Bacteria–bacteria dialogue

Within the sponge holobiont, bacterial cells do not only interact with the sponge cells, but also with bacteria of the same or other species. Figure 4.3 is an illustration of the density of microbes within the sponge mesohyl matrix. Competition for space and resources, or initiation of biofilm formation, as well as secondary metabolite production are among the well-studied topics of bacterial communication (Abisado et al. 2018). Thereby, competition and cooperation are facilitated within the proximity

The sponge extracellular matrix is densely populated by sponge cells, as well as by diverse and abundant bacterial symbionts

FIGURE 4.3 The sponge extracellular matrix is densely populated by sponge cells, as well as by diverse and abundant bacterial symbionts. Light microscopy images of semi-thin sponge tissue sections (A) of Plakortis simplex stained with Richardson solution and (B) of Acantheurypon spinispinosum. An epithelium-like outer cell layer, (pinacoderm, visible in (B)) surrounds the extracellular matrix (mesohyl) and the inorganic skeleton made up of spicules. Only pinacocytes are connected by tight junctions (Draper et al. 2019). C. choanocytes in Ch, choanocyte chamber; P, pinacoderm; B. bacterial cells in mesohyle; S, sponge cell; W. water canal. (Images kindly- provided by Kathrin Busch [GEOMAR Helmholtz Centre for Ocean Research Kiel].) of micrometers and are often a matter of balance (Nadell et al. 2016; Rakoff-Nahoum et al. 2016). Modeling can aid in predicting the metabolic interactions between bacteria, either based on co-occurrence models from relative abundance data (Thomas et al. 2016) or from metabolic models as inferred from metagenomic data (Slaby et al. 2017).

Quorum sensing

Bacteria-bacteria communication within the sponge extracellular matrix is mediated by quorum-sensing (QS). QS is a universal principle that aids interbacterial communication and is known from free-living as well as host-associated marine bacteria (reviewed in Hmelo 2017). Quorum sensing relies on the use of diffusible chemical signals in a population density-dependent manner. With increasing bacteria population size, the concentration of released QS molecules increases accordingly and eventually reaches a level (quorum) that initiates coordinated responses at the population level. QS mediates cellular mechanisms such as cell division, secondary metabolite production, plasmid transfer, and biofilm formation (Fuqua et al. 1994; Venturi and Subramoni 2009).

One of the most studied QS active molecule classes are N-acyl homoserine lactones (AHLs). AHLs are produced via the synthase LuxI family and interact with the LuxR cognate receptor proteins to initiate transcriptional activators and gene expression (Fuqua et al. 1994). The AHLs were first discovered in the marine bacterium Vibrio fisherii (Fuqua et al. 1994), and were detected in bacteria isolated from sponges for the first time in 2004 (Taylor et al. 2004). A great variety of AHLs have been recovered from sponge-derived bacterial isolates of different phylogenetic affiliations, including Gammaproteobacteria, Alphaproteobacteria, Firmicutes, and Flavobacteria (Mohamed et al. 2008; Bin Saidin et al. 2017; Mangano et al. 2018).

While bacterial isolates allow a thorough characterization of AHLs and their producer, only a small fraction of the sponge symbionts is cultivable, making it difficult to interpret the relevance of AHLs within the holobiont. Metagenomic data from Theonella swinhoei revealed an AHL synthase of an uncultured member of the Rhodobacterales family (Britstein et al. 2016). When heterologously expressed in E. coli, the synthase produced three different AHLs, demonstrating its function in vitro (Britstein et al. 2016). The first in vivo evidence of AHL production within the sponge comes from a study on Suberites domuncula (Garderes et al. 2012). AHLs were found in extracts of the whole sponge but not in extracts from the sponge cells, suggesting that the sponge itself does not produce AHLs (Garderes et al. 2012).

AHL production seems to be dependent on host species and varies over time (Britstein et al. 2018). Out of four investigated sponge species, one showed AHL production year- round, one showed no production at all, and two species displayed periodic production of AHLs (Britstein et al. 2018). For the sponge with constant AHL production, 14 different AHL molecules were identified, while only nine were present in the three replicate individuals. However, it is still unclear what drives this diversity. AHL patterns were neither related to LMA/HMA dichotomy nor correlated to microbiome composition (Britstein et al. 2018). One possibility is that constantly expressed AHLs derive from the core microbiome while varying AHL molecules result from transient seawater bacteria. Britstein et al. (2018) propose that microbial activity (i.e. gene expression) rather than microbial composition could account for AHL variability.

On the contrary, a single sponge-associated bacterium can produce a high diversity of AHLs, as in the case of a member of the family Rhodobacteraceae (Alphaproteobacteria), Pamcoccus sp. Ss63 isolated from Sarcotragus sp. (Saurav et al. 2016). Paracoccus sp. Ss63 is present in low abundances in seawater, sediment, and other sponges. A diverse array of AHL molecules may provide the possibility to sense various environmental cues aiding the free-living versus host-associated lifestyle (Girard et al. 2019). For example, the pH gradient between seawater and sponges might benefit the accumulation of AHLs within the host and thus aid symbiosis establishment (Saurav et al. 2016). The clear role of AHLs within the sponge holobiont remains unknown, however, they are likely relevant for the bacteria- bacteria dialogue within the sponge holobiont.

Quorum quenching

Quorum quenching (QQ) and quorum sensing inhibition (QSI) refer to mechanisms by which QS molecules are degraded or inactivated and communication is interrupted (reviewed in Borges and Simoes 2019). Sponges have recently been mined for both, QSI and QQ molecules (reviewed in Saurav et al. 2017), while several sponge extracts showed QSI orQQ activity which inhibited biofilm formation and/or population growth (Annapoorani et al. 2012; Mai et al. 2015; Britstein et al. 2016; Gutierrez-Barranquero et al. 2017). In some cases, these molecules were able to disrupt established biofilms (Gutierrez-Barranquero et al. 2017). Plakofuranolactone (^-lactone) is one of the few well-described QQ active molecules and was isolated from the sponge Plakortis cf. lita (Costantino et al. 2017). The bacterial origin of this molecule has been proven, but the microbial producer remains unidentified. A dual QS/QSI activity was described for bacteria isolated from sponges (Gutierrez-Barranquero et al. 2017), highlighting the complexity of bacterial interactions within the sponge holobiont.

The exchange of molecules between bacteria might ultimately interfere with the communication between sponge cells, or between sponge and bacteria. QS was shown to not only work in bacteria-bacteria interactions, but also to be involved in interkingdom communication in both animals and plants (Gonzalez and Venturi 2013; Pietschke et al. 2017; Weiland-Brauer et al. 2019). In primmorph-cultures and adult Suberitus domuncula sponges, short-term stimulation with bacterial N-3- oxododecanoyl-L-homoserine lactone affected gene expression of the sponge host, while cell viability and morphology remained unaffected (Garderes et al. 2014). More specifically, genes related to immunity and apoptosis were downregulated, as assessed by qRT-PCR. potentially aiding the sponge to monitor and regulate bacterial populations (Garderes et al. 2014). This is a fascinating example of the interlinked dialogue between sponges and bacteria, as molecules that have originally evolved for bacteria-bacteria interactions may eventually be adopted by the sponge as a means to detect and respond to microorganisms.

Phage-bacteria–host dialogue

Phages are the most abundant and diverse entities in the oceans (Rohwer 2003) and, along with their role as major bacterial killers, significantly impact global biochemical cycles (Suttle 2007), bacterial fitness, and diversity (King et al. 2018). In terms of numbers, each milliliter of seawater contains on average about 10 million virus particles. As filter-feeding animals, sponges pump up to 24,000 liters of seawater through their system per day (Weisz et al. 2008), exposing them to up to an estimated ~2.4 x 1013 viruses daily. The very high exposure to viruses prompts the question whether viruses interact in any way with either the sponge host or with its associated microbial symbionts. Interestingly, defense mechanisms against invading phages were identified previously as enriched features of microbial sponge symbionts by metagenomics (Fan et al. 2012; Slaby et al. 2017). These defense mechanisms are based on self-nonself-discrimination (i.e., restriction-modification system) or prokaryotic adaptive immunity (i.e., CRISPR-Cas system), representing major strategies against viral infection. While sponges are clearly exposed to massive amounts of viruses, little is known about their potential dialogue with the sponges and its associated microbial symbionts.

Phage diversity and host-specificity

The presence of virus-like particles w'ithin sponge tissues was already described in 1978 (Vacelet and Gallissian 1978) and was confirmed recently by electron- microscopy (Pascelli et al. 2018). In order to capture the molecular diversity of the viral associates, sponge virome sequencing was performed on several Great Barrier Reef sponges (Laffy et al. 2018). Interestingly, the identified patterns indicated species-specific viral signatures. Taxonomically, many of the recovered sponge associated viruses were dominated by clades of bacteriophages such as by tailed bacteriophages of the order Caudovirales (dsDNA) and Microviridae (ssDNA), as well as viruses including members of Megavirales and Parvoviridae (Laffy et al. 2018). High viral diversity and novelty was also found in a recent study by Jahn et al. (2019) who used metagenomics to characterize the viral diversity of three Mediterranean sponge species along with seawater controls. The extent of novelty in the sponge viromes was astonishing: only 3% were known on the taxonomic family level. The identified virome signatures (“fingerprints”) were highly specific to their host sponges in that each individual displayed its own unique virome signature (Jahn et al. 2019). The observation of viruses being individual specific is consistent with similar findings in humans (Moreno-Gallego et al. 2019).

Ankyphages aid symbionts in immune evasion

Jahn et al. (2019) further described a group of phages (hereafter termed “Ankyphages”) that suppresses immune cell function and phagocytosis in eukaryotic cells (reviewed in Leigh 2019). These Ankyphages encode a novel symbiont phage-encoded protein, ANKp, that modulates eukaryote-bacterium interaction by altering the eukaryotes’ physiology in response to bacteria. Specifically, it appears that the phage-encoded Ankyrin protein is secreted from the bacterial cell and downregulates eukaryotic proinflammatory cytokines and phagocytosis in response to ANKp. These experiments were performed in murine cell lines as an experimentally tractable model for sponge-microbe interactions is still lacking. Murine macrophages display many features of a major class of sponge cells (archaeocytes) which, much like macrophages, are single, amboeboid cells that patrol the sponge matrix in search for bacteria to be phagocytosed. Moreover, the major elements involved in mammalian immune signaling were found to be present in sponges. The resulting data show, to our knowledge for the first time, that phage ANKp modulates the eukaryote response to bacteria by downregulating proinflammatory signaling along with reduced phagocytosis rates.

Surprisingly, homology searches revealed that Ankyphages are widely distributed in host-associated environments, including the human oral cavity, gut, and stomach. It is thus tempting to speculate that the role of Ankyphages in mediating the dialogue between bacteria and animal hosts is much more widespread than in the context of marine sponges. In summary, ANKp represents the first secreted phage effector protein that downregulates eukaryote immunity upon exposure to bacteria. This is of relevance to host-microbe symbiosis research in that it provides the functional underpinnings for tripartite phage-bacteria-eukaryote dialogue. Moreover, this finding is of interest in the context of phage therapy as mechanisms to temper host immune responses are urgently sought-after in clinical and medical settings.

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