Legionella pneumophila, a Gram-negative intracellular bacterium that infects and replicates within alveolar macrophages, causes Legionnaires’ disease or Pontiac fever. L. pneumophila Philadelphia-1 is firstly isolated after an explosive pneumonia outbreak in 1976 in Philadelphia. The Legionnaires’ mortality rate is ranging from 1 to 80% depends on risk factor such as age, transmission factor, and immunity. In the other hand, the Pontiac fever is considered as self-limiting flu-like disease. Legionella infects humans through aerosol inhalation, although human-to-human transmission is generally not established (Diederen, 2008; Yao et al., 2024). L. pneumophila inhabits both natural and man-made aquatic ecosystems, such as water fountains, foot spas, and cooling towers (Head et al., 2021). Well-known as a pathogen with a broad range of protozoan hosts—Paramecium (Watanabe et al., 2016), Acanthamoeba (Kim et al., 2023), and Tetrahymena thermophila—the latter has emerged as a favorable host for L. pneumophila. Moreover, L. pneumophila is efficiently ingested but poorly digested by the ciliate T. thermophila. Coculturing this ciliate with L. pneumophila increases the survival and rapid multiplication of Tetrahymena (Berk et al., 2008; Boamah et al., 2017). These protozoan hosts are likely responsible for the environmental persistence of Legionella by providing a platform for survival and protection from various environmental stresses.

As previously reported, the L. pneumophila Ofk308 strain, an environmental isolate, exhibits cytotoxicity toward the Paramecium host (Watanabe et al., 2016). Paramecium infected with Ofk308 undergoes cell lysis and avoids phagosome–lysosome fusion (Watanabe et al., 2016). However, its cytotoxicity toward T. thermophila remains unclear. The findings of the study with Tetrahymena are important in clarifying whether the cytotoxicity of Ofk308 strain is very specific to Paramecium or whether it is widely exerted in other protozoan hosts. The Ofk308 strain harbors several well-characterized virulence factors, including dot, lvh, rtx, and hsp60 (Tachibana et al., 2013). Notably, the Dot/Icm type IV secretion system is essential for intracellular replication, allowing Legionella to form a replication-permissive compartment, i.e., the Legionella-containing vacuole (LCV), via this system (Chauhan and Shames, 2021). We have also identified several other bacterial factors involved in interactions with protozoan hosts and analyzed their functions (Watanabe et al., 2016, 2018; Nishida et al., 2018). Interestingly, however, when comparing the gene profiles of general Legionella strains capable of utilizing protozoa as hosts with those of the cytotoxic Ofk308 strain, no marked differences were observed in the presence of these known genes. These findings suggest that an analysis focusing on expression levels or the timing of expression is required. In this study, we explored factors relatively highly expressed in the Ofk308 strain using RNA-seq and hypothesized that they may be novel candidate factors involved in cytotoxicity toward the protozoan host. Among the identified candidates, fosA1, encoding a putative vicinal oxygen chelate (VOC) protein family, and msrA1, encoding a bifunctional methionine sulfoxide reductase B/A (msrB/A), were found to contribute to cytotoxicity in T. thermophila. Understanding the interaction between these organisms from a public health perspective may help identify new environmental infection risks and control strategies.

RNA-Seq was initially performed to uncover genes with higher expression in the Ofk308 strain (Figure 2 and Table 2), and several candidate genes such as archease, DMT, lpg2541, mscL, msrB/A, and VOC were identified for further construction into deletion mutant strains. Archease is a 16 kDa protein involved in DNA or RNA processing. This was followed by the identification of DMT, a large group of membrane transporters. The next gene, lpg2541, belongs to the Dot/Icm T4SS effector family, contributing to bacterial conjugation, gene transfer, and delivery of virulence factors into host cells. Finally, mscL, along with mscS, forms part of the mechanosensitive channel proteins that provide limited protection against hypoosmotic shock in bacteria (Pivetti et al., 2003; Gomez-Valero et al., 2011; Desai et al., 2014; Tsuchiya et al., 2016). Additional candidates include four hypothetical proteins: Ofk_A, Ofk_B, Ofk_C, and Ofk_D, with unknown functions. We expect that the genes of these candidates contribute during Legionella infection within CU427 cells, as further explained.

The biphasic life cycle of Legionella has been widely associated with a broad range of protozoan hosts that exhibit highly specific host-pathogen interactions. Tetrahymena survives during co-culture with Legionella Phil-1, showing an almost twofold increase in number within 2 h postinfection. Naturally, under optimal conditions, Tetrahymena demonstrates a rapid growth rate of less than 2 h (Cassidy-Hanley, 2012). This suggests that CU427 may gain mutualistic symbiosis during infection with Phil-1. Interestingly, as previously reported, Ofk308 exhibits cytotoxicity toward several Paramecium strains in an MOI-dependent manner (Watanabe et al., 2016), which aligns with our findings that Tetrahymena maintains a similar relationship with Ofk308. Furthermore, in our study, CU427 was infected using Legionella cultures at the postexponential phase. This growth phase is correlated with increased virulence, cytotoxicity, motility, and the capacity to evade phagosome–lysosome fusion (Molmeret et al., 2004). Thus, our findings provide a more accurate prediction of Ofk308’s cytotoxicity. In contrast to ΔVOC and msrB/A, which significantly altered the relative number of viable CU427 (Figure 3), deletion of archease, DMT, lpg2541, mscL, Ofk_C, and Ofk_D remained cytotoxic to CU427 within 48 hpi. It is possible that these genes are not directly related to Ofk308’s cytotoxicity toward the host, or that Ofk308 may harbor alternative genes that compensate for the deletions, resulting in no change in cytotoxicity in the mutant strains.

Despite Legionella growing optimally at 37°C, this bacterium can replicate at temperatures ranging from 25°C–42°C, leading to a broad range of intracellular growth within protist hosts (Richards et al., 2013). A previous study also showed that co-culturing Legionella with Tetrahymena can increase Legionella numbers in environmental water samples (Fields et al., 1984). This aligns with our findings, where the intracellular CFU count of Phil-1 within CU427 increased over 48 h at the optimal growth temperature for T. thermophila (30°C). In contrast, due to the death of the Tetrahymena host, it is difficult to accurately assess changes in intracellular CFUs for Ofk308 or the deletion mutants that did not reduce cytotoxicity. Notably, each deletion mutant showed a distinct trend in intracellular bacterial numbers. A previous study reported that, in Legionella and other Gram-negative bacteria, growth temperature strongly influences gene expression and may trigger pathogenicity (Aroori et al., 2013; Hochstrasser and Hilbi, 2022). However, no studies definitively demonstrate the involvement of our target genes in intracellular growth at 30°C. Future comparative studies across different temperatures may clarify the specific roles of these genes.

Protozoa uptake bacteria through traditional or coiling phagocytosis and confine them to phagosomes, which subsequently fuse with lysosomes (Yang et al., 2022). Legionella was taken up into CU427 as a food vacuole and developed into an LCV compartment over time. Intracellular bacterial loads of Legionella Phil-1 and Ofk308 were analyzed by counting the LCVs. In Figure 5, a higher number of LCVs containing Ofk308 with enlarged size and the release of Ofk308 into the host cell cytoplasm before host cell lysis indicate that the environmental wild type exhibits greater virulence within CU427. Modified Legionella, such as genetic knockdowns or mutants, are known to show reduced growth and egress ability within host cells compared to the wild type (Mraz and Weir, 2022). In Figure 5, the pellet expelled from CU427 after 2 hpi contains viable Legionella. This expelled pellet elevates Legionella’s virulence, enabling it to persist longer in the environment and form biofilm. Additionally, the expelled pellet is also believed to function as a defensive mechanism of Tetrahymena IB against Legionella infection or as a food reserve in T. thermophila (Hojo et al., 2012; Swart et al., 2018; Kawashiro et al., 2021; Personnic et al., 2021). The fact that the Ofk308 strain is cytotoxic and less able to provide these benefits to Tetrahymena hosts suggests significant variation in the relationship between Legionella and Tetrahymena.

In this study, we continued to focus on the VOC and msrB/A mutant strains. VOCs are members of the 10 metalloenzyme superfamilies, which play roles in various biological reactions, including antibiotic resistance, and are able to utilize metal ions for their catalytic activity. The structural member of VOC is including fosfomycin resistance protein A; fosA that is key enzyme for a broad-spectrum antibiotic, fosfomycin. It facilitate the conjugation of glutathione to the exposide ring of fosfomycin that is crucial step for fosfomycin inactivation, thus it conferring resistance (He and Moran, 2011; Ito et al., 2017). Legionella is a catalase-positive bacterium; deleting the VOC gene may disrupt its catalytic function (Bandyopadhyay and Steinman, 2000). Despite lack of information about Legionella resistance to fosfomycin, the parental strain Ofk308 is originated from water environment posses highly expression in VOC (Table 2, Figure 2). It could be believed that such a horizontal gene transfer from any resistance pathogen is exist and contribute to its survival ability in the environment. Therefore, it may relate to the observation that deleting VOC decreases the intracellular growth of Legionella within CU427, exhibiting smaller LCV formation compared to Ofk308 at 2, 24, and 48 hpi. msrB/A is known for its role in protecting cells against oxidative stress. Consisting of two major enzymes, msrA and msrB, it repairs oxidative protein damage by converting methionine sulfoxides (Met-SO) in proteins back to methionine (Met) (Zhao et al., 2010; Cudic et al., 2016). Single deletions of msrB and both msrB/A in other intracellular bacteria, such as Francisella novicida, reduce cytotoxicity in mice due to impaired bacterial growth (Saha et al., 2017). Taken together, a similar effect is observed in CU427 after infection by msrB/A, where LCV formation remains small within 24 hpi due to Legionella becoming more sensitive to reactive oxygen stress. To confirm these findings, we constructed VOC: VOC and msrB/A: msrB/A, where both genes reduce the relative number of viable CU427 host cells similarly to the parental strain Ofk308 (Figure 3). In addition, the CFU counts of both VOC and msrB/A are statistically reduced within 24 h. Our study demonstrates, for the first time, the roles of VOC and msrB/A in Legionella and highlights their significance in the virulence of this pathogen. Understanding the factors involved in the Legionella lifecycle within various hosts offers a new perspective on Legionnaire’s disease control and prevention.

The VOC and msrB/A contribute to the intracellular growth of Legionella within T. thermophila. The significance of these genes would become clearer as their detailed functions and expression regulation mechanisms are elucidated in the future. We highlighted the latest findings in host-pathogen interactions involving Legionella and emphasized that maintaining the symbiotic relationship with protist hosts in aquatic ecosystems should be considered to help control the environmental risk of Legionella infection.