CsrA and CsrB are required for the post-transcriptional control of the virulence-associated effector protein AvrA of Salmonella enterica

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International Journal of Medical Microbiology 299 (2009) 333–341 www.elsevier.de/ijmm

CsrA and CsrB are required for the post-transcriptional control of the virulence-associated effector protein AvrA of Salmonella enterica Tobias Kerrinnesa, Zohar Ben-Barak Zelasb, Wiebke Streckela, Franziska Fabera, Erhard Tietzea, Helmut Tscha¨pea, Sima Yaronb, a

National Reference Centre for Salmonellae and other Enterics, Robert Koch-Institute, Wernigerode, Germany Faculty of Biotechnology and Food Engineering, Technion–Israel Institute of Technology, Haifa 32000, Israel

b

Received 24 June 2008; received in revised form 17 August 2008; accepted 16 September 2008

Abstract The virulence-associated effector protein AvrA of Salmonella enterica is an ubiquitin-like acetyltransferase/cysteine protease, which interferes with the first line of immune response of the target organism. In contrast to translation of the AvrA protein in S. enterica strains, which takes place either constitutively (class 1 strains), or after acid induction (class 2 strains), or not at all (class 3 strains); the constitutive transcription of the respective avrA genes occurs regardless of these defined expression classes. When the number of avrA genes and mRNA molecules is raised experimentally using plasmids carrying the respective cloned avrA genes together with their promoter regions, the translation of avrA mRNA takes place very strongly in all respective AvrA expression classes. This kind of copy-dependent, posttranscriptional control of AvrA was shown to be dependent on the regulatory action of the CsrA/CsrB system since the deletion of both genes completely abolished the translation in the tested S. enterica strains, whereas the transcription remained unaffected. Moreover, AvrA production in strains carrying the cloned avrA genes on plasmids remained dependent on the presence of CsrA but unaffected in csrB mutant strains. On the other hand, overproduction of the regulatory molecules CsrA and CsrB in S. enterica strains carrying cloned csrA and csrB genes on plasmids ceased the expression of AvrA again. Therefore, the expression of avrA is suggested to be regulated in a post-transcriptional manner by critical and effective concentrations of CsrA (see-saw regulation), which is achieved through the sequestering activity of CsrB. r 2008 Elsevier GmbH. All rights reserved. Keywords: AvrA; Virulence; Salmonella enterica; Transcription; Post-transcriptional regulation

Introduction Salmonella enterica (S. enterica) infections appear either with enteric (inflammatory and/or excretory, selflimiting diarrhoea) or with systemic (generalised, typhoid-like) clinical syndromes. A set of various Corresponding author. Tel.: +972 4 8292940; fax: +972 4 8293399.

E-mail address: [email protected] (S. Yaron). 1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2008.09.001

virulence-associated proteins have been identified which are involved in these different infectious pathways (Raffatellu et al., 2005; Zhang et al., 2003). Two type III secretion systems (T3SS-1, T3SS-2) translocate such virulence-associated proteins (effector proteins) into the target cells of the hosts, with the consequence that the effector proteins modulate, impede, or destroy a multitude of cellular functions and defence mechanisms (Galan, 2001; Hensel, 2000; Wallis and Galyov, 2000).

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The effector protein AvrA is a member of a family of ubiquitin-like acetyltransferases/cysteine proteases produced by animal pathogenic bacteria [e.g. Yersinia: YopP, YopJ (Orth, 2002)] and plant pathogens [e.g. Pseudomonas syringae: AvrPpig1 (Alfano and Colmer, 2004)]. In animals, AvrA of Salmonella interferes with the NFkB pathway of the target cells by removing ubiquitins from ub-IkBa (Ye et al., 2007) or by acetylation of specific mitogen-activated protein kinase kinases (MAPKKs) (Jones et al., 2008), it helps to inhibit the IL-8 production and in turn the inflammatory response of the host against infectious agents and also affects the programmed cell death (Collier-Hyams et al., 2002; Ye et al., 2007). In addition, it activates the b-catenin transcription factor (Sun et al., 2004) and stabilizes cell permeability and tight junctions in intestinal epithelial cells (Jones et al., 2008; Liao et al., 2008). Therefore, AvrA may function as an antiinflammatory protein, whereas the other effector proteins like SopB, SopE, and SopE2 do the opposite (Friebel et al., 2001; Huang et al., 2004; Steele-Mortimer et al., 2000; Zhang et al., 2003). The AvrA protein appears as a frequently occurring member in the virulence network of salmonellae, particularly among epidemic strains of S. enterica serovar Typhimurium (S. Typhimurium) and S. Enteritidis, whereas among several other salmonellae like S. Typhi the avrA gene is missing (Hardt and Galan, 1997; Prager et al., 2000; Streckel et al., 2004). Moreover, in analysing the production of the AvrA protein, 4 different expression classes have been identified (Ben-Barak et al., 2006): Salmonella strains which do not contain the avrA gene were designated class 0, strains belonging to class 1 express constitutively AvrA, strains of class 2 need an acid signal for AvrA induction (pH 5.0–6.0), and strains of class 3 reveal no expression of the AvrA protein under a broad range of growth conditions including temperature, excess or deficiency of calcium, iron, magnesium, phosphates, cellular surface factors (HeLa cell lysates), or pH (Streckel et al., 2004). The different expression profiles of class 1, 2, and 3 strains were found to be independent of any DNA sequence variations of the promoter or structural part of the avrA genes (Ben-Barak et al., 2006). In this communication, data are summarised concerning transcription, translation, and post-transcription control of the S. enterica effector protein AvrA. It was found that the transcription of avrA takes place constitutively in all S. enterica strains belonging to class 1, 2, or 3, but that the translation is regulated positively in a post-transcriptional manner by CsrA/CsrB of the csr regulatory system. The csr system of Escherichia coli and Salmonella consists of 4 components: RNA-binding protein CsrA which has to be adjusted to an effective level by its counteracting, noncoding RNAs (ncRNA), CsrB and CsrC, and the CsrD protein that specifically targets both ncRNAs for degradation by

RNase E (Liu et al., 1997; Suzuki et al., 2006; Weilbacher et al., 2003). This system controls post-transcriptionally (by either activation or repression) the expression of a wide variety of genes (Romeo, 1998), but its effect on the expression of AvrA had not been described.

Materials and methods Bacterial strains All bacterial strains used throughout the experiments are listed in Table 1. For each of the 4 AvrA expression classes of the 405 S. enterica strains studied, one representative strain was chosen for the here reported studies. Other strains belonging to the respective expression classes were listed by Streckel et al. (2004) and Ben-Barak et al. (2006). All strains were derived from the type culture collection of the National Reference Centre of Salmonellae and other Enterics (NRC), Wernigerode, Germany. Moreover, several deletion mutant derivatives (see Table 1) from class 0 strain S. Paratyphi B EPV-2 (e.g. TK102, TK103), class 1 strain S. Typhimurium DT193 (TK105, TK110), class 2 strains S. Paratyphi B EPV-1 (TK117, TK118), and class 3 strain S. Typhimurium DT44 (TK107, TK108) were applied throughout the experiments. All respective site-specific deletion mutants were constructed essentially according to Datsenko and Wanner (2000) using the primer pairs H1P1csrASalmo 50 atg ctg att ctg act cgt cga gtt ggt gag acc tca tga GCG ATT GTG TAG GCT GGA GCT 30 and H2P2csrASalmo 50 tta gta act gga ctg ctg gga ttt ttc agc ctg gat acg cCC ATG GTC CAT ATG AAT ATC C 30 for inactivating the csrA gene according to ACC. No. AAL21706.1, as well as H1P1csrBSalmo 50 caa aaa aaa ggg agc act gta taa aca gtc ctc ccg gtt GCG ATT GTG TAG GCT GGA GCT30 and H2P2csrBSalmo 50 ctg gac tgc tgg gat ttt tca gcc tgg ata cgc tgg taC CAT GGT CCA TAT GAA TAT CC 30 for inactivating the gene csrB according to ACC. No. 1254489. Small letters represent the homology region for site-specific recombination with the target gene and capital letters represent the primer part for the PCR with the plasmid DNA pKD3 and pKD4. All deletion mutants were primarily obtained in S. Typhimurium ATCC 14028a by inserting the resistance cassettes and subsequently transduced by P22H as had been described by Schmieger (1972). The correctness of the mutations was confirmed by sequencing. All strains were stored as glycerol (20%) cultures at 70 1C.

Plasmids The plasmids pSV220 and pSV230 (both originated from PCR cloning of avrA from class 2 S. Virchow

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Table 1.

335

Bacterial strains applied throughout the study.

Salmonella enterica

No.

Origin

Relevant genetic properties

S. S. S. S. S. S. S. S. S. S. S. S.

00-1035 TK102 TK103 04-0217 TK105 TK110 00-1205 TK117 TK118 05-3915 TK107 TK108

Enteritis (wt-0) Deletion mutant of 00-1035 Deletion mutant of 00-1035 Enteritis (wt-1) Deletion mutant of 04-217 Deletion mutant of 04-217 Enteritis (wt-2) Deletion mutant of 00-1205 Deletion mutant of 00-1205 SL1344 reference strain (wt-3)b Deletion mutant of SL1344 Deletion mutant of SL1344

DavrA (AvrA class 0)a DavrA , DcsrA DavrA , DcsrB avrA (AvrA constitutively expressed ¼ class1)a avrA, DcsrA avrA, DcsrB avrA (acid-inducible AvrA ¼ class 2)a avrA, DcsrA avrA, DcsrB avrA (no AvrA expression ¼ class 3)a avrA, DcsrA avrA, DcsrB

Paratyphi B (EPV-2) Paratyphi B (EPV-2) Paratyphi B (EPV-2) Typhimurium/DT193 Typhimurium/DT193 Typhimurium/DT193 Paratyphi B (EPV-1) Paratyphi B (EPV-1) Paratyphi B (EPV-1) Typhimurium/DT44 Typhimurium/DT44 Typhimurium/DT44

a The classification of the AvrA expression classes (0, 1, 2, and 3) according to Streckel et al. (2004); other class 0, 1, 2, and 3 strains (see Streckel et al., 2004; Ben-Barak et al., 2006) gave rise to similar or identical results as indicated here for the selected strains. b According to Hardt and Galan (1997), the avrA gene in SL1344 is very weakly but constitutively expressed while the here applied SL1344 strain was found negative in its AvrA synthesis (class 3). Since avrA nucleotide sequences of both strains are identical (compare GL032066 and AY769769) and also the negative AvrA production has been confirmed using both procedures applied by Hardt and Galan (1997) as well as by Streckel et al. (2004), it is suggested that both strains reveal slight differences concerning the here reported regulation system. Therefore, we prefer to designate our SL1344 variant with the laboratory number 05-3915 and regard it as a class 3 strain similar to the others published earlier (Ben-Barak et al., 2006; Streckel et al., 2004).

Table 2.

Plasmids used in this study.

Designation Propertiesa pSV220 pSV230 pSTM330 pSTM350b pCA71 pCA114

Replicon: pcDNA3.1A; PavrA, avrA, amp, his tag Replicon: pCS21; PavrA, avrA, kan Replicon: pCS21; PavrA, avrA, kan Replicon: pNW33N; PavrA, avrA, cat Replicon: pUC; PBAD, araC, csrB, amp Replicon: pUC; PBAD, araC, csrA, amp

Source of genes

Origin

S. Virchow SV23, LT31, (class 2 strain ) S. Virchow SV23, LT31, (class 2 strain ) S. Typhimurium SL1344 (class 3 strain) S. Typhimurium SL1344 (class 3 strain) S. Typhimurium ATCC14028 (class 3 strain) S. Typhimurium ATCC14028 (class 3 strain)

Ben-Barak et al. (2006) This study This study This study Altier et al. (2000a) Altier et al. (2000a)

a Abbreviations used: PavrA, promoter of avrA gene; PBAD, promoter of the araBAD operon; kan, kanamycin resistance gene; cat, chloramphenicol resistance gene; amp, ampicillin resistance gene; PT7, promoter of the phage T7; lacO, operator gene for b-galactosidase determinant; his-tag, C-terminal His-tag and V5 epitop. b The plasmid pSB869 (Hardt and Galan, 1997) was also used in the experiments, but no differences in the expression of AvrA were observed with respect to the here used plasmids (see also Ben-Barak et al., 2006).

SV23), as well as pSTM330 and pSTM350 (originating from PCR cloning of avrA from class 3 strain S. Typhimurium SL1344) were applied in the experiments (Table 2). PCR cloning was carried out as described earlier (Ben-Barak et al., 2006). The plasmids pCA71 and pCA114 (Altier et al., 2000a) were also applied in the experiments described here (Table 2). All plasmids were transferred to the respective S. enterica strains by electroporation (Ausubel et al., 1997) and characterised by PCR and subsequently by Western blots.

DNA techniques All DNA techniques such as PCR, sequencing, transformation, and plasmid identifications were carried

out as described earlier (Ben-Barak et al., 2006; Prager et al., 2000).

Quantitative real-time reverse transcription PCR (qRT-PCR) analysis of avrA transcription For each strain (wild type and mutant) three independent RNA preparations from three independent cultures were made with the RNeasys Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. To avoid DNA contaminations, a second DNA digestion was made with DNase (Roche Applied Science, Mannheim, Germany) for 2 h. The QuantiTects SYBRs Green RT-PCR Kit (Qiagen) was used to amplify the cDNA product of the avrA

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transcripts (using the Primer pair mwg256 50 CTA AAC ACC GAA GCA TTG ACC 30 and mwg257 50 GGA AAC AAG CTC ATG GAC TGA C 30 ) and gyrA (using the Primer pair mwg260 50 AGC TCC TAT CTG GAT TAT GCG AT 30 and mwg 261 50 CGA TTA CGT CAC CAA CGA CA 30 ). The relation between the transcript amounts of avrA and gyrA was evaluated based on results of DNA standard curves with known DNA concentrations (for comparison see Fey et al., 2004). In representative samples, we also analysed the amounts of 16S rRNA and invA mRNA as described (Fey et al., 2004; Tabak et al., 2007) to determine if the amounts of gyrA were not changed in the mutants. The one-step real-time RT-PCR was done in an ABI PRISM 7000 real-time Cycler (Applied Biosystems, Darmstadt, Germany). The data were analysed with the 7000 System SDS Software (Version 1.2) (Novell), according to the manufacturer’s suggestions. In addition, the RT-PCR products were checked on agarose gels and by melting curves as recommended by the manufacturer.

Western blot analysis of AvrA and SopB translation and secretion The culture conditions for the production of the effector proteins AvrA and SopB, their production and harvesting, the SDS-PAGE, the Western blots analysis using monovalent but polyclonal a-AvrA and a-SopB antibodies, and the semi-quantitative estimation of AvrA and SopB production were carried out as earlier described (Ben-Barak et al., 2006), The results of total AvrA presented here are given as the sum of mg AvrA protein per ml of supernatant (SUP) of bacterial culture and of whole cell lysates (WCL) of washed bacterial cells originating from 1 ml culture.

Expression of the plasmid-encoded csrA/csrB genes Overexpression of CsrA and CsrB was obtained by induction of the Salmonella strains harbouring either pCA114 or pCA71, respectively, with 0.5% arabinose as described (Altier et al., 2000a).

Results The post-transcriptional control of AvrA in S. enterica S. enterica strains belonging to the 4 AvrA expression classes 1, 2, 3, and 0 (Table 1) were submitted to mRNA analysis using the qRT-PCR and to gene product analysis using Western blots in order to characterise transcription and translation. As demonstrated in Table 3

all S. enterica strains of class 1, 2, and 3 were found positive for avrA mRNA irrespective of their classification into the 3 AvrA protein expression classes. Only one strain of each class is listed as a representative of the other additionally tested strains and all revealed similar qRT-PCR and Western blot data (Table 3). As expected, in class 0 no mRNA was identified. The qRT-PCR results are in high correlation with previous results using GFP as a marker protein that also indicated similar transcription rates in S. enterica strains of class 1, 2, and 3 (Ben-Barak et al., 2006). In contrast to these qRT-PCR results, the Western blot signals indicating the AvrA protein presence (Table 3) confirm our earlier findings (Ben-Barak et al., 2006) of the different expression profiles of S. enterica strains: strains of class 1 gave rise to a positive Western blot signal, strains of class 2 need an acid induction for their AvrA expression, and strains of class 3 reveal no expression of AvrA under a broad range of growth conditions (see also Streckel et al., 2004). These data point to a kind of post-transcriptional control of the AvrA protein. When AvrA was expressed, the majority of the protein was found in the supernatant and not in the cells. In order to understand the constitutive transcription of avrA in contrast to the lack of translation of the AvrA protein, several site-specific deletion mutant strains were constructed, which might reveal changes in avrA transcription or expression. Mutant strains carrying deletion rhyB, rpoS, sprB, iroN, phoP, and fur genes were found to keep the AvrA protein production unaffected, and deletion of invA resulted in inhibition of the secretion of AvrA to the SUP without any effect on the total amount of AvrA (unpublished results), whereas deletion in csrA as well as csrB stopped the AvrA protein production in strains of classes 1 and 2 (Table 3; for hfq see footnote g). Since the transcription of avrA genes of classes 1, 2, and 3 remains unaffected by deletion in csrA or csrB, the results in Table 3 demonstrate that CsrA in concert with CsrB allow the translation of avrA mRNA. In contrast, neither csrA nor csrB mutations affect the transcription (data not shown) as well as the translation (Fig. 1) of the effector protein SopB. Since the CsrA/CsrB control system acts copydependently (Altier et al., 2000a, b), it was questioned if its observed regulatory influence on AvrA protein production could be abolished by a higher copy number of avrA-mRNA molecules. In order to enhance the copy numbers of avrA mRNA, the respective avrA genes together with their own promoters (derived from classes 1, 2, and 3) were cloned into the plasmid pCS21 (Table 2), and tested subsequently concerning their expression in S. enterica strains belonging to classes 1, 2, 3, and 0. As seen in Table 3, all tested S. enterica strains are able to transcribe and translate all plasmid-encoded avrA genes. This might speak in favour of that a higher copy number of

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Table 3.

337

Transcription and translation levels of chromosomal- and plasmid-encoded AvrA in S. enterica mutant strains.

Strains

Culture conditions (pH)

Transcriptiona/translation levelsb Plasmid freec

S. Paratyphi B 00-1035, class 0e TK102(DcsrA)f TK103(DcsrB)f,g S. Typhimurium 04-0217, class 1e TK105 (DcsrA)f TK 110(DcsrB)f S. Paratyphi B 00-1205 class 2e TK 117(DcsrA)f TK 118(DcsrB)f S. Typhimurium SL1344, class 3e TK107 (DcsrA)f TK108 (DcsrB)f,g

7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0 7.4 5.0

pSV220d

qRT-PCRa

WBb

qRT-PCRa

WBb

0 0 0 0 0 0 0.85 0.60 1.52 nt 0.94 nt 0.48 0.49 nt 1.09 nt 0.64 0.35 0.34 0.78 nt 0.19 nt

0 0 0 0 0 0 125 120 0 0 0 nt 0 172 0 0 0 0 0 0 0 0 0 nt

0.81 1.33 3.57 nt 3.80 nt 1.80 1.82 1.60 nt 1.14 nt 2.09 1.22 0.97 1.38 nt nt 0.84 1.57 nt nt nt nt

160 180 0 0 150 140 200 180 0 0 180 nt 180 170 0 0 100 120 220 200 0 0 160 nt

a

Transcription (qRT-PCR; QavrA/gyrA: amount of avrA mRNA normalised to the amount of gyrA mRNA). The minimal detectable level of normalised avrA mRNA was 0.05. b Translation [sum of the western blot levels from whole cell lysates (WCL) and supernatants (SUP)]. The numbers correspond to mg AvrA/ml of cell cultures according to semiquantitative Western blots (see Material and methods). The minimal detectable level of AvrA was 2 mg/ml of culture supernatant [see Streckel et al. (2004) and Ben-Barak et al. (2006)]. c Similar results were achieved with the plasmids pcDNA3.1A and pCS21 without insert (data not shown). d Similar results were achieved with the plasmids pSB869, pSV230, and pSTM330 (data not shown). e Other S. enterica strains belonging to the respective classes 0, 1, 2, and 3 revealed comparative results. Other mutants derived by the Datsenko/ Wanner procedure (Datsenko and Wanner, 2000) such as rpoS, phoP, fur, rhyB, sprB etc. did not change the AvrA phenotype significantly (unpublished results). f csrA and csrB mutants remain positive for translation and secretion of effector protein SopB (see Fig. 1). g The S. Typhimurium Dhfq mutation [hfq: chaperon of several ncRNA types (Geissmann and Touati, 2004)] in SL1344 (strain WR1959) revealed similar results as the DcsrB mutation in TK108. nt, not tested; WB: Western blot.

avrA mRNA can abolish its post-transcriptional inhibition in class 2 and 3 strains of S. enterica.

The post-transcriptional control factor is the CsrA protein together with the ncRNA CsrB The qRT-PCR and Western blot results obtained with the csrA and csrB deletion mutants (Table 3) point to the nature of the post-transcriptional regulation: the DcsrA mutations in TK102, TK105, TK107, and TK117 strains inhibit the AvrA protein production, but have no effect on the transcription of the respective avrA genes. This effect was observed with the chromosomal as well

as the plasmidal-encoded AvrA (Table 3). In contrast, the DcsrB mutants, TK103, TK108, TK110, and TK118 allow the translation of plasmidal but not of chromosomal-encoded AvrA production, which might be understood as a CsrA overproduction due to its absent sequestering (Altier et al., 2000a). A representative picture of the differences between the AvrA and SopB protein amounts is given in Fig. 1. Based on these observations, it can be concluded that the CsrA protein is essential for the translation of the chromosomal as well as plasmidal-transcribed avrA mRNA, and that CsrB counteracts this activation by sequestering the CsrA protein to a critical or effective concentration (Table 3). If this is the case, an overexpression of CsrA

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1

2

3

4

5

6

S

SopB 45 kD

30 kD 1

2

3

4

5

6

S

AvrA

30 kD

Fig. 1. Western blots of the virulence-associated effector proteins SopB and AvrA from S. enterica mutant strains. (A) SopB. (B) AvrA. Lane 1: supernatant of S. Paratyphi B EPV-2 class 0 strain 00-1035 (see Table 1) carrying the plasmid pSTM330 (see Table 2). Lane 2: Supernatant of S. Paratyphi B EPV-2 TK102 (DcsrA) carrying pSTM330. Lane 3: supernatant of S. Paratyphi B EPV-2 TK103 (DcsrB) carrying pSTM330. Lane 4: Supernatant of S. Typhimurium SL1344 (04-3915) TK107 (DcsrA) carrying pSTM330. Lane 5: Supernatant of S. Typhimurium SL1344 (04-3915) TK108 (DcsrB) strain carrying pSTM330. Lane 6: Supernatant of S. Typhimurium SL1344 (04-3915). S, molecular standard. Table 4. 7.4.

Influence of overexpression of CsrA and CsrB on the expression (transcription and translation) of the AvrA protein at pH

Strains

Transcriptionala/translational/levelb pSV220c

Chromosomal avrA

S. Paratyphi B 00-1035, class 0d 00-1035d carrying pCA114 00-1035d carrying pCA71 S. Typhimurium DT193 04-0217, class 1e 04-0217, carrying pCA114 04-0217, carrying pCA71 S. Typhimurium SL1344, class 3f SL1344 carrying pCA114 SL1344 carrying pCA71

qRT-PCRa

WBb

qRT-PCRa

WBb

0.0 0.0 0.0 0.85 0.80 0.75 0.55 0.50 0.65

0.0 0.0 0.0 100 0 0 0 0 0

1.90 1.30 1.60 1.80 1.60 1.65 1.15 1.20 1.25

180 0 0 200 0 0 200 0 0

a

Real-time RT-PCR quotient of avrA/gyrA-RNA. mg AvrA protein/ml of cell culture [sum of the Western blot levels from whole cell lysates (WCL) and supernatants (SUP)]; data correspond to the results of 3 independent experiments. c Similar results were achieved with the plasmids pSB869, pSV230, and pSTM330. d Similar data were obtained from S. Paratyphi B (SPV) 99-9330 [for strains see Ben-Barak et al. (2006)]. e Similar data were obtained from S. Typhimurium/DT104, 97-1208 [for strains see Ben-Barak et al., (2006)]; see also Table 3. f Similar data were obtained from S. Typhimurium/DT204, 00-2379 [for strains see Ben-Barak et al. (2006)]; see also Table 3. b

and CsrB should again compensate the higher copy number of the plasmid-encoded avrA mRNA. Using the CsrA and CsrB overexpressing plasmids pCA114 (CsrA) and pCA71 (CsrB) in class 0, class 1, and class 3 strains carrying AvrA plasmids (pSV220, pSTM330 etc.) and submitting them to test the AvrA production by Western blots, it was found (Table 4) that the overproduction of CsrA as well as of CsrB shuts down the AvrA production similarly as it could be observed with the deletion mutant strains. The results summarised in Table 4 confirm the findings of Table 3

that only a critical or effective concentration of CsrA allows the post-transcriptional activation of avrA mRNA to be translated into the AvrA protein which is achieved by the counteracting ncRNA CsrB.

Discussion The virulence-associated effector protein AvrA of S. enterica, which interferes with the first line of immune

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response of the target organism (Collier-Hyams et al., 2002), is an important partner in the virulence phenotype of this pathogen (Ben-Barak et al., 2006; Streckel et al., 2004). Our earlier studies revealed that epidemic or clinical strains of S. enterica can be grouped into 4 classes with respect of their AvrA expression. Since differences between the avrA expression classes 1, 2, and 3 strains are not due to different transcription profiles but to different post-transcriptional regulatory events, and since plasmid-encoded avrA is translated in all classes (Tables 3 and 4), a post-transcriptional activation factor was anticipated. In the present results, a post-transcriptional activation factor for the AvrA protein production, the CsrA protein, has been identified, since csrA deletion mutants do not allow the translation of chromosomal as well as of plasmidal-encoded AvrA. CsrA is a post-transcriptional regulator that binds the target mRNA molecules and alters their stability or blocks the Shine-Dalgarno sequence from recognition by ribosomes (Wei et al., 2001). Looking at the 50 UTR region of avrA, it is very likely that the CsrA action takes place at the upstream untranslated region of the mRNA itself, maybe by binding to the GGA motif on positions 8, 12, and 25 NT of 50 UTR region proximal to the first ATG of the avrA open reading frame (AY769769), or by blocking or opening the RBS and ATG for translation (for review see Gottesman, 2004), depending on the above-mentioned critical concentration. In Salmonella, CsrA activates the expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes and other effector proteins secreted by T3SS1 as well as the flagellar synthesis genes, the operon for synthesis of vitamin B12 and genes of metabolic pathways likely to be used by Salmonella in the intestinal milieu (Lawhon et al., 2003). These genes are affected by CsrA (probably indirectly) at the transcription level (see also Lawhon et al., 2003), in opposition to avrA that is affected by CsrA at the post-transcriptional level. Thus, it is suggested here that the CsrA protein can directly regulate the avrA mRNA, and this regulation (activation or inhibition) depends on a critical or effective relative concentration of CsrA (in connection with the avrA-mRNA concentration). This effective concentration of CsrA is quantitatively adjusted by the untranslated RNA CsrB, the second element of the csr control system, which sequesters or ‘neutralises’ the action of CsrA (up to 18–20 CsrA molecules bind to a single CsrB molecule) (Liu et al., 1997). Indeed, deletions in csrB inhibit the translation of chromosomal-encoded but not of plasmidal-encoded AvrA. We suggest that csrB mutations enable an overproduction of CsrA which in turn affects an inhibition of the AvrA protein production of chromosomal-encoded avrA, but not of plasmidal-encoded avrA due to an increased avrA-mRNA copy number in such

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strains (Table 3; see also Altier et al., 2000a, b; Lawhon et al., 2003). Similarly, an inhibition of the AvrA expression by an overproduction of either CsrA or CsrB can also be achieved using the plasmids pCA114 (CsrA) and pCA71 (CsrB), respectively. These plasmids overexpress CsrA or CsrB to such an extent that also the AvrA plasmid-containing (pSV220, pSTM330 etc.) strains ceased with their AvrA production, indicating that the bacteria must tightly control the levels of CsrA, CsrB and avrA mRNA to achieve expression of the AvrA protein. Such a kind of a ‘see-saw’ regulation had already been observed for S. Typhimurium ATCC 14028a when the inhibitory regulation of invasion proteins (such as InvF, SipC, and PrgH) by CsrA was found either by its absence or by its overproduction (Altier et al., 2000a, b). It can be suggested therefore that any post-transcriptional regulation by the cooperative action of CsrA and CsrB either through stabilising or destabilising the avrA mRNA provides a rapid and extremely sensitive means to alter virulence gene expression in response to environmental conditions (see also Lawhon et al., 2003). A very similar post-transcriptional control system as a kind of see-saw regulation was identified for the virulence make-up and/or biofilm formation in Vibrio cholerae, E. coli, Pseudomonas aeruginosa, and Erwinia carotovora (Kay et al., 2005; Lee and Galan, 2004; Lenz et al., 2005; Valverde et al., 2004; Ventre et al., 2006; Wang et al., 2005). Nevertheless, although CsrA/B and their homologues exist in a number of bacterial species, too, their substructure and subsequently the functions that they regulate might be quite diverse (Lawhon et al., 2003). The avrA gene is located in the SPI-1 region, together with other invasion genes, but the function of AvrA as an anti-inflammatory and as a stabilizer of the TJ structure differs from the function of the other effector proteins (Liao et al., 2008). The regulation of AvrA was also found to be unique, because except for the low pH in the class 2 strains, none of the environmental signals known to activate the invasion proteins regulate AvrA (Streckel et al., 2004). Moreover, SPI-1 regulators like the HilA, InvF, and PhoP/PhoQ that activate the invasion proteins (Bajaj et al., 1996; Eichelberg and Galan, 1999; Ellermeier and Slauch, 2007) have no effect on AvrA (Eichelberg et al., 1999). Here we show, for the first time, a global system that also controls the expression of AvrA. However, while the csr system directly regulates the expression of AvrA, its effect on the other effector proteins of SPI-1 and T3SS was shown to be through other regulators such as HilA or InvF (Lawhon et al., 2003). The results summarised in this paper underline our earlier suggestion that the posttranscriptional regulation of the effector protein AvrA can be regarded as a critical step in fine tuning the virulence network in different Salmonella serovars. As

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other effector proteins (but not all) also seem to be posttranscriptionally regulated, this might contribute to our understanding how S. enterica strains can display such a broad range of virulence patterns and clinical syndromes.

Acknowledgements The generous gift of the plasmids pCA71 and pCA114 from C. Altier, Cornell University, USA, and the E. coli strain TR1-5 from D. Haas, Lausanne, Switzerland, as well as the technical help of Ute Siewert, Ute Strutz, Petra Haas, and Dina Shachar are highly appreciated. We thank Dr. Rita Prager for helpful discussion and Dr. Wolfgang Rabsch, both RKI, for his support of one of us (T.K.) to introduce him into the DatsenkoWanner- technique. We thank also Dr. Rabsch for providing the deletion mutant strains WR1924 (rhyB), WR1932 (rpoS), WR1963 (sprB), WR1920 (iroN), WR1926 (phoP), and WR1928 (fur) for testing them with respect to their AvrA production. This work was supported in part by the Israel Science Foundation Grant no 1275/04 and by the BMBF Grant 01Kl9901 of the Bundesministerium fu¨r Bildung und Forschung ‘Emerging food-borne pathogens in Germany’.

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