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

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Bergsten et al., 2007; Hultgren et al., 1985). The weak host response to ABU is therefore consistent with the loss of adherence and functional fimbriae. Our results thus suggest that the host response may drive co-evolution, and that virulence-associated genes with proinflammatory effects may be targeted for inactivation. In this way, ABU isolates may succeed in persisting without inducing a bactericidal inflammatory response.

Classical studies (Haldane, 1949) proposed that microbes evolve to increase their virulence.

The theory was based mainly on the observation that virulence increases pathogen transmission between hosts thereby increasing the number of available multiplication sites for the microbe. Virulence for the urinary tract may in part fit this theory, but does not mainly serve to increase the number of infected hosts, but rather the number of sites in a given host.

By expressing fimbriae and other virulence factors, UPEC establish a monoculture in the urinary tract, with less competition than in the complex and competitive intestinal microflora.

Unfortunately, virulence is only partially successful, due to the brief time window between the establishment of bacteriuria and the activation of a host defence, which in most cases eliminates the infection. ABU is an interesting model to study the evolution of commensalism rather than virulence. The ABU strains, in contrast, avoid provoking a host response that leads to their elimination and instead, they establish long-term persistence. The loss of virulence may therefore be a preferred evolutionary strategy and there may be positive selection for variants, which are adapted for growth in the urinary tract. Advantages include a rich source of nutrients and the potential for transmission to new hosts. This is in contrast to acute pyelonephritis, which is associated with mortality, premature delivery and reduced fertility and thus with a potential loss of the ecologic niche. Our results clearly support the notion of reductive evolution as an attenuation mechanism converting virulent uropathogenic E. coli to asymptomatic carrier strains. While there was no common or specific set of genes that was inactivated or lost by all ABU isolates relative to virulent UPEC, the ECOR group B2 and D

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isolates showed distinct mutations in virulence-associated genes rather than a large overall genome loss, which is consistent with an ongoing host-bacterial evolution.

6.3. Genome reduction and evolution of ST 73 ABU strains This study suggests that ABU is caused by E. coli strains of different backgrounds, which share the ability to establish bacteriuria and to persist in the urinary tract, but the molecular details are poorly understood. Sequence type 73 represents an important and successful phylogenetic lineage within ECOR group B2, which also includes the prototypic UPEC strain E. coli CFT073 and the non-pathogenic E. coli strain Nissle 1917. The genomic and phenotypic diversity among members of ST73 reflects the genome plasticity of E. coli.

Although four ABU strains as well as UPEC isolate CFT073 and non-pathogenic strain Nissle 1917 belong to the same ST, they differ in the presence of functional fimbrial determinants as well as in their LPS and hemolytic phenotype (Grozdanov et al., 2002; Grozdanov et al., 2004; Welch et al., 2002). The DNA sequence diversity of their fim, pap and foc genes is consistent with the phenotypic heterogeneity within this group of identical or very closely related organisms. However, genome size assessment could show that differences in between isolates exist. Accordingly, the E. coli ST73 includes highly virulent uropathogenic, ABU as well as non-pathogenic variants, which may have arisen from a common ancestor by reductive evolution (Fig. 50). Our results confirm recent findings (Johnson et al., 2006; Wirth et al., 2006), that the current MLST schemes do not reliably predict the genotypes or phenotypes of individual isolates.

145   Discussion 

  Fig. 50: Geno- and phenotypic diversity among closely related members of E. coli clonal group (ST 73). The high E. coli genome plasticity results in a marked phenotypic variability among individual members of the same sequence type, which thus includes pathogenic and non-pathogenic variants.

Genome reduction/loss of function contributes to the evolution of these ABU variants from uropathogenic ancestors. ST, sequence type; fim, type 1 fimbrial determinant; pap, P fimbrial determinant; papG, P fimbrial adhesin encoding gene; foc, F1C fimbrial determinant; focD, F1C fimbrial usher encoding gene.

6.4. Host immune response during bacterial colonisation

ABU development is affected by the quality of the host response. In the murine UTI model, an ABU-like state is created when the innate immune response is disturbed. The innate response is controlled by TLR4 and mutations, which disturb TLR4 signalling, result in an asymptomatic carrier state resembling human ABU. The infected mice fail to recruit the inflammatory cells, which are crucial for bacterial clearance (Frendeus et al., 2001a; Frendeus et al., 2001b; Hagberg et al., 1984) and as a result, bacteria persist in the urinary tract. In another study, the response of human epithelial cells, obtained from urinary tract surgery, to infection with either fimbriated or non-fimbriated bacteria was examined (Samuelsson et al., 2004). It was shown that production of IL 6 and IL 8 correlated with bacterial fimbriation.

Human uroepithelial cells possess the molecular machinery to respond to UPEC. It was speculated that differential expression of the membrane-bound receptors regulates its sensitivity to infection and allows discrimination between more-virulent (UPEC) and lessvirulent (ABU) strains. Therefore, the combination of the expression of different bacterial

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surface-associated molecules and the host ability to sense them is critical for the development of symptoms and bacterial clearance or the asymptomatic carrier state.

The lack of inflammation prevents the symptoms and tissue damage that are associated with symptomatic UTI in a fully responsive host. Recently, children with ABU were shown to express lower amounts of TLR4 than controls without a history of UTI and children with primary ABU had even lower levels than those who had an ABU recurrence after a prior symptomatic UTI episode (Ragnarsdottir et al., 2007). The TLR4 variations add an essential variable to the understanding of ABU. Independently of the host genetics, a “commensal” ABU strain would not be expected to cause symptomatic UTI, but a more virulent strain might cause symptoms and an attenuating host response in patients with normal TLR4 expression, would eventually lead to ABU. Patients with low TLR4 levels would be expected to have a reduced innate response to infection, and would develop ABU also upon infection by more virulent. A good example for such strain is ABU 64, which in contrast to the other examined isolates was able to express many UPEC-associated virulence factors (Table 9).

Bacteria were reported to actively modulate host immune response (Klumpp et al., 2001;

Klumpp et al., 2006). UPEC strain NU14 suppresses both TNF-α- and LPS-mediated NF-KB activation and IL 6 secretion in urothelial cell cultures. Additionally, NU14 can inhibit IL 6 secretion induced by nonsuppressor strain K-12 strain from urothelial cells in a mixed culture.

Furthermore, examination of a panel of clinical E. coli isolates, broadly representing different phylogenetic groups, revealed that 15 of 17 strains also possessed the ability to suppress cytokine secretion (Billips et al., 2007). In addition, modulation of cytokine secretion was independent of the presence of type 1 fimbriae and 21 other known virulence factors. Another example how bacteria subvert host defences is expression of TIR (Toll/interlucin-1 receptor) domain containing proteins (Tcps). These proteins were found to be common in the virulent UPEC and were termed TcpC. TcpC acts by inhibiting Toll-like receptor (TLR) and myeloid differentiation factor 88 (MyD88) specific signalling, thus suppressing innate immunity and increasing virulence (Cirl et al., 2008).

Asymptomatic bacteriuria, i.e. colonization of the urinary tract without causing significant host responses could be a combination of different strategies. First of all, it is patientdependent, and as already mentioned, due to variations of the uroepithelial host receptors (Ragnarsdottir et al., 2007; Samuelsson et al., 2004). Secondly, as a result of this study

147   Discussion 

(Zdziarski et al., 2008), bacteria might prevent recognition by their host and subsequent innate immune response activation due to point mutations in genes encoding for virulence factors. Finally, ABU isolates might actively modulate the immune system by expressing so far unknown molecules, which are independent from still functional or already deactivated virulence factors. A high diversity among ABU strains suggests that a successful ABU state is a sophisticated process and is a combination of above mentioned components. Therefore, future experiments should include sequencing of ABU genomes, looking for interkingdom crosstalk and a more detailed characterisation of ABU isolates regarding host response modulation.

6.5. Host-bacterium interactions

Already in 1989, Hansson and colleagues reported that long term carriage of bacteria in the urinary tract seemed to induce changes in bacterial surface antigens. However, at that time methods to analyse bacterial relatedness were not yet very well developed. Using multilocus enzyme electrophoresis and O antigen analysis they found that 10 out of 25 O-typeable strains converted to non-typeable while retaining the same electrophoretic type. It was suggested that the loss of surface antigens occurs during long term exposure of E. coli to the components of the urinary tract. This study was a consequence of the discovery that untreated asymptomatic bacteriuria in young school girls protected against invasion by other bacterial strains, often leading to symptomatic infections (Lindberg et al., 1978; Savage et al., 1975). Interestingly, in the next years one of the isolates, E. coli strain 83972, was used for deliberate patient colonisation (Andersson et al., 1991; Sunden et al., 2006). Thanks to the close collaboration with Catharina Svanborg and Björn Wullt (Lund), the consequences of host–bacterium interaction could be analyzed in more details.

6.5.1. Bacterial variability and host response

Consecutive re-isolates of strain 83972 derived from the patient colonisation study were analysed regarding their genome structure. It turned out that several bacterial clones have changed their restriction pattern indicative of DNA rearrangements, deletions or point mutations. One of the frequently observed DNA modifications are point mutations resulting in single nucleotide polymorphisms (SNPs). It is rather difficult to detect SNPs by the PFGE

148   Discussion 

approach, because it would then have to be located in the restriction site of the used enzyme.

So far, only genome sequencing allows detection of unknown point mutations. However, in the genomics era with sequencing techniques becoming cheaper and faster, this might give us more insights into bacterial microevolution during host colonisation. Second, and most likely easier to detect by genomic fingerprints are DNA rearrangements mediated by mobile DNA elements. This includes IS element transposition, prophage insertions and excisions as well as acquisition of larger DNA stretches called genomic or pathogenicity islands (Dobrindt et al., 2004; Hacker and Kaper, 2000).

When bacteria enter a new host, as it was also the case for strain 83972 during deliberate colonisation studies, they enter a new environment and must start to grow and reproduce. The growth rate, the same colonization success, depends on several factors. Among the most important are the availability of nutrients, physical conditions like urodynamics in the bladder (Wullt et al., 1998), competitiveness against other microbes and antimicrobial host defences (Bergsten et al., 2005; Wullt et al., 2003). Even if the bacterium is able to initially multiply, due to its former life in the urinary bladder of a young girl for three years (Andersson et al., 1991), its fitness is probably be suboptimal in the new niche. Optimal fitness has to be reached through a process of adaptation within this environment by modifications of preexisting genes. Thus, point mutations, gene loss and acquisition, IS element-mediated transposition may contribute to improve bacterial fitness. The cycles of natural selection will be repeated until the bacterium reaches an optimal adaptation state in the bladder of a currently colonised patient.

Bacterial persistence in the urinary tract is not without an effect on the bladder physiology.

Depending on the bacterial epitope, innate immune response is induced to a certain extent.

Uropathogenic E. coli strains activate IL 6 and IL 8 chemokines. Activation is much stronger when bacteria express functional fimbriae, however, non-fimbriated isolates cause lower and delayed response (Samuelsson et al., 2004). Likely, in most cases asymptomatic bacteriuria isolates belong to the second group of ‘activators’. The same bacterium might have distinct activating abilities, what could be seen among colonized patients with strain 83972 (Fig. 21 and Fig. 22), where the mean of IL 8 expression was very diverse, and in the strongest ‘responder’ was over 8-fold higher than in the lowest one. On the other hand, the level of immune response may vary due to patients susceptibility to UTI and theirs prelevance to a group of low- or high-responders (Svanborg et al., 2006).

149   Discussion 

IL 6 may cause fever and triggers the acute phase response, while IL 8 recruits inflammatory cells to the site of infection (Hedges et al., 1995). The most important are neutrophils (PMN), which play a pivotal role in host defence against microbial infection (Engel et al., 2006;

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