In times long past, highly evolved eukaryotes, for example biochemists, occasionally quipped that bacteria were nothing more than a bag of enzymes. Well, from the perspective of bacteria, one could return the compliment: eukaryotes, for example single-celled amoebae, are little more than a bag of bacteria. A hyperbole for the sake of a pun? Not, if it comes to Acanthamoeba species.
"Acanthamoeba spp. are among the most prevalent protozoa found in the environment. They are distributed worldwide, and have been isolated from soil, air, sewage, seawater, chlorinated swimming pools, domestic tap water, bottled water, dental treatment units, hospitals, air-conditioning units, and contact lens cases. Additionally, they have been isolated from human skin, nasal cavities, throats, and intestines, as well as plants and other mammals." (Wikipedia)
There are presently 30+ described species in the genus Acanthamoeba (phylum Amoebozoa, see Figure 1), and Shi et al. (2021) list in their review of The Ecology and Evolution of Amoeba-Bacterium Interactions a whopping 35 bacterial species from various phyla that are known for non‑predatory interactions with one or more Acanthamoeba species. "Non‑predatory interactions" means that these bacteria do not simply serve the amoebae as food but have been found either as intracellular commensals, or as mutualists, or as pathogens. Or, depending on growth conditions, in any of these roles ─ including eventually serving as food, mind you.
A hint for medical microbiologists interested in human pathogens: You will spot some of the "usual suspects" in the list of bacterial species... but skip it and continue to the next paragraph if you deem "35 bacterial species" sufficient to attest diversity:
· Actinobacteriota: Mobiluncus, Mycobacterium
· class Alphaproteobacteria: Rickettsia, Afipia, Jidaibacter, Caedibacter, Paracaedibacter, Odysella
· Campylobacterota, formerly Epsilonproteobacteria: Campylobacter, Arcobacter, Helicobacter
· Bacillota, formerly Firmicutes: Staphylococcus, Procabacter, Listeria, Bacillus
· class Gammaproteobacteria, formerly Betaproteobacteria: Burkholderia, Ralstonia
· class Gammaproteobacteria: Escherichia, Salmonella, Yersinia, Pseudomonas, Stenotrophomonas,
Coxiella, Francisella, Berkiella
· Bacteroidota: Prevotella, Amoebophilus, Porphyromonas, Flavobacterium
· Chlamydiota: Chlamydophila, Parachlamydia, Protochlamydia, Waddlia, Neochlamydia, Simkania
The list may not even be complete, as I miss, for example, Legionella pneumophila (class Gammaproteobacteria) known to survive and replicate within both Acanthamoeba castellanii and A. polyphaga, and "Candidatus Nucleicultrix amoebiphila" (class Alphaproteobacteria) that infects A. castellanii and makes itself at home in its nucleus (see here in STC).
Also missing from the list are the Vibrios. Van der Henst et al. (2015) observed that V. cholerae can resist intracellular killing by A. castellanii. Non‑digested bacteria were either released or established a replication niche within the contractile vacuole. The bacteria were maintained therein even upon encystment of the amoebae (in the literature, you also find "amoebas" or "amebas", reflecting English's preference for the 3'-s to indicate the plural and its reluctance to use the œ), and ultimately returned to the aquatic habitat via lysis of their hosts. It's more complicated for Vibrio parahaemolyticus that seems to resist predation by A. castellanii but depends on an unidentified diffusible factor secreted by the amoebae for long-term survival in a co‑culture (microbiologists have no special term for such an interaction that smells a bit like spooky interaction at a distance).
To prevent any misunderstandings I should add that one usually finds one or a few bacterial species in a single amoeba, not the whole zoo. In fact, most of the human pathogens in the above list were found in a direct test of whether they can "non‑predatory interact" (in the above sense) with easily cultured Acanthamoebae, for example A. castellanii. Generalized, such studies have led to a paradigm shift in epidemiology over the past two decades, insofar as environmental amoebae, not just Acanthamoebae, are now considered as natural reservoirs of many human pathogens. As one example, the plague bacterium, Yersinia pestis, that is known to circulate in the environment in rodents and is transmitted among them by fleas, was found to survive and propagate for days in the vacuole of Dictyostelium amoebae (better than in A. castellanii).
But how does it actually look like in the wild? Which bacterial endosymbionts are found in environmental Acanthamoeba isolates? Schmitz-Esser et al. (2008) addressed this question in their study of of ten novel Acanthamoeba strains isolated from soil and lake sediment samples from Austria, Tunisia, and Dominica. Eight of the ten isolates, tentatively assigned to the Acanthamoeba genus based on morphology, contained intracellular bacteria as revealed by DAPI staining (fluorescent DNA dye). 18S rRNA sequencing confirmed that all isolates belong to known 18S-subtypes of Acanthamoeba (see here for a tree).
Schmitz‑Esser et al. found by 16S rRNA sequencing that all eight Acanthamoeba isolates contained just one endosymbiont, and these are closely related to known Acanthamoeba endosymbionts. Two endosymbionts are most closely related to Paracedibacter (Alphaproteobacteria) and Procabacter (Betaproteobacteria), three are more closely related to Amoebophilus (Bacteroidota), and three related to Protochlamydia amoebophila and Parachlamydia (Chlamydiota), respectively (see here for the trees).
They localized the endosymbionts in their hosts by fluorescence in situ hybridization (FISH) and found them distributed in the cytoplasm with no indication of a preference for certain organelles and in considerably varying numbers (Figures 2 + 3). Only the two Parachlamydia spp. showed cell clustering, which may point to their residing in large vacuoles resembling the host-derived inclusion characteristic for known Chlamydiae (Figure 2A, Figure 3F). Note that Schmitz‑Esser et al. confirmed the presence of just one endosymbiont in each of their different hosts as they used a dual-labeling strategy for the bacteria (a bacteria-specific probe (blue) and a species-specific probe (red) and found only a single signal type (blue+red=yellow) in every sample besides the eukaryote-specific probe for the host (blue).
This study by Schmitz‑Esser et al. points to ─ in their own words ─ "the existence of only a limited number of phylogenetically different groups of obligate bacterial endosymbionts of Acanthamoeba spp., showing a global distribution. This might suggest that adaptation of bacteria to long-term intracellular symbiosis with Acanthamoebae has originated only a few times during evolution." I stumble over the "obligate" because they did not show this experimentally in their study and only refer to comparable studies. The "obligate" will become debatable even for Chlamydiae, which until not so long ago were known exclusively as obligate intracellular pathogens of vertebrates: in a study from 2020, Dharamshi et al. describe their discovery of an astonishing variety of Chlamydiae in deep anoxic marine sediments from the Arctic Mid-Ocean Ridge, many of them potentially free-living.
That said, I agree with the concluding remarks of Schmitz‑Esser et al. that the (in 2008) ongoing genome sequencing projects for Acanthamoeba castellanii Neff and several Chlamydiae "will help to understand similarities and differences between these symbionts and the interactions with their Acanthamoeba hosts, as well as the role of free-living amoebae as evolutionary training grounds for facultative intracellular bacteria."