FurA did not regulate genes of the 38 kb island but is crucial for survival of MAP

In gram-negative as well as gram-positive bacteria Fur homologues regulate amongst others genes involved in virulence and stress response. Furthermore, Fur is described as a regulator for the iron homeostasis (BURY-MONÉ et al., 2004; GANCZ et al., 2006; HORSBURGH et al., 2001; PALYADA et al., 2004; REA et al., 2004). In MAP, the role of FurA is not completely understood. Since it is similar organized with KatG a similar function in stress response like in other mycobacteria is suggested (MASTER et al., 2001; MILANO et al., 2001; PYM et al., 2001; SANTOS et al., 2008;

ZAHRT et al., 2001). Due to the fact that two putative fur boxes were located in the promoter region of mptA it appears that FurA regulates the mpt operon and is therefore suggested to be involved in the iron homeostasis of MAP (STRATMANN et al., 2004). But, no characterization of FurA was performed in MAP so far. Thus, a MAP∆furA strain was constructed and characterized in the presented thesis.

The characterization of the MAP∆furA strain by transcriptome analysis indicated no differential expression of the mpt operon or genes located on the 38 kb island. The fur box analysis, however, was performed with the fur box sequence of Pseudomonas aeruginosa (P. aeruginosa) using the database PRODORIC (prokaryotic database of gene regulation, http://www.prodoric.de, 2010; MÜNCH et al., 2003, 2005; STRATMANN et al., 2004). This fur box located in the promoter region of mptA shared only 26 % identity to the furA box in the promoter of furA in MTB (data not shown). A functional characterization of FurA in MTB by electrophoretic mobility shift assays (EMSA) indicated a weak binding to its own fur box (LUCARELLI, 2006). Additionally, no conserved palindromic consensus

sequences could be revealed via FurA box analysis in MTB (LUCARELLI, 2006). The low identity between fur boxes of MTB and P. aeruginosa suggested that mycobacterial furA boxes have a different sequence compared to other fur boxes.

This appears to be also true for MAP and could therefore explain that our data indicated no regulatory function for the mpt operon in MAP.

The presumed function of FurA to be involved in the iron homeostasis was disproved since only two genes were assigned to the iron metabolism - mbtE and mbtF. Both genes belong to the mycobactin synthesis operon and were upregulated in MAP∆furA. Previous studies on the iron homeostasis revealed IdeR as the regulator for iron uptake and storage in MAP (JANAGAMA et al., 2009, 2010). It was demonstrated that IdeR regulates the mbtB genes, involved in iron uptake, as well as the bfrA gene, involved in iron storage. Additionally, it was reported that IdeR regulates mbtB in vitro as well as in vivo. Moreover, in this study an upregulation for mbtE was found in vivo but not in vitro (JANAGAMA et al., 2009). The different regulatory mechanisms of mbtB and mbtE suggested that IdeR regulates mbtB, but not mbtE. In turn, mbtE is suggested to be regulated by FurA and only necessary in vivo. The finding of a putative furA box via in silico analysis using the furA box of MTB in the mbtE supported this hypothesis (data not shown).

A putative function could not be identified for 86 differentially expressed genes in the MAP∆furA strain. The remaining 86 genes, however, belong to the intermediary metabolism such as amino acid, carbohydrate or fatty acid as well as to the secondary metabolism like stress response, signal transduction, transcription and regulation or virulence. The deletion of furA resulted in different expression pattern of eight transcriptional regulators such as lysR and whiB2 as well as regulators of the TetR family. Homologues of whiB were also found in many other Actinomycetales (CHATER and CHANDRA, 2006; HUTTER and DICK, 1999; SOLIVERI et al., 1992).

Molecular genetic characterization of WhiB3, for instance, showed that it is involved in the maintenance of redox homeostasis and virulence in MTB (BANAIEE et al., 2006; SAINI et al., 2012; SINGH et al., 2007, 2009; ZHENG et al., 2012). A recent study reported that WhiB2 is stress-induced and upregulated 2 h after infection of murine J774.A1 macrophages with a MAP K-10 strain (GHOSH et al., 2013). In line

with this, our study revealed a downregulation of the whiB2 regulator in MAP∆furA.

This indicated a regulation of WhiB2 by FurA in response to oxidative stress.

In this context, four proteins ahpC, ahpD, sodC, and katG were found to be regulated in MAP∆furA. The homologues of katG and sodC in MTB are described as major virulence factors since they counteract the phagocyte oxidative burst (DUSSURGET et al., 2001; VINCENT et al., 2004). The homologues of ahpC and ahpD in MTB belong to an essential antioxidant defense system (HILLAS et al., 2000; KOSHKIN et al., 2004; MASTER et al., 2002). In MAP, both genes were described as major antigens and therefore an important role in virulence was suggested (OLSEN et al., 2000). Furthermore, it was reported that the ahpC/ahpD system is upregulated in MAP after oxidative and nitrosative stress as well as at any times (2h and 24h) during infection of naϊve or INF-γ activated murine J774.A1 macrophages (GHOSH et al., 2013; KAWAJI et al., 2010). Moreover, it was demonstrated that the ahpC/ahpD system is regulated by the oxyR gene in MAP (GHOSH et al., 2013). In MTB, OxyR is characterized as a redox sensor, which regulates a group of genes including AhpC, KatG as well as FurA (PAGA et al., 2006). In MAP, however, katG, sodA, and sodC as well as FurA were not activated during infection of murine macrophages, which suggested an indirect regulation of the AhpC/AhpD system by OxyR (GHOSH et al., 2013). Due to the fact that ahpC as well as ahpD are upregulated in MAP∆furA it appears that the ahpC/ahpD system in MAP is indirectly regulated by OxyR but directly regulated by FurA. In addition to this, the regulation of important oxidative stress response proteins such as SodC and KatG highlights the crucial role of FurA in the antioxidant defense system of MAP.

As already stated above, previous studies reported a critical function of the lipid metabolism in the intracellular survival and virulence of mycobacteria. Our transcription analysis of MAP∆furA revealed thirteen genes belonging to the lipid metabolism. Among these genes three enoyl-CoA hydratases (echA), MAP0576 (echA13), MAP3087c (echA17), and MAP1482, catalyze the second step of the degradation in ß-oxidation – the hydrolation of the trans-double-bound (BAHNSON et al., 2002). Enoyl-CoA hydratases are enzymes involved in many pathways including the fatty acid metabolism, the valine, leucine, and isoleucine degradation, as well as

the lysine degradation (www.genomes.jp/kegg, 2008). In MTB, enoyl-CoA hydratases such as echA10 were described to be involved in the biosynthesis of mycolic acid, an important virulence factor of mycobacteria (BARRY et al.; LAMARCA et al., 2004;

TAKAYAMA et al., 2005). It appears that FurA regulates genes for the biosynthesis of mycolic acids in MAP. Additionally, three genes were assigned to the MMC pathway, MAP2437, MAP0673, and MAP1226 (mutB). This pathway is essential for the already mentioned detoxification of propionyl-CoA in pathogenic mycobacteria, which results from the in vivo degradation of cholesterol for energy production (CHANG et al., 2009; MINER et al., 2009; WEIGOLDT et al., 2013; YANG et al., 2011). Overall, these data suggest a crucial role of FurA in the virulence of MAP, which is characterized by oxidative stress response, lipid metabolism such as biosynthesis of mycolic acids, and detoxification of propionyl-CoA.

Since the expression pattern of MAP∆furA showed significant changes in genes involved in the survival within macrophages, a mice study was conducted to investigate the consequences of these transcriptional changes in MAP∆furA in vivo.

Despite to the fact that most of the measured parameters showed similar results to the MAPwt strain, a two-third times lower bacterial load of viable MAP∆furA was detected in the liver of infected mice. This clearly indicated a lower biological fitness of MAP∆furA in the animal model.

It is not clear, why the other parameter such as body weight, weight of liver and spleen, as well as granuloma area revealed a similar biological fitness between MAPwt and MAP∆furA. These similarities might be explained by the presence of MAP particles, since killed mycobacteria act as a very potential adjuvant to boost an immune response in the host (FREUND and THOMSON, 1948; FREUND et al., 1937; STILS, 2005). The similar numbers of granuloma in both strains indicated a similar acute infection of the murine liver. But the decreased amount of viable MAP∆furA displayed a reduced survival over a period of four weeks.

These data confirmed that FurA displayed an essential virulence factor in MAP.