Three’s a crowd: Saccharibacteria episymbiosis modulates phage predation of host bacteria

The realization that microbiology is more diverse and complex than appreciated is both sobering and exciting in equal measure. Such was the case beginning in the mid-1990s with the discoveries of the Candidate Phlya Radiation (CPR) bacteria and the DPANN (an acronym of the names of the initial phyla: Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota) archaea, massive radiations of organisms which are distributed widely in multiple environments (1). CPR and DPANN are ultrasmall organisms, some less than 200 nm, with a stripped-down genome and lacking many essential biosynthetic pathways, often including those for fatty acids, nucleotides, and peptidoglycan (2). While some CPR and DPANN organisms may be capable of a free-living lifestyle, most are thought to depend on other species for the basic resources they are incapable of synthesizing themselves and are obligate epibionts, or ectosymbionts, living on the surface of their host (2). Possibly as a further adaption to their dualistic nature, CPR and DPANN organisms can possess the biosynthetic machinery for the production of secondary metabolites including lantibiotics and other ribosomally synthesized and posttranslationally modified peptide (RiPP) products (3, 4). The potential for CPR and DPANN to impact microbial ecosystems is therefore enormous; however, it hardly bears mentioning that such studies are impeded the difficulty of laboratory culture (5). Culture-independent studies showed that the oral cavity is teeming with the CPR constituent, Saccharibacteria, particularly during inflammatory disease (5). Moreover, the relationship between Saccharibacteria and the human mouth is an ancient one, and the organism has been identified in the dental calculus of Neolithic hunter-gatherers (6). The question thus arose as to the role of the Saccharibacteria in the mouth. While their numbers increase in periodontitis, in turned out that they cause attenuation the virulence of their hosts in an in vivo model of the disease (7). Any relationship with pathogenicity, then, is not a straightforward one. In PNAS, Zhong et al. (8) uncover an entirely novel role for Saccharibacteria, that of protecting biofilms of the host organism from lytic phage predation. This interaction will thus promote long-term coexistence of phage and host while avoiding a Red Queen style evolutionary arms race (Fig. 1).

Fig. 1.

In biofilm communities of XH001 (brown rods) TM7x (green cocci) episymbiosis reduces CWP expression and promotes resistance to LC001 phage predation. TM7x-free XH001 cells express CWP and are permissive to LC001 thus facilitating phage and host coexistence. Planktonic XH001 daughter cells or cells shed from the biofilm have lower CWP expression, regardless of TM7x episymbiosis, and are resistant to LC001 infection.

The study reported by Zhong et al. (8). arose from the convergence of two research themes under investigation in their laboratories. This group had previously coisolated the first cultivable representative of Saccharibacteria, Nanosynbacter lyticus type strain TM7x, with its host organism Schaalia odontolytica (formerly Actinomyces odontolyticus) strain XH001 (9). Episymbiotic growth of XH001 with TM7x promoted biofilm formation through autoinducer-2 (AI-2) quorum sensing (10). Independently, they identified a lytic phage designated LC001 that targets biofilm, but not planktonic, XH001 cells (11). The surface grown as opposed to planktonic distinction is significant because in the high shear force conditions of the oral cavity, a biofilm mode is considered essential for long-term persistence. In contrast, planktonic organisms, either new arrivals or shed from biofilms, are in a race against time to find a surface to colonize. In their PNAS study, Zhong et al. (8) found that TM7x episymbiosis conferred resistance of XH001 cells to LC001 phage-induced lysis, with protection being the result of decrease in adsorption of LC001 to XH001. Loss of phage binding could be due to a genetic mutation or a transient change in expression, and this was addressed by establishing mixed cocultures in which all TM7x-free XH001 were derived from TM7x-attached XH001 single cells. Surface-grown TM7x-free XH001 cells which were derived from TM7x-attached XH001 could be bound by LC001, strongly implicating transient modulation of a XH001 surface structure as the basis of phage resistance.

To begin to define the molecular basis of TM7x-dependent resistance to LC001 phage adsorption Zhong et al. (8) performed an RNA sequencing (RNA-Seq) comparison of XH001 with TM7x/XH001 (12). With relevance to phage adsorption, a gene cluster encoding proteins with predicted functions in cell wall polysaccharide (CWP) biosynthesis was down-regulated in TM7x/XH001. This region included a series of enzymes responsible for the synthesis of glycerol-phosphate (GroP) repeats substituted with N-acetyl-galactosamine (GlaNAc), major constituents of cell wall anchored teichoic acids (WTA). In Gram-positive bacteria, CWP are often receptors for phage binding, and GroP repeats in particular constitute a receptor for Siphovirdae phage (13), a family that includes LC001.

In PNAS, Zhong et al. uncover an entirely novel role for Saccharibacteria, that of protecting biofilms of the host organism from lytic phage predation.

Given the correlation among TM7x episymbiosis, expression of genes encoding CWPs and susceptibility to LC001 predation, Zhong et al. (8) sought to provide more directive evidence for the role of CWP regulation using an overexpression approach. A predicted operon comprising CWP-related genes and which was differentially regulated by TM7x, was overexpressed in XH001 cells. The authors went on to show that transcription in this operon was no longer reduced by association with TM7x. With the necessary tools established, the team was able to show that overexpression of CWP leads to significantly enhanced LC001 binding and sensitivity to phage lysis even when the XH001 cells have formed episymbiosis with TM7x. The fundamental elements of the mechanism of TM7x action are thus in place: episymbiosis of TM7x with XH001 cells down-regulates expression of CWP at the transcriptional level and the consequent loss of the CWP receptor for LC001 adsorption confers resistance to phage predation.

The emerging model for the interconnectivity of TM7x, XH001 CWP, and sensitivity to LC001 phage had one more test to pass. Namely, it must accommodate the observation that planktonic XH001 cells are resistant to phage adsorption and infection. The simplest explanation would be that planktonic cells express lower levels of CWP. An RNA-Seq analysis indeed confirmed this situation, and further overexpression of CWP rendered planktonic XH001 cells susceptible to LC001 infection.

Having defined the underpinning of the role of TM7x in modulating phage sensitivity, Zhong et al. (8) then turned their attention to the broader biological picture and asked how the interaction may regulate the persistence of phage and host. First, the authors showed that with serial passage XH001 monocultures could develop resistance to infection by phage which were isolated at the beginning of the experiment. Further cycles of phage infection and lysis produced phage capable of infecting the newly resistant XH001, the classical result indicating that the phage is evolving as well as the host bacteria. With the establishment of a TM7x/XH001 episymbiosis, the situation changed and the onset of phage resistance was considerably slowed in TM7x-free XH001 allowing XH001 to take a break from the evolutionary arms race.

Phage are a long-recognized player in microbial ecology. This study by Zhong et al. (8) reveals that our concepts of the nature of the phage-host relationship need updating to incorporate tripartite relationships. In the case of S. odontolytica XH001, surface-grown cells can escape phage predation in the absence of dedicated defense systems by entering into a symbiosis with the epibiont Saccharibacteria TM7x. In contrast to mutation-driven resistance, TM7x-induced resistance is transient and reversible. XH001 cells lose their phage resistance either when detached from TM7x or derived from TM7x-associated XH001 through cell division. Heterogenous populations of phage-sensitive and phage-resistant organisms promote long-term coexistence of bacteria and phage. Another implication is that as XH001 cells develop in biofilm mode and resist the physical shearing forces of the oral cavity, association with TM7x will relieve pressure from lytic phages. As discussed by Zhong et al. (8), the tripartite dynamics with two antagonistic partners may be an additional way to create a “source-sink” situation for a bacterial–phage interaction (14). Infection of TM7x-free XH001 allows phage replication and is the “sink” component. Episymbiotic XH001 are resistant to this sink, but the continual production of phage-susceptible TM7x-free XH001 generates the “source” for LC001 predation.

Given the abundance of Saccharibacteria in the nature, future study in this area is eagerly anticipated. Topics to address, as alluded to by Zhong et al. (8), include the structure of the CWP binding moiety for the phage; the mechanistic basis for downregulation of CWP-associated genes by TM7x; control of association/dissociation of TM7x with XH001; the ubiquity of this mechanism across other episymbiotic pairs; and the extent to which this occurs in vivo. Further advances are important to more fully understand the multidimensionality of interspecies and interkingdom interactions that drive the emergent properties of polymicrobial communities.