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«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»

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Chemostat-cultured E. coli exhibit distinct regulatory responses upon exposure to either NO or GSNO (Flatley et al., 2005; Pullan et al., 2007). While the genes hmpA and ytfE were described to be affected under both conditions, the Fnr and Fur regulon were exclusively deregulated during exposure to NO. The GSNO-specific response includes methionine biosynthesis, multidrug transport (mdtC) and amino acid transport (yhaO) (Pullan et al., 2007). Analysis of the transcriptome of in vivo re-isolates indicated that the strains SR12 and KA25 exhibited, with minor exceptions, an expression pattern that resembled that of GSNO

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exposed bacteria. While in strain SR12 none of the genes from Fnr and Fur regulon were deregulated, in strain KA25 transcript levels of the nrfHIEF gene cluster were up-regulated. In addition, norV and narV transcript levels were down-regulated.

Interestingly, our study indicates for the first time that in E. coli methionine biosynthesis and protection against nitrosative stress might be linked to FrmA expression. As already mentioned above, FrmA (also designated as AdhC) belongs to the family of class III glutathione-dependent alcohol dehydrogenase and is reported to protect eukaryotic cells from antimicrobial nitric oxide (Hedberg et al., 2003; Liu et al., 2001). Kidd and colleagues (2007) discovered that the adhC gene in Haemophilus influenza is required for defence against nitrosative stress. Salmonella enterica mutants in adhC, however, were not impaired in NO detoxification (Bang et al., 2006). Up to date, nothing is known regarding the function of AdhC during urinary tract colonisation of E. coli, and therefore, a new model is proposed as

follows (Fig. 52):

  Fig. 52: Model of nitric oxide detoxification based on hierarchical cluster analysis of genes differently de-regulated in in vivo re-isolates SR12 and KA25 relative to their parent strain 83972. The different genes and proteins found to be up-regulated in the transcriptome and proteome profiles of strains SR12 and KA25 are indicated in blue. Black dotted lines show known regulatory responses. Red dotted lines with a question mark indicate proposed new functional relationships. NO – nitric oxide, iNOS - inducible nitric oxide synthase, GSH – glutathione, GSNO - S-nitrosoglutathione, GSSG – glutathione disulphide, Hcy - homocysteine.

162   Discussion 

It has been proven that NO and GSNO exhibit distinct chemical reactivities (Wink and Mitchell, 1998). NO is well soluble in water, diffuses easily through membranes and interacts with biomolecules within its immediate environment to form other reactive nitrogen species (RNS), such as GSNO and SNOs through interactions with glutathione (GSH) and thiols, respectively (Fang, 2004). GSNO has been shown to be dependent on the Dpp dipeptide ABC transporter (Abouhamad et al., 1991). The transcriptome analysis of in vivo re-isolates SR12 and KA25 indicated, however, only one gene dppA from the entire dppABCDF operon to be up-regulated relative to strain 83972. Therefore, it is not clear whether in re-isolates SR12 and KA25 GSNO is only intracellularly formed by interaction of NO with GSH or whether it is also transported from the environment into the cell. The ex vivo character of the study increases the possibility of false-negative artefacts and future in situ experiments would be needed to address this question.

Current experimental approaches tend to distinguish NO from GSNO response mechanisms (Flatley et al., 2005; Pullan et al., 2007). However, there are some implications that both of them take place at the same time depending on the redox state of the cell (Fang, 2004). Either imported or intracellularly formed GSNO nitrosates homocysteine, thereby withdrawing an intermediate product from the methionine biosynthetic pathway (Flatley et al., 2005).

Homocysteine depletion results in free MetR, and this protein activates methionine biosynthesis and Hmp expression (Fig. 52). Hmp detoxifies nitric oxide. However, GSNO is still being formed and needs to be neutralized. Although, GSNO-induced gene expression has been analysed, it was not shown how this compound is metabolized by E. coli.

Ammonia and glutathione disulphide (GSSG) are the main products of the AdhC enzyme.

The yield depends on the free GSH pool in the cell (Liu et al., 2001), however, in the transcription profiles of in vivo re-isolates SR12 and KA25 the glutathione synthesis pathway was not affected. Interestingly, ammonium generated from the NO detoxification reaction might be incorporated to 2-oxoglutarate resulting in L-glutamate. This reaction is performed by methionine aminotransferase YbdL.

Not much is known about the molecular basis of regulation of frmRAB expression. So far, only one report describes that an amber suppressor tRNA inactivates the repressor FrmR resulting in de-repression of the frmRAB operon (Herring and Blattner, 2004). These authors also identified another seven genes to be de-regulated by the same mechanism. Among those

163   Discussion 

were rbsDAC coding for proteins involved in ribose metabolism. However, the whole transcriptional unit consists of the rbsDACBKR genes. In our study only rbsB was significantly up-regulated, while the other genes were not de-regulated. Performed hierarchical cluster analysis of deregulated genes in strains SR12 and KA25 implies a functional relatedness of the metR, frmRAB, hmp and probably ybdL genes. Therefore, future studies are needed in order to uncover molecular mechanisms of frmA expression in E. coli.





The expression level of this alcohol dehydrogenase in E. coli seems to be host-dependent. All analysed re-isolates from the same patient exhibited similar frmA transcript levels which varied from patient to patient. Interestingly, independent inoculation events of patient SR pinpointed that up-regulation of frmRAB transcription was not an accidental event and was not inherited to the next bacterial generations but occured upon exposure to the specific conditions existing in the bladder of patient SR.

6.9. Implications and Outlook

I this study, several aspects of asymptomatic bacteriuria were investigated. First of all, it was found that strains with different phylogenetic backgrounds have the ability to establish asymptomatic bacteriuria. Among those were strains that resembled rather commensal-like isolates as well as degenerated UPEC strains with inactivated virulence factors. It was also shown that ABU isolates belong frequently to a certain phylogenetic lineage (ST73), that also includes pathogenic and commensal strains. Furthermore, this study showed that the host plays an important role in bacterial micro-evolution. Future efforts might consider the detailed analysis based on complete genome sequences of several such closely related organisms and the identification of DNA regions that undergo modification upon exposure host defence mechanisms. This, in combination with drug development, might help to improve the quality of life of people frequently suffering from urinary tract infections.

Another, very important implication of the presented study is the impact of host variability in the establishment of asymptomatic bacteriuria. It was documented that bacterial gene expression depends on the host background and is permanently regulated what allows the bacterium to persist in the bladder. Depending on the patient, E. coli strain 83972 was able to

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take up and metabolize diverse carbon sources, thus being able to colonize this usually sterile niche and to avoid colonization by uropathogens causing symptomatic urinary tract infection.

ABU strain 83972 was carefully characterized regarding many virulence and fitness factors, however, flagella has never been considered as an important factor during the establishment of asymptomatic bacteriuria. We were able to show that flagella are still functional and are expressed in several in vivo re-isolates. This supports its possible function during ABU.

Thereby mutant analysis would be needed to define the importance of this still remaining intact bacterial surface-associated organelle.

It has never been shown that host defence might trigger bacterial evolution. Here, due to the carefully designed study, we were able to show that in vivo growth in the human bladder but not in vitro growth in human urine triggered genomic changes in E. coli. Using single strains re-isolated from different host backgrounds bacterial co-evolution was captured. Future analyses should include the comparison of genome sequences of the parent strain and its consecutive in vivo re-isolates.

Finally, the direct exposure of strain 83972 to host defence factors led to discovery that similar strategies of antimicrobial nitric oxide detoxification take place, both in humans and E. coli grown in the urinary tract. It has already been shown for Haemophilus influenza that AdhC is required for defence against nitrosative stress (Kidd et al., 2007) and this protein is conserved among many organisms (Liu et al., 2001), but it was never documented that uropathogenic or ABU E. coli isolates take advantage of it to detoxify reactive nitrogen species. Recently, Richardson et al. (2008) proved that Staphylococcus aureus, one of the most successful human pathogens, evades the antimicrobial activity of nitric oxide by expressing an NO-inducible L-lactate dehydrogenase (Ldh1), implicating the role of adaptive metabolism in microbial defence mechanisms. Therefore, much effort is needed to further assess the molecular basis of regulation of adhC expression by uropathogenic bacteria and its role during bladder colonisation.

In summary, the presented work on the characterisation of asymptomatic bacteriuria E. coli isolates also addresses important aspects of commensalism, host-driven bacterial evolution and the impact of individual host repsonses during bladder colonisation.

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7. References   Abouhamad, W.N., Manson, M., Gibson, M.M., and Higgins, C.F. (1991) Peptide transport and chemotaxis in Escherichia coli and Salmonella typhimurium: characterization of

the dipeptide permease (Dpp) and the dipeptide-binding protein. Mol Microbiol 5:

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Agace, W., Hedges, S., Andersson, U., Andersson, J., Ceska, M., and Svanborg, C. (1993a) Selective cytokine production by epithelial cells following exposure to Escherichia coli. Infect Immun 61: 602-609.

Agace, W.W., Hedges, S.R., Ceska, M., and Svanborg, C. (1993b) Interleukin-8 and the neutrophil response to mucosal gram-negative infection. J Clin Invest 92: 780-785.

Akaike, T. (2001) Role of free radicals in viral pathogenesis and mutation. Rev Med Virol 11:

87-101.

Aktories, K. (1997) Rho proteins: targets for bacterial toxins. Trends Microbiol 5: 282-288.

Amor, K., Heinrichs, D.E., Frirdich, E., Ziebell, K., Johnson, R.P., and Whitfield, C. (2000) Distribution of core oligosaccharide types in lipopolysaccharides from Escherichia coli. Infect Immun 68: 1116-1124.

Anderson, G.G., Palermo, J.J., Schilling, J.D., Roth, R., Heuser, J., and Hultgren, S.J. (2003) Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301: 105Anderson, G.G., Dodson, K.W., Hooton, T.M., and Hultgren, S.J. (2004) Intracellular bacterial communities of uropathogenic Escherichia coli in urinary tract pathogenesis.

Trends Microbiol 12: 424-430.

Andersson, P., Engberg, I., Lidin-Janson, G., Lincoln, K., Hull, R., Hull, S., and Svanborg, C.

(1991) Persistence of Escherichia coli bacteriuria is not determined by bacterial adherence. Infect Immun 59: 2915-2921.

Andrews, S.C., Robinson, A.K., and Rodriguez-Quinones, F. (2003) Bacterial iron homeostasis. FEMS Microbiol Rev 27: 215-237.

Anfora, A.T., Haugen, B.J., Roesch, P., Redford, P., and Welch, R.A. (2007) Roles of serine accumulation and catabolism in the colonization of the murine urinary tract by Escherichia coli CFT073. Infect Immun 75: 5298-5304.

Arber, W. (1993) Evolution of prokaryotic genomes. Gene 135: 49-56.

Bang, I.S., Liu, L., Vazquez-Torres, A., Crouch, M.L., Stamler, J.S., and Fang, F.C. (2006) Maintenance of nitric oxide and redox homeostasis by the salmonella flavohemoglobin hmp. J Biol Chem 281: 28039-28047.

Barraud, N., Hassett, D.J., Hwang, S.H., Rice, S.A., Kjelleberg, S., and Webb, J.S. (2006) Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 188: 7344-7353.

Barua, S., Yamashino, T., Hasegawa, T., Yokoyama, K., Torii, K., and Ohta, M. (2002) Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxinproducing Escherichia coli O157:H7. Mol Microbiol 43: 629-640.

Baumler, A.J., Tsolis, R.M., van der Velden, A.W., Stojiljkovic, I., Anic, S., and Heffron, F.

(1996) Identification of a new iron regulated locus of Salmonella typhi. Gene 183:

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Berger, H., Hacker, J., Juarez, A., Hughes, C., and Goebel, W. (1982) Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli. J Bacteriol 152: 1241-1247.

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Bergsten, G., Samuelsson, M., Wullt, B., Leijonhufvud, I., Fischer, H., and Svanborg, C.

(2004) PapG-dependent adherence breaks mucosal inertia and triggers the innate host response. J Infect Dis 189: 1734-1742.

Bergsten, G., Wullt, B., and Svanborg, C. (2005) Escherichia coli, fimbriae, bacterial persistence and host response induction in the human urinary tract. Int J Med Microbiol 295: 487-502.

Bergsten, G., Wullt, B., Schembri, M.A., Leijonhufvud, I., and Svanborg, C. (2007) Do type 1 fimbriae promote inflammation in the human urinary tract? Cell Microbiol.

Beutler, B. (2000) Tlr4: central component of the sole mammalian LPS sensor. Curr Opin Immunol 12: 20-26.

Billips, B.K., Forrestal, S.G., Rycyk, M.T., Johnson, J.R., Klumpp, D.J., and Schaeffer, A.J.

(2007) Modulation of host innate immune response in the bladder by uropathogenic Escherichia coli. Infect Immun 75: 5353-5360.

Birch, A., Hausler, A., Ruttener, C., and Hutter, R. (1991) Chromosomal deletion and rearrangement in Streptomyces glaucescens. J Bacteriol 173: 3531-3538.



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