Bipolar Biogeographical Distribution of Parafrancisella Bacteria Carried by the Ciliate Euplotes

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The Version of Record is available online at: https://doi.org/10.1007/s00248-023-02263-1 ” 1 Bipolar biogeographical distribution of Parafrancisella bacteria carried by the ciliate Euplotes 1 Annalisa Candelori1, Graziano Di Giuseppe2, Eduardo Villalobo3, Andreas Sjödin4,5, Adriana Vallesi1 2 1School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC), Italy 3 2Department of Biology, University of Pisa, Pisa, Italy 4 3Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain 5 4Department of Clinical Sciences, Pediatrics, Umeå University, Umeå, Sweden 6 5Division of CBRN Security and Defense, FOI – Swedish Defense Research Agency, Umeå, Sweden 7 8 ORCID ID: Graziano Di Giuseppe, 0000-0002-9999-7650; Eduardo Villalobo, 0000-0002-0331-115X; Andreas Sjödin, 9 0000-0001-5350-4219; Adriana Vallesi, 0000-0002-4127-090X 10 Corresponding author: Adriana Vallesi, e-mail: [email protected] 11 Abstract 12 Parafrancisella adeliensis, a Francisella-like endosymbiont, was found to stably reside in the 13 cytoplasm of an Antarctic strain of the bipolar ciliate Euplotes petzi. To inquire whether Euplotes 14 cells collected from distant Arctic and peri-Antarctic sites host Parafrancisella bacteria, wild-type 15 strains of the congeneric, bipolar species, E. nobilii, were screened for Parafrancisella by in situ 16 hybridization and 16S gene amplification and sequencing. Results indicate that all Euplotes strains 17 analyzed contained endosymbiotic bacteria with 16S nucleotide sequences closely similar to the P. 18 adeliensis 16S gene sequence. This finding suggests that Parafrancisella/Euplotes associations are 19 not endemic to Antarctica, but are common in both the Antarctic and Arctic regions. 20 Keywords: Francisella-like endosymbiont, Parafrancisella, Euplotes, microbial associations, 21 polar microorganisms 22 The genus Francisella comprises facultative intracellular γ-protobacteria which are well known for 23 their ability to enter, persist and proliferate inside different hosts [1]. F. tularensis is a specialized 24 2 pathogen of both invertebrates and vertebrates, and in humans it causes tularemia [2]. 25 F. noatunensis is the etiological agent of the fish disease known as francisellosis [3], while the 26 generalist species F. philomiragia and F. novicida may harm human beings with a compromised 27 immune system [4]. In recent years, the genus was expanded by the inclusion of ‘environmental’ 28 bacteria isolated from marine and freshwater environments, cooling water systems and other 29 environmental sources, and Francisella-like endosymbionts of ticks and ciliates [5-7]. The 30 inclusion of these species required a redesigning of the original organization of the genus, which is 31 currently subdivided into four genera: Francisella, Parafrancisella, Allofrancisella, and 32 Pseudofrancisella [7]. 33 The newly established genus Parafrancisella is represented by one species, which was isolated 34 from an Antarctic strain of the bipolar unicellular ciliate Euplotes petzi [8]. Gene sequences from 35 this species, originally designed as Francisella adeliensis and now re-named Parafrancisella 36 adeliensis, were also found in a metagenomic analysis of a bacterial consortium associated with 37 another Antarctic ciliate species, E. focardii [9], suggesting that P. adeliensis can colonize different 38 Euplotes species living in sympatry in the Antarctic marine environment. This endosymbiont has a 39 remarkable adaptability in terms of growing at temperatures ranging from 4 to 30 °C, but not at 37 40 °C, or at salinities ranging from 0 to 35 ‰. Its genome includes genes of the Francisella 41 pathogenicity island needed for intracellular growth but lacks genes for the biosynthesis of essential 42 amino acids. These two genetic traits are in common with pathogenic Francisella species and imply 43 a host dependency for essential amino acids [8]. 44 To date, little is known about the distribution of P. adeliensis, in particular whether this species is 45 endemic to Antarctica or a secondary colonizer carried by its host. In this latter case, free-living 46 ciliates with a bipolar distribution deserved research interest as their natural reservoirs and global 47 vectors. Since live strains of E. petzi were no longer available in our laboratory, we addressed this 48 question by analyzing an alternative bipolar Euplotes species, i. e. E. nobilii, which inhabits the 49 3 same marine environment and shares food sources and behavior with E. petzi. In addition, this 50 species is represented by Arctic and Antarctic populations characterized by various degrees of 51 trans-tropical gene flow and genetic connection [10-12]. Five Arctic and one peri-Antarctic wild 52 type strains, assigned to E. nobilii based on morphological and genetic grounds [12,13], were 53 selected: two (denoted Bess-B, Bess-F) were collected at two different coastal sites of Bessel Fjord, 54 Greenland; two (4Pyrm4 and Lyg sp. B) from the coasts near Pyramiden and Longyearbyen, 55 Svalbard Islands; one (Barrow) from Barrow, Northern Alaska; one (PN-1) from Puerto Natales, 56 Chilean Patagonia (Fig. 1). Each strain was derived from a single specimen, isolated in pasteurized 57 natural or artificial seawater and maintained in cold rooms, at 4-6 °C, using the green algae 58 Dunaliella tertiolecta as a food source. 59 In order to determine whether these strains harbor Parafrancisella bacteria within their cytoplasm, 60 two fluorescent probes were used in in-situ hybridization assays (Supplementary information). One 61 green-fluorescent probe (EUB338) was specific to a 16S rRNA-sequence conserved in most 62 bacterial species, while the other red-fluorescent probe (Bwall1448) was specific to a 23S rRNA 63 region of Francisella which is conserved also in P. adeliensis 23S gene [8,14]. The two probes 64 yielded merged yellow-fluorescent signals in the cytoplasm of the six E. nobilii strains (Fig. 2). 65 Only in cells of the strain Lyg sp. B, merged fluorescent signals indicated that Parafrancisella co66 existed with other bacteria; in all the other strains Parafrancisella represented the only guest. 67 DNA preparations from each E. nobilii strain were then used to amplify the Parafrancisella 16S 68 gene sequence, following a two-step amplification protocol (Supplementary information). In all the 69 strains analyzed, a single 16S gene sequence was obtained (Supplementary Table S1). Pairwise 70 sequence comparisons showed that these 16S gene sequences shared high level of identity, ranging 71 from 99 to 100%, with the 16S gene sequence of P. adeliensis, suggesting that Parafrancisella 72 bacteria represent common guests of Euplotes species living in polar marine environments. 73 4 The 16S gene sequences of E. nobilii endosymbionts and representative species within the four 74 genera of the Francisellaceae family were next used to generate a phylogenetic tree 75 (Supplementary information). As expected, the 16S gene sequences of the E. nobilii endosymbionts 76 cluster together with the 16S gene sequence of P. adeliensis in a well-supported clade (Figure 3A). 77 The genetic relationships between the E. nobilii endosymbionts and P. adeliensis were further 78 analyzed by constructing a haplotype network (Figure 3B). Five haplotypes, differentiated by 1 to 8 79 mutational events, were identified. The haplotypes of the endosymbionts from Arctic strains of E. 80 nobilii form two distinct lineages which separate from the one shared by P. adeliensis and the 81 endosymbiont of the Patagonian E. nobilii strain PN-1. 82 Taken together these findings provide evidence that the Parafrancisella/Euplotes associations are 83 common in the polar marine environment and that these associations have a bipolar distribution. 84 The occurrence of Parafrancisella in polar Euplotes collected from distant regions implies that their 85 symbiotic relationship is stable. This suggests that both microbial partners benefit from their stable 86 partnership [15]. On the one hand, the bacterial endosymbiont finds the right conditions to survive 87 and replicate by obtaining the essential amino acids it cannot synthesize [8]. On the other hand, the 88 eukaryotic host may use the many enzymes that bacteria secrete into its cytoplasm through a type 89 VI secretion system [8]. These enzymes include antioxidant enzymes useful for coping with the 90 oxidizing marine environment of the polar regions. Further studies are now needed for improving 91 our knowledge of this microbial relationship and its ecological role in cold environments. 92 Acknowledgements 93 We would like to thank Prof. P. Luporini (University of Camerino) and Prof. F. Dini (University of 94 Pisa) for collecting ciliate specimens in various Antarctic and Arctic expeditions, and for isolating 95 and maintaining Euplotes cultures. 96 97 Funding 98 5 This work was financially supported by the PNRA (Programma Nazionale di Ricerca in 99 Antartide) from the Italian Ministero dell’Università e della Ricerca (MUR), grant number 100 PNRA18_00152. 101 102 Conflict of interests The authors declare no competing interests. 103 104 References 105 1. Colquhoun DJ, Larsson P, Duodu S, Forsman M (2014) The family Francisellaceae. In: Rosenberg E, DeLong EF, 106 Lory S, Stackebrandt E, Thompson F (eds) The Prokaryotes. Springer, Berlin Heidelberg, pp 287–314 107 2. Sjöstedt A (2007) Tularemia: history epidemiology pathogen physiology and clinical manifestations. Ann N Y 108 Acad Sci 1105:1–29 109 3. 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In: Dharumadurai D (ed) Microbial Symbionts, Academic Press, pp 419-439 145 146 7 Figure legend 147 Fig. 1 Geographic origin and denomination of the E. nobilii strains used in this study. Localities are 148 indicated by black flags and numbers indicate the collection sites. The gray flag marks the Antarctic 149 origin of E. petzi, from which P. adeliensis was first isolated. 150 151 Fig. 2 Intracellular localization of Parafrancisella bacteria in cells belonging to different polar E. 152 nobilii strains. Fluorescent in situ hybridization assays were performed with the green fluorescein153 labeled probe EUB338 (top panels) for all eubacteria and the red Cy3-labeled probe Bwall1448 154 (middle panels) specific for Francisella [28]. The co-localization of the two signals (yellow) is 155 shown at the bottom panels. Only in cells of the strain Lyg sp. B, Parafrancisella bacteria co156 existed with another type of bacteria, from which it differed for a more peripheral cytoplasmic 157 localization. Scale bar = 20 μm 158 159 Fig. 3 Phylogenetic analysis and genetic diversity of Parafrancisella. (A) Phylogenetic tree of the 160 family Francisellaceae. Bootstrap values for Maximum Likelihood, Maximum Parsimony, and 161 posterior probability values for Bayesian Inference are provided for each node. Values below 50% 162 and 0.90 for bootstrap and posterior probability, respectively, are not shown. The scale bar 163 corresponds to two hundred substitutions per 1,000 nucleotide positions. The accession numbers of 164 the sequences used to generate the tree are indicated in brackets; the newly obtained sequences are 165 shown in bold. The 16S gene sequence of Escherichia coli was used as an outgroup. (B) Median166 joining haplotype network. Each circle represents a unique haplotype, its size reflects the number of 167 individuals expressing that haplotype, and black and white colors denote the Antarctic/Patagonian 168 and Arctic location, respectively. The denomination of E. nobilii strains containing Parafrancisella 169 bacteria belonging to each haplotype is shown. Crosshatches indicate the number of nucleotide 170 differences between haplotypes. 171 172 8 FIGURE 1 173 174 175