Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

Abstract

Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes.

A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper

“…S1 and S2), we were interested in examining whether this island is silenced by NAPs. The H-NS NAP is not present in Legionella, but three Fis paralogs (Fis1, Fis2, and Fis3), which are also NAPs, were previously shown to directly repress the expression of EEGs (32). Examination of the lciRS-lciE intergenic region led to the identification of four potential Fis regulatory elements (TG-N 13 -C), two close to or overlapping the Ϫ10 promoter element of lciE (Fig.…”

“…Glycerol was added to a concentration of 50%, and the purified protein was then stored at -20°C. A gel mobility shift assay was performed as previously described (32), with a few modifications. The regulatory region lciE (176 bp) was amplified by PCR using the primers listed in Data Set S1 and 3= end labeled with digoxigenin (DIG) by using DIG-11-ddUTP (Roche).…”

“…To date, five regulatory systems have been shown to directly regulate the expression of effector-encoding genes (EEGs) in L. pneumophila: (i) the PmrAB two-component system (TCS) activates the expression of about 40 EEGs (18,19); (ii) the CpxRA TCS activates or represses the expression of about 30 EEGs and also regulates the expression of several icm/dot genes (20)(21)(22)(23); (iii) the LetAS-RsmYZ-CsrA regulatory cascade represses the expression of about 40 EEGs during exponential phase (24)(25)(26)(27)(28)(29)(30)(31); (iv) two Fis regulators repress the expression of about 20 EEGs during exponential phase (32); and (v) the Fur regulator controls the expression of a single EEG (mavN) as well as several other proteins involved in iron acquisition (33)(34)(35)(36)(37). In addition, these regulatory systems have been shown to assemble into an interconnected regulatory network using accessory components, such as modulators (AckA-Pta and PTS Ntr [22,38]) and connectors (LetE and LerC [22,39,40]).…”

The Legionella pneumophila CpxRA two‐component regulatory system: new insights into CpxR's function as a dual regulator and its connection to the effectors regulatory network

“…An additional type of interplay between regulators is the direct regulation of a single effector by multiple regulators. Several such combinations were thus far described for effector‐encoding genes regulated by CpxR: (i) A single effector (Ceg33) was shown to be activated by both CpxR and PmrA (Altman and Segal, ); (ii) Four effectors (CegC2, Ceg7, Ceg18 and Ceg29) were shown to be activated by PmrA and repressed by CpxR ([Altman and Segal, ] and this study); (iii) Three effectors (CegC1, MavT and Lpg2461) were shown to be repressed at the level of transcription by CpxR and at the level of translation by CsrA ([Rasis and Segal, ] and this study); and (iv) Two effectors (SidM/DrrA and CegC4) were shown to be repressed by both CpxR and the Fis regulators (Altman and Segal, ; Zusman et al ., ). Interestingly, there are combinations of dual direct regulation that were not observed: regulation by both PmrA and CsrA, as well as activation by CpxR and repression by CsrA.…”

A Unique cis -Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner

“…We propose that binding of the cis -encoded Anti-hfq sRNA to hfq mRNA in exponential growth leads to low translation of Hfq, whereas when the expression of Anti-hfq sRNA decreases in the transmissive phase, high expression of Hfq is possible. This leads to the expression of several Dot/Icm secreted substrates, global regulators like Fis1 and Fis2 that are implicated in the regulation of virulence traits (44) and probably of several of the many growth phase dependently regulated sRNAs that we identified earlier (13) (Fig. 8).…”