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negatively affect the survival of the insect cell. In Drosophila, Wolbachia replication was indicated to be tissue-dependent as growth rates differ significantly between head and ovaries (McGraw et al., 2002). Wolbachia replication is also dependent on the stage of the host life cycle. In A. albopictus, Wolbachia replication stops during the diapause of mosquito eggs in which no host cell division occurs, highlighting the dependence of Wolbachia proliferation on host cell division (Ruang-Areerate et al., 2004). In the filarial nematode B. malayi, Wolbachia numbers are low and remain constant in microfilaria and insect-borne larval stages, but proliferation increases suddenly after the infection of a vertebrate host (McGarry et al., 2004;
Taylor et al., 2013). This seems to play an essential role in larval development as demonstrated by the arrested larval growth and development in response to antibiotic treatment (Taylor et al., 2012). B. malayi and B. pahangi infective-stage larvae co-cultured in vitro with the yeast R.
minuta have been shown to support consistent and reproducible molting to the fourth larval stage (Smith et al., 2000). It was suggested that the larvae are benefiting from an unknown secreted product of the yeast. Since proliferation rates of Wolbachia severely increase in this phase of the larvae (McGarry et al., 2004; Taylor et al., 2013), the bacteria might also benefit from secretion products of R. minuta. In this thesis, cell-free wAlbB were co-cultured with viable R. minuta. The co-cultivation harmed the Wolbachia leading to a concentration-dependent decrease of bacteria rather than enhanced growth. Thus, it might be assumed that the secreted yeast product might be more beneficial for the viability of filarial larvae than for Wolbachia. In a future experiment, R. minuta lysate in different concentrations might be used.
This would prevent that bacteria are overgrown by the yeast. Notably, it was recently shown that wAlbB is able to grow in artificially infected Saccharomyces cerevisiae (Uribe-Alvarez et al., 2018). Compared to controls, infected yeast lost viability early, but this system might potentially provide a promising future model of interactions that occur in a naturally infected eukaryote host (Uribe-Alvarez et al., 2018).
Summing up, these findings provide insight into the complexity of Wolbachia replication and endosymbiont-host dependency. Further research will be necessary to elucidate the multiple mechanisms that influence and regulate Wolbachia replication and to enhance growth as well as stability of the wAlbB cell-free culture.
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target or because Wolbachia are indeed resistant. The cell-free system meets all requirements to examine antibiotics directly applied to Wolbachia. Thus, another part of this thesis was the application of antibiotics to the cell-free wAlbB culture. Different antibiotics were supplemented to the cell-free culture medium, gDNA was prepared and growth rates were monitored via qPCR of the 16S rRNA gene. To exclude that penicillin or streptomycin, which are supplements of the normal cell culture medium, have an inhibitory effect on cell-free wAlbB proliferation, cultures with and without these antibiotics were tested. Here, no differences in growth rates were observed supporting previous findings that Wolbachia are resistant to β-lactams (O'Neill et al., 1997). Proliferation rates were also examined with the
Wolbachia-affecting antibiotics corallopyronin A, doxycycline, fosfomycin and rifampicin.
However, cell-free wAlbB growth rates were not decreased in the presence of corallopyronin A, doxycycline or fosfomycin. Only treatment with rifampicin led to decreased cell-free growth, but a complete inhibition of growth was not observed here as well. It might be assumed that an effect on growth would only be observed using antibiotics with bactericidal activity like rifampicin, but this assumption does not hold true as fosfomycin also acts bactericidal (Michalopoulos et al., 2011). Generally, it should be considered that gDNA was prepared and measured from the whole culture and it is possible that DNases are lower or absent in the cell-free culture. Thus, gDNA fragments of dead cells were potentially measured by qPCRs leading to a seemingly increase of Wolbachia. To solve this issue, expression levels of the 16S rRNA gene prepared from the cell-free culture were measured. However, no differences between antibiotic treated and untreated cultures were revealed due to low expression after twelve days also in untreated controls. A study in wMel confirmed that rRNA expression levels are high and variable among samples (Gutzwiller et al., 2015). Thus, this approach is not suitable to reliably compare antibiotic treated and untreated wAlbB cultures.
Another approach to determine bacteria numbers is counting of cells under a microscope and discrimination between living and dead bacteria by LIVE/DEAD® staining. This was applied to the cell-free Wolbachia, but in the majority of experiments, only few cells were detected after twelve days also in the controls cultures without antibiotic treatment. Other previous attempts to count cell-free Wolbachia also turned out to be inappropriate as a high variance between measured cells per qPCR and actually counted cells was revealed (J. Vollmer, pers. communication). Therefore, this approach was rejected and the impact on the morphology of antibiotic-treated wAlbB was investigated to reveal potential antibiotic-effects on Wolbachia. Cell-free Wolbachia were fixed, stained and visualized under a microscope. In a previous study, wAlbB residing in C6/36 insect host cells were treated with the lipid II-synthesis
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blocking antibiotic fosfomycin, which led to enlarged bacteria (Vollmer et al., 2013). This finding indicated that lipid II is essential for cell division in Wolbachia. Moreover, fosfomycin treatment on Wolbachia revealed a perturbed localization of wPal suggesting an interaction of this lipoprotein with lipid II or its processed form (Vollmer et al., 2013). This assumption is supported by the results of the interaction assays in this thesis (see chapter 4.4). The incubation of cell-free wAlbB with fosfomycin also showed an aberrant phenotype with enlarged cells and delocalized wPal appearing in a spot-like pattern. None of the other antibiotics tested revealed a visible aberrant phenotype. Ciprofloxacin, clindamycin, corallopyronin A, doxycycline, rifampicin, sulfamethoxazole and trimethoprim do not target cell wall biosynthesis, thus an impact on cell morphology was unlikely. For antibiotics targeting the cell wall or synthesis steps (ampicillin, bacitracin, vancomycin) an aberrant phenotype was more likely. For example, ampicillin-treated C. trachomatis have aberrant, enlarged reticulate bodies (Liechti et al., 2014).
Here, no changes compared to the control cells were observed after twelve days of incubation with the respective antibiotic. On the one hand, it can be proposed that Wolbachia are indeed resistant to these antibiotics and therefore no change of morphology was detected. On the other hand, it might be assumed that antibiotics were unstable and thus ineffective. Since stability, solubility and shelf life of antibiotics were considered while preparing the assays, this possibility can be neglected. Another explanation for the inefficacy of the substances might be an inaccessibility of their targets. For instance, most Gram-negative bacteria are naturally resistant to vancomycin as this molecule cannot pass the outer membrane (Geraci, 1977).
Wolbachia likely have an unusual outer membrane since they are unable to synthesize lipid A, a key moiety of lipopolysaccharide (Foster et al., 2005). Thus, certain compounds, which are too large to pass the outer membrane of Gram-negative bacteria, might pass the unique Wolbachia outer membrane. This was already demonstrated for corallopyronin A which normally depletes only Gram-positive bacteria, but is also highly active against Wolbachia (Schiefer et al., 2012). In contrast, it should be considered that compounds that normally pass the Gram-negative outer membrane might not be able to reach their target in Wolbachia due to their unique outer membrane.
In conclusion, the investigation of Wolbachia cultures in terms of antibiotic susceptibility remains challenging. To establish rapid and reliable tools to analyze antibiotic assays, several attempts could be beneficial. The amplification of genes like the wolbachial surface protein (wsp) WD1063 might be more eligible than 16S rRNA to measure replication or depletion of cell-free Wolbachia. Wsp is stably-expressed in Wolbachia making it suitable to compare expression levels of antibiotic treated and untreated cells (Gutzwiller et al., 2015).
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Wolbachia growth inside yeast cells can be determined by PCR of wsp and this approach might be applied to the cell-free wAlbB culture (Uribe-Alvarez et al., 2018). Additionally, Wolbachia replication or depletion can also be estimated by the detection of wsp by Western Blot and analysis of band intensity (Uribe-Alvarez et al., 2018). As another approach, fluorescence-activated cell sorting of LIVE/DEAD® stained Wolbachia after antibiotic treatment might lead to more detailed and accurate results instead of analysis with a microscope.