NATURAL AND MAN-MADE ENVIRONMENTAL RESERVOIRS FOR LEGIONELLA 

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A new concept in antisense RNA-mediated regulation: the excludon

Recently a sophisticated asRNA-dependent mechanism that achieves a regulatory connection between neighbouring genes that often have opposite functions was reported in L. monocytogenes.
The name ‘excludon’ has been coined to describe this new regulatory mechanism. A single transcript serves in parallel as an antisense repressor of one gene or group of genes and as an mRNA promoting the expression of the adjacent genes. This results in the regulation of adjacent genes of opposite functions (Sesto et al. 2013).
The ‘excludon’ in L. monocytogenes takes part in the control of flagellum biosynthesis. The asRNA, Anti0677 has a proximal region that is antisense to the lmo0675–lmo0676–lmo0677 locus and a distal region that is part of the gene encoding the regulator MogR (Toledo-Arana et al. 2009) (Table 1). The lmo0676– lmo0677 locus encodes FliP and FliQ that together with FliR (encoded by the adjacent gene lmo0678) constitute the flagellum export apparatus. The MogR regulator binds the fli operon promoter and down-regulates the flagellum and thus motility in L. monocytogenes. When Anti0677 is expressed, it inhibits the synthesis of lmo0675 (its function is not known yet) and the entire flagellum export apparatus and leads simultaneously to the expression of MogR. Thus the flagellum apparatus is concurrently turned off by the increasing expression of MogR and by the repression of the lmo0676–lmo0677 locus driven by the asRNA Anti0677. Furthermore, the inhibition of the flagellumexport apparatus is likely to involve the formation of a double-stranded RNA when Anti0677 is transcribed and the subsequent cleavage by RNase III (Wurtzel et al. 2012). The above-described asRNA Anti0677 was the first example for the excludon concept; however, at least 13 additional, putatively excludon-like regulated genomic lociwere detected through comparative transcriptomic analysis of L. monocytogenes, suggesting that excludon-mediated regulation might represent a frequent mechanism. Furthermore, similar organizations have been found in other bacteria such as L. pneumophila (unpublished data), thus this mechanism of antisense regulation is probably not restricted to the genus Listeria but extends to other bacteria.

Cis-encoded sRNAs: regulation of virulence gene expression

Interestingly, a long asRNA of 1.2 kb, named AmgR, was identified in the antisense strand of the mgtCBR operon and complementary to the 5′ terminus of this polycistronic mRNA in S. enterica serovar Typhimurium (Table 1). AmgR is transcribed from a promoter that is located in the intergenic region betweenthe mgtC and mgtB genes. The aforementioned operon encodes the MgtC protein implicated in Mg2+ homeostasis and virulence, the Mg2+ transporter MgtB and the 30 amino acid peptide MgtR that mediates degradation of MgtC by the FtsH protease. Intriguingly, the transcription of the polycistronic mgtCBR mRNA and the AmgR sRNA is under the positive control of the two-component regulatory system PhoP/PhoQ. In detail, when PhoQ senses low extracytoplasmatic Mg2+, it phosphorylates PhoP, which then binds to the mgtC and amgR promoters, mediating the transcription of the mgtCBR mRNA and the long sRNA.
As such, the long RNA regulatory element limits the MgtC and MgtB protein levels, promoting the MgtR binding to the MgtC protein and the subsequent degradation by the FtsH protease.
Markedly, AmgR serves as a timing device to attenuate virulence in mice mediated by the sense-encoded MgtC protein (Lee and Groisman 2010). The finding that the AmgR asRNA is located inside a polycistronic region revealed that the repertoire of regulatory sRNA elements might be larger than thought and raises the possibility that additional asRNAs exist which have been overlooked.
Another example of an asRNA, named lesR-1, encoded within the pSLT virulence plasmid has been reported recently in the bacterial pathogen S. enterica serovar Typhimurium (Gonzalo-Asensio et al. 2013) (Table 1). RNA expression has been studied using oligonucleotide microarrays containing probes in each of the two strands of every intergenic region testing a variety of culture conditions. Remarkably, lesR-1 was preferentially expressed in non-growing dormant bacteria during colonization of fibroblasts. The deletion of this asRNA not only influenced the control of bacterial growth within the fibroblast cell lines but also impaired virulence in a mouse infection model. As an as- RNA that is overlapping the PSLT047 transcript, the lesR-1 regulatory mechanism involves the direct interaction of the 3′ end of the RNA molecule leading to the subsequent remodelling of the PSLT047 protein levels. Given the virulence phenotype, this suggests that PSLT047 is important for the intracellular lifestyle of S. enterica serovar Typhimurium (Gonzalo-Asensio et al. 2013).

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DYNAMIC EXPRESSION CHANGES OF sRNAs DURING INTRA- AND EXTRACELLULAR GROWTH

The use of whole-genome tiling arrays and RNA deep sequencing has revealed that sRNAs, similar to protein-coding genes, show dynamically changing expression profiles during growth in laboratory media but also during infection. An example is L. pneumophila as this intracellular pathogen is known to have a pronounced biphasic life cycle. Schematically, pathogenic L. pneumophila encode one set of genes dedicated to transmission that includes all major virulence traits and another set that promotes replication in phagocyte vacuoles, a characteristic that is reflected in a major shift of gene expression during the switch from replicative to transmissive bacteria (Molofsky and Swanson 2004; Bruggemann et al. 2006). Recently, RNAseq analyses showed that ∼700 sRNAs are expressed in the L. pneumophila genome. Similar to the protein-coding genes, >60% of the sRNAs are growth phase dependently regulated (Sahr et al. 2012). Most interestingly, transcriptional start site mapping identified in addition a high number of sRNAs with tandem promoters, 30% of which are used growth phase dependently (Sahr et al. 2012). These strong changes in sRNA expression profiles indicate an important role for sRNAs in regulating the biphasic life  cycle of L. pneumophila and thus virulence. Similarly, transcriptome data using a 70mer oligonucleotide array of the S. enterica Typhimurium genome revealed that ∼2% (98 genes) of all genes are differentially expressed in nongrowing intracellular bacteria, when the fibroblast infection model was used (Nunez-Hernandez et al. 2013). Later work also identified sRNAs that are differentially expressed. As an example, SraL shows higher expression levels in non-growing bacteria than in actively growing ones (Ortega, Gonzalo-Asensio and Garcia-del Portillo 2012). A comprehensive picture of growth phase- and condition-dependent expression of sRNAs was reported for L. monocytogenes. The analyses of L. monocytogenes expression profiles using tiling arrays and bacteria grown in exponential phase, stationary phase, hypoxia and low temperature or isolated from the intestine of axenic mice or bacteria grown in the blood of human donors provided important information about their expression conditions and thus hints of their putative functions (Toledo-Arana et al. 2009). Similarly, RNA sequencing of L. monocytogenes grown in macrophages showed that 29 of the 150 described sRNAs are specifically induced during intracellular growth, pointing to functional roles during infection. Indeed, knockout mutants of three of these RNAs, named rli31, rli33-1 and rli50, were defective in intracellular growth (Mraheil et al. 2011). Recently the non-coding genome was characterized in multiple growth conditions that are relevant for the infection process using RNA deep sequencing. This led not only to the characterization of condition-dependent expression of sRNAs but also to the identification of new regulatory mechanisms such as the excludon described above (Wurtzel et al. 2012).

Table of contents :

LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER ONE: GENERAL INTRODUCTION 
1.1 ADAPTATION TO ENVIRONMENTAL CHANGES
1.2 THE GENUS LEGIONELLA
1.2.1 CHARACTERISTICS OF THE GENUS LEGIONELLA
1.2.2 NATURAL AND MAN-MADE ENVIRONMENTAL RESERVOIRS FOR LEGIONELLA
1.3 LEGIONNAIRES’ DISEASE
1.3.1 SYMPTOMS AND CLINICAL MANIFESTATIONS
1.3.2 DIAGNOSIS AND TREATMENT
1.3.3 RISK FACTORS
1.3.4 EPIDEMIOLOGY AND INCIDENCE
1.4 ASSOCIATION WITH FREE-LIVING AMOEBAE
1.5 INTRACELLULAR LIFE CYCLE WITHIN PHAGOCYTIC CELLS
1.5.1 HIJACKING HOST CELL DEFENSES BY THE DOT/ICM MACHINERY
CHAPTER TWO: HFQ AND SMALL REGULATORY RNAS 
2.1 IMPLICATION OF SMALL REGULATORY RNAS AND THEIR REGULATORS IN VIRULENCE AND ADAPTATION OF INTRACELLULAR BACTERIA
2.2 THE RNA CHAPERONE HFQ
2.2.1 GENERAL PROPERTIES AND STRUCTURE OF HFQ
2.2.2 THE RNA BINDING-FEATURES OF HFQ
2.2.3 MECHANISMS OF HFQ RIBOREGULATION
2.2.4 REGULATION OF HFQ EXPRESSION
2.2.5 ROLE OF HFQ IN BACTERIAL PATHOGENS
2.2.6 THE RNA BINDING PROTEINS HFQ AND CSRA MAY WORK TOGETHER
CHAPTER THREE: REGULATION OF L. PNEUMOPHILA VIRULENCE 
3.1 THE L. PNEUMOPHILA LIFE CYCLE
3.2 REGULATORY NETWORK GOVERNING LEGIONELLA DIFFERENTIATION
3.2.1 METABOLIC TRIGGERS
3.2.2 TRANSCRIPTIONAL CONTROL BY SIGMA FACTORS
3.2.3 POST-TRANSCRIPTIONAL REGULATION OF THE TRANSMISSIVE TRAITS
3.2.4 IMPLICATION OF REGULATORY SRNAS ON L. PNEUMOPHILA VIRULENCE
3.2.5 REGULATORY NETWORK GOVERNING L. PNEUMOPHILA BI-PHASIC LIFE CYCLE  AIM OF THE PH.D. THESIS
CHAPTER FOUR: RESULTS- REGULATION OF HFQ IN L. PNEUMOPHILA 
4.1 L. PNEUMOPHILA HFQ- CHROMOSOMAL ORGANIZATION
4.2 ARTICLE PUBLISHED IN THE JOURNAL MBIO
4.3 SUPPLEMENTARY RESULTS
CHAPTER FIVE: CONCLUSIONS AND PERSPECTIVES 
REFERENCES

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