Spontaneous Aberrant Bodies Formation in Human Pneumocytes Infected with Estrella lausannensis

Estrella lausannensis, a Chlamydia-related bacterium isolated from a Spanish river, is considered as a possible emerging human pathogen. Indeed, it was recently demonstrated to multiply in human macrophages, resisting oxidative burst and causing a strong cytopathic effect. In addition, a preliminary study highlighted a correlation between antibody response to E. lausannensis and pneumonia in children. To clarify the pathogenic potential of these bacteria, we infected a human pneumocyte cell line with E. lausannensis and assessed its replication and cytopathic effect using quantitative real-time PCR and immunofluorescence, as well as confocal and electron microscopy. Our results demonstrated that E. lausannensis enters and replicates rapidly in human pneumocytes, and that it causes a prompt lysis of the host cells. Furthermore, we reported the spontaneous formation of aberrant bodies, a form associated with persistence in Chlamydiae, suggesting that E. lausannensis infection could cause chronic disorders in humans.


Introduction
The Chlamydiae phylum encompasses several families of obligate intracellular Gramnegative bacteria with varied ecological niches [1,2].They have been isolated from a wide variety of hosts, ranging from humans to arthropods or protozoa.[3][4][5][6].The phylum is divided into bacteria belonging to the Chlamydiaceae family, which includes well-known human and animal pathogens such as C. trachomatis, C. pneumoniae or C. psittaci, and Chlamydia-related bacteria, which encompass emerging pathogens as well as endosymbionts of insects, arthropods, or amoebae [4,7,8].The scientific and medical interests of characterizing Chlamydia-related bacteria are multiple, including the discovery of potential emerging pathogens [9][10][11][12][13] and improved knowledge about infection mechanisms and about the evolution of these strict intracellular bacteria [1,[14][15][16][17].
Estrella lausannensis, a member of the Criblamydiaceae family, was isolated in 2011 from a water sample of the Llobregat river (Spain), [7].It is an excellent model organism for studying Chlamydia-related bacteria as it can replicate in a wide range of cell lines, including human macrophages, Vero cells, insect cells and fish cells, as well as in various amoebae such as Acanthamoebae castellanii, Dictyostelium discoideum and Hartmannela vermiformis [7,[18][19][20][21].Like other Chlamydiae, E. lausannensis multiplies using a biphasic life cycle alternating between the infectious form of the bacterium, the elementary body (EB), and the actively dividing form, the reticulate body (RB), which replicates through binary fission [22,23].A third form of development, the aberrant body (AB), occurs when bacteria are exposed to stress factors such as iron or tryptophan starvation or to growth-inhibiting agents such as interferon-gamma or antibiotics [24].
Several studies identified E. lausannensis as a possible emerging human pathogen.In particular, a correlation between the presence of anti-E.lausannensis antibodies and Microorganisms 2023, 11, 2368 2 of 9 pneumonia in children was documented (Lienard et al., unpublished data), and it was demonstrated that E. lausannensis resist oxidative burst of human macrophages, and even cause a strong cytopathic effect in this cell line [18].E. lausannensis genome sequencing also highlighted important chlamydial virulence factors such as a Type 3 Secretion System (T3SS) and a catalase (KatA) [25,26].
In this context, and to determine the pathogenic potential of E. lausannensis towards humans, we assessed its ability to grow in the human pneumocyte cell line A549 and evaluated the potential cytopathic effect caused by infection.

Infection Procedure
Cells were infected as described in de Barsy et al. [27] with a 1/500 dilution of E. lausannensis grown in A. castellanii.This dilution represents an MOI of 0.1.

Quantitative PCR
Cells were harvested at different time points post infection (0 h, 3 h, 8 h, 24 h, 32 h, 48 h and 72 h post infection) in 1 mL of DMEM.Genomic DNA was extracted from 100 µL following the manufacturer's instructions (Wizard SV Genomic DNA purification kit, Promega, Madison, WI, USA).qPCR targeting the 16S rRNA gene was performed as described previously by Lienard et al. [8].

Electron Microscopy
1 × 10 9 A549 cells were seeded in a 25 cm 2 flask 24 h before infection, infected as described above with a 1/50 dilution of E. lausannensis and harvested 24 h post infection.Cells were fixed and washed as described in de Barsy et al. [27].Thin sections on grids were prepared by the Electron Microscopy Facility of Geneva University and observed under a Philips EM 201 transmission electron microscope (Philips, Eindhoven, The Netherlands).

Results
Originally isolated from an amoeba, E. lausannensis was previously shown to multiply in a variety of cell lines, including human macrophages [18,20,21,27], and to be associated with pneumonia in children (Lienard et al., unpublished data) To further clarify its potential to cause lower respiratory tract infections in humans, we assessed its ability to grow in the human pneumocyte cell line A549 and evaluated the cytopathic effect of infection.

E. lausannensis Efficiently Replicates in Human Pneumocytes
E. lausannensis growth in A549 cells was evaluated using a specific qPCR developed by Lienard et al. [8].As shown in Figure 1, genomic DNA copies increased by about 2 logs in 72 h, with an exponential replication phase between 8 h and 24 post infection.We observed a lag phase between 0 h and 8 h post infection.During this time, EBs are internalized, differentiate into RBs, and create a replicative niche.After 48 h pi, the number of bacteria reaches a plateau since only a few cells are available for a second round of infection.
under a Philips EM 201 transmission electron microscope (Philips, Eindhoven, The Netherlands).

Results
Originally isolated from an amoeba, E. lausannensis was previously shown to multiply in a variety of cell lines, including human macrophages [18,20,21,27], and to be associated with pneumonia in children (Lienard et al., unpublished data) To further clarify its potential to cause lower respiratory tract infections in humans, we assessed its ability to grow in the human pneumocyte cell line A549 and evaluated the cytopathic effect of infection.

E. lausannensis Efficiently Replicates in Human Pneumocytes
E. lausannensis growth in A549 cells was evaluated using a specific qPCR developed by Lienard et al. [8].As shown in Figure 1, genomic DNA copies increased by about 2 logs in 72 h, with an exponential replication phase between 8 h and 24 post infection.We observed a lag phase between 0 h and 8 h post infection.During this time, EBs are internalized, differentiate into RBs, and create a replicative niche.After 48 h pi, the number of bacteria reaches a plateau since only a few cells are available for a second round of infection.In Table 1, we compare the doubling time of different Chlamydia-related bacteria and of Chlamydia trachomatis in mammalian (human pneumocytes, human macrophages, Vero cells) and insect cell lines (S. frugiperda and A. albopictus) as well as in two different freeliving protozoa (A.castellanii and D. discoideum).E. lausannensis replication is rapid and efficient in A549 pneumocytes, although slightly slower than in in other mammalian cell lines [18,,27].Its doubling time in pneumocytes is longer than that of Waddlia chondrophila [19], but much shorter than that of Simkania negevensis [28].Indeed, E. lausannensis shows a longer doubling time than W. chondrophila in almost all cell lines tested, except in insect cells, but it grows much faster than S. negevensis and C. trachomatis.In Table 1, we compare the doubling time of different Chlamydia-related bacteria and of Chlamydia trachomatis in mammalian (human pneumocytes, human macrophages, Vero cells) and insect cell lines (S. frugiperda and A. albopictus) as well as in two different free-living protozoa (A.castellanii and D. discoideum).E. lausannensis replication is rapid and efficient in A549 pneumocytes, although slightly slower than in in other mammalian cell lines [18,27].Its doubling time in pneumocytes is longer than that of Waddlia chondrophila [19], but much shorter than that of Simkania negevensis [28].Indeed, E. lausannensis shows a longer doubling time than W. chondrophila in almost all cell lines tested, except in insect cells, but it grows much faster than S. negevensis and C. trachomatis.

E. lausannensis Spontaneously Form Aberrant Bodies in Human Pneumocytes and Epithelial Cells
We followed the growth of E. lausannensis in human pneumocytes using immunofluorescence and confocal microscopy as well as using electron microscopy.As shown in Figure 2A, at 0 h post infection, EBs are localized on the surface of pneumocytes, and at 3 h pi, they start to differentiate into RBs, which are characterized by a larger size and a decondensed chromatin.At 8 h pi, RBs start to replicate, and inclusions with multiple bacteria are visible.At 16 h pi, inclusions are very large and contain multiple bacteria.As Interestingly, we observed the spontaneous formation of aberrant bodies (ABs) in some pneumocytes as early as 16 h pi (Figure 3A,B).In these cells, bacteria stopped di viding and their size increased significantly, being around 2 μm when several ABs ar present in one inclusion and even exceeding 5 μm when one single aberrant body i present in the inclusion.Such large ABs were also observed using electron microscopy They appeared as large areas of low electron density, chromatin being possibly localized  Electron microscopy images taken 24 h post infection (Figure 2B) showed the typical star shape of E. lausannensis EBs with their condensed chromatin [8].RBs appear brighter due to chromatin decondensation.Interestingly, we observed the spontaneous formation of aberrant bodies (ABs) in some pneumocytes as early as 16 h pi (Figure 3A,B).In these cells, bacteria stopped dividing and their size increased significantly, being around 2 µm when several ABs are present in one inclusion and even exceeding 5 µm when one single aberrant body is present in the inclusion.Such large ABs were also observed using electron microscopy.They appeared as large areas of low electron density, chromatin being possibly localized outside the cut.Interestingly, RBs and ABs were sometimes observed in the same inclusion, although this situation was not predominant.
Using immunofluorescence and confocal microscopy, we could also detect this intriguing spontaneous appearance of aberrant bodies in E. lausannensis-infected human epithelial (HEp-2) cells as of 16 h pi (Figure 3C).
We measured the cytopathic effect of E. lausannensis infection in human pneumocytes through propidium iodide incorporation and trypan blue labelling.Both methods revealed a strong cytopathic effect as early as 24 h post infection but failed to quantify host cell mortality at later stages of infection.
Using immunofluorescence and confocal microscopy, we could also detect this intriguing spontaneous appearance of aberrant bodies in E. lausannensis-infected human epithelial (HEp-2) cells as of 16 h pi (Figure 3C).
We measured the cytopathic effect of E. lausannensis infection in human pneumocytes through propidium iodide incorporation and trypan blue labelling.Both methods revealed a strong cytopathic effect as early as 24 h post infection but failed to quantify host cell mortality at later stages of infection.

Discussion
In this study, we demonstrated that E. lausannensis replicates rapidly in human pneumocytes and that it induces a strong cytopathic effect in this cell line, like what was observed when infecting macrophages or insect cells [18,20].As observed through con-

Discussion
In this study, we demonstrated that E. lausannensis replicates rapidly in human pneumocytes and that it induces a strong cytopathic effect in this cell line, like what was observed when infecting macrophages or insect cells [18,20].As observed through confocal microscopy, infected cells undergo strong lysis as of 24 h post infection.This phenomenon of rapid lysis reduces the possible number of replication cycles and limits E. lausannensis overall growth (2-log increase in bacterial DNA copy number in A549, compared with 3-log for W. chondrophila in the same cell line [19]).In addition, methods such as propidium iodide uptake or trypan blue incorporation, which rely on dye entry into cells with damaged membranes, were unable to quantify dead cells later than 24 h pi.This inability could be explained by the violent cell fragmentation caused by E. lausannensis infection and the resulting absence of an intermediate stage between living and completely burst cells.A previous work from Kebbi-Beghdadi et al. demonstrated that E. lausannensis replicates in two insect cell lines [20].In this earlier study, the measurement of propidium iodide incorporation also did not correlate with immunofluorescence observations, suggesting that the same phenomenon of abrupt lysis of host cells occurred in these insect cell lines.
Our results also highlight the fact that E. lausannensis multiplication in mammalian cells, like the multiplication of W. chondrophila, is much faster than that of the recognized human pathogen C. trachomatis.Both bacteria, again unlike C. trachomatis, are also able to grow in a wide variety of host cells, an ability that could be linked to the expression of a large number of OmpA proteins involved in the adhesion to the host cell [25,29].This hypothesis is reinforced by the observation that another Chlamydia-related species, Parachlamydia acanthamoebae, which encodes a single Omp protein, multiplies efficiently in only a few cell lines [30].
The most interesting finding of this study is the spontaneous formation of aberrant bodies in human pneumocytes and epithelial cells.Aberrant bodies are defined as enlarged forms of RBs that are persistent and viable, but not proliferative [24].They appear when bacteria undergo stress such as antibiotics or IFN-gamma treatment [31,32], during nutrient deprivation such as iron deficiency [33,34] or when host cells are co-infected with herpes simplex virus [35].These forms are reversible, meaning that bacteria resume their replication cycle when the stress stimulus disappears [36].Spontaneous formation of aberrant bodies is regarded as a form of persistence associated with chronic infection [37].Aberrant bodies have been observed in vivo in pig enterocytes infected with Chlamydia suis [38] and in vitro in human endometrial cells infected with W. chondrophila [19].E. lausannensis ABs have also been observed during Vero cell infection in presence of penicillin G but not in untreated cells [27].
Scherler et al. demonstrated for W. chondrophila that different stress stimuli induce morphologically distinct aberrant bodies [24].These aberrant bodies are either numerous and small, or very large and few in number It is interesting to note that the morphology of E. lausannensis aberrant bodies observed in human pneumocytes and epithelial cells is heterogeneous, ranging from numerous small ABs to a single very large one, and that both types can be present in the same cell.When observed using electron microscopy, E. lausannensis ABs appear clearer than EBs and RBs, similarly to what was observed with ABs of Chlamydia suis [38].Furthermore, E. lausannensis infection in pneumocytes induces a very strong cytopathic effect, despite the presence of ABs, contrarily to what happens with W. chondrophila, where infection in human endometrial cells causes a lesser cytopathic effect than in cell lines in which this species does not form ABs [19].Because of this cytopathic effect, the evolution of E. lausannensis ABs is difficult to study in A549 cells, and it is not possible to determine whether ABs have the potential to resume their cycle and in which conditions this could occur.
If E. lausannensis ABs spontaneously appear in the context of a natural infection, they could contribute to pathogenicity by reducing bacterial visibility by the immune system [37].They could also provoke a chronic asymptomatic infection, as is the case in 70-90% of C. trachomatis infections [39], or cause a latent infection capable of reactivating under certain conditions, such as co-infection with another microorganism, similar to what was described in pigs infected with C. suis and Salmonella sp [38].Given the rapid lysis of infected pneumocytes by E. lausannensis, this aberrant form could also be a bacterial mechanism to avoid a rapid and detrimental destruction of host cells.
To conclude, this study supports the hypothesis that E. lausannensis is an emerging pathogen potentially able to cause lower respiratory tract infections in humans.The spontaneous formation of aberrant bodies in human pneumocytes and epithelial cells is intriguing and should be further studied.It would be particularly interesting to know if spontaneous aberrant bodies formation is restricted to human cells or if it can also be observed in other mammalian cell lines.Ultimately, the presence of these aberrant forms should also be investigated in vivo in an animal model of E. lausannensis infection.

Figure 1 .
Figure 1.Growth kinetics of E. lausannensis in A549 pneumocytes.Bacterial replication is measured using qPCR for 72 h following infection.The results are the mean +/− SD of 4 independent experiments.

Figure 1 .
Figure 1.Growth kinetics of E. lausannensis in A549 pneumocytes.Bacterial replication is measured using qPCR for 72 h following infection.The results are the mean +/− SD of 4 independent experiments.

Figure 2 .
Figure 2. E. lausannensis growth in A549.(A) Immunofluorescence staining and confocal micros copy at different time points after infection.Bacteria are labelled in green, pneumocytes in red an DNA in blue.(B) Electron micrography 24 h post infection showing an inclusion containing EBs RBs and ABs (1), a pneumocyte with multiple inclusions (2), star-shaped elementary bodies (3) an a dividing RB (4).

Figure 2 .
Figure 2. E. lausannensis growth in A549.(A) Immunofluorescence staining and confocal microscopy at different time points after infection.Bacteria are labelled in green, pneumocytes in red and DNA in blue.(B) Electron micrography 24 h post infection showing an inclusion containing EBs, RBs and ABs (1), a pneumocyte with multiple inclusions (2), star-shaped elementary bodies (3) and a dividing RB (4).

Figure 3 .
Figure 3. Spontaneous formation of aberrant bodies in human pneumocytes and epithelial cells.(A) Immunofluorescence and confocal microscopy 24 h post infection in pneumocytes showing ABs of different sizes (2 to 5 μm) and inclusions containing RBs. White arrow indicates an inclusion containing multiple RBs and one AB.Bacteria are labelled in green, pneumocytes in red and DNA in blue.(B) Electron micrography at 24 h post infection showing large ABs in inclusions.(C) Spontaneous formation of ABs in HEp-2 cells 16 h pi (image 1) and 24 h pi (image 2).

Figure 3 .
Figure 3. Spontaneous formation of aberrant bodies in human pneumocytes and epithelial cells.(A) Immunofluorescence and confocal microscopy 24 h post infection in pneumocytes showing ABs of different sizes (2 to 5 µm) and inclusions containing RBs. White arrow indicates an inclusion containing multiple RBs and one AB.Bacteria are labelled in green, pneumocytes in red and DNA in blue.(B) Electron micrography at 24 h post infection showing large ABs in inclusions.(C) Spontaneous formation of ABs in HEp-2 cells 16 h pi (image 1) and 24 h pi (image 2).

Table 1 .
Doubling times (in hours) of Estrella lausannensis, Simkania negevensis, Waddlia chondrophila and Chlamydia trachomatis in different cell lines.Nd = not determined.For E. lausannensis in A549, the doubling time was calculated with Doubling Time Calculator using values of 8 h and 24 h post infection.It is interesting to note that in inclusions, DAPI only stained EBsDNA.Decondensed chromatin in RBs is not visible and only late-cycle inclusions when RBs reverted to EBs are clearly labelled with this dye.