observed normally shaped TM7x bacteria growing on the cell surface of the host bacteria by FISH (Figs. 4, S7). Consistent with our previous findings, TM7x attached to bacterial hosts had simple dot/cocci or teardrop-like morphology, shown in green (Figs. 4, S7) [17]. Remarkably, compared to all tested bacterial hosts, only on ICM58, many TM7x localized to the cell poles (Fig. 4f). The polar localization was previously not observed in the close relatives of TM7x, but was shown in a distant lineage (HMT-351) that grows on Actinomyces sp. HMT-897 [32]. Exactly how and why pole localization occurs is yet to be determined. Typically, gram-positive bacteria have significant long-axis polarization in terms of protein composition and cell wall structure [33], and TM7x could be targeting those areas. The polar localization of TM7x on ICM58 suggests a different mechanism for attachment compared to other hosts.

Genome content separates permissive and nonpermissive hosts

As genomes of twenty-three out of the twenty-seven tested Actinomyces strains are publicly available, we downloaded them for comparative genomic analyses. To place the currently unnamed genomes (e.g., Actinomyces sp. F0310) in context with named species, we first related genomes by average AAI and constructed a phylogenomic tree from concatenated core genes (Fig. S8a, b). From the AAI data, clear patterns emerged: the thirteen TM7x-susceptible genomes, including XH001, span the two closely-related species A. odontolyticus and A. meyeri (>83% AAI to XH001) and a few unnamed strains ranging from 74 to 85% AAI to XH001 (Fig. S8a). These relationships were confirmed by a phylogenomic tree generated with PhyloPhlAn based on 387 concatenated core genes (Fig. S8b). The phylogenomic tree revealed an A. odontolyticus clade including four A. odontolyticus strains and A. sp. ICM39, which is sister to a monophyletic clade of the three nonpermissive strains, and another clade containing two A. meyeri strains and A. sp F0310.

We then performed a pangenome analysis to compare the genome content of these strains (Fig. 5) to identify genomic signatures associated with different susceptibility to TM7x infection. By grouping genomes based on gene content (Fig. 5, top right dendrogram), the resistant strains (concentric layers colored red) are clearly separated from the susceptible strains (permissive (blue) and nonpermissive (purple); Fig. 5), agreeing closely with the phylogenomic tree (Fig. S8b). Remarkably, within the susceptible strains the nonpermissive strains (purple) form an internal subgroup distinct from permissive strains (blue) (Fig. 5). All phylogenomic analyses and AAI are consistent with the observed separation of groups (heatmap in Fig. 5), while the 16S rRNA gene phylogeny fails to indicate that the purple group of nonpermissive hosts is distinct (Fig. 1). As the susceptible strains span at least two phylogenetically classified species (A. odontolyticus and A. meyeri) and potentially other closely related but unnamed species, the genome grouping by gene content broadly reflects the previously observed phylogenomic and AAI distinctions (Figs. 5, S8). F0310 was the only strain that shifted places from being similar to A. meyeri species based on genome sequence (in phylogenomic tree) to being in middle of the A. odontolyticus species. Based on the gene content and phylogenomic tree, the nonpermissive genomes are a genetically distinct group most closely related to A. odontolyticus and less so to A. meyeri.

Furthermore, core gene clusters for the various groups can be readily discerned, with 346 gene clusters forming the core of all 23 genomes, 464 exclusively shared by all susceptible strains, and 51 and 28 gene clusters exclusively shared by the resistant and nonpermissive strains, respectively (Fig. 5, Table S2). For context, each genome contains ~1700–2800 gene clusters (Fig. 5, light gray bar chart on right). While most genomes are estimated to be nearly complete and a handful are closed, most of the genomes are not closed and may be missing genes for methodological rather than biological reasons (Fig. 5, bar charts of genome statistics). Yet, the correlation of gene content with response to TM7x raises the possibility that certain shared genomic features may underly the observed phenotypes.

Comparative genomics reveal functional characteristics of different groups

We observed clades of strains defined by phylogeny and response to TM7x, e.g., permissive hosts. Ranking the predicted functions found across genomes for each TM7x response category (permissive, nonpermissive, or resistant) and combinations thereof can reveal functions enriched for each response type. The differentially enriched functions for these groups span multiple functional categories, from central metabolism to cell wall synthesis to regulation and recombination (Table 1).

For resistant vs. susceptible Actinomyces, numerous gene functions were exclusive to each (Table S2), potentially due to the strong genetic distinction between the two groups. Most pronounced of all functions were cell wall modification associated genes. Within the top five scored genes, we found Mur ligase family [34] and bacitracin resistance [35] proteins associated with resistant strains, and glycosyl transferase family [36] and O-mannosyltrasferase [37] proteins from susceptible strains (Table 1). These genes may directly or indirectly influence the TM7x attachment to the host. In addition, a key gene in the arginine deaminase (ADI) pathway, amidinotransferase arcA, was found in all ten of the resistant strains but none of the susceptible strains (Table 1). The ADI pathway can facilitate growth in acidic environments by increasing the pH, raising the possibility that TM7x, which encode a complete ADI pathway, could complement their ADI-less hosts [38]. Given the drastic oral pH shifts [39, 40] as well as localized pH stress from streptococcal neighbors [41], pH modulation and tolerance could be key for oral Actinomyces [40].

Permissive and nonpermissive genomes also contained distinctive functions (Table 1). For example, permissive strains are enriched for a GlcNAc-PI de-N-acetylase [42] and family 4 glycosyl hydrolase [43], which could be putatively involved in the hydrolysis of cell envelope glycoproteins, and may have the potential to regulate TM7x attachment levels. Interestingly, resistant and nonpermissive strains also share some functions not found in any permissive strains, such as a cytidine triphosphate (CTP) synthase.

Amino acid variants reveal genes phylogenetically correlated with TM7x response

While comparing gene presence can reveal major traits that may be involved in the observed phenotypes, it cannot distinguish between subtle but potentially critical variations in the sequence of shared proteins. If TM7x susceptibility is not due to clade-specific genes but instead distinct sequence variants of certain core genes, those sequence variants should correlate with TM7x sensitivity.

Thus, we employed a phylogenetic approach to look for core genes with sequence variants that match the observed phenotypes. This is a powerful way to identify shared genes in a pangenome that are correlated with an ecological phenotype [44], though sometimes prone to false positives and noise. From each of the 291 and 419 gene clusters with a single copy in each of the 23 genomes and the 13 susceptible genomes, respectively (Fig. 6a), we created a phylogenetic tree and compared it against topologies that differentiated sequence variants from nonpermissive (purple) vs. permissive (blue) vs. resistant (red). Fifteen gene clusters produced such topologies that distinguished each response type (Fig. 6b–d). While some are almost certainly noise (e.g., ribosomal protein rplR), many functionally interesting genes are identified including several cell envelope-associated proteins like the protein translocase secA, the ABC transporter sn-glycerol-3-phosphate ugpC, and an L,D-transpeptidase (Fig. 6b–d). The genes listed here represent a relatively short list of hypotheses that await future experimental investigation before any confident assertions can be made about their relevance to Actinomyces/TM7x associations.

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