Introduction
============

*Pseudomonas aeruginosa* is a ubiquitous Gram-negative bacterium that can cause both acute and chronic infections in individuals with compromised immunity such as cancer patients and those with cystic fibrosis ([@B7]; [@B49]; [@B38]; [@B53]).

The type III secretion system (T3SS) is an important virulence factor of *P. aeruginosa*, through which effector proteins are directly injected into the cytosols of eukaryotic host cells, inhibiting host defense by inducing cell death in polymorphonuclear phagocytes, macrophages, and epithelial cells ([@B8]; [@B21]; [@B32]). Expression of the T3SS confers an increased virulence in *P. aeruginosa* and is associated with poor clinical outcomes ([@B14]), whereas strains with defective T3SS display attenuated virulence in mouse acute infection models ([@B51]). To date, four effector proteins have been identified and well characterized in *P. aeruginosa*, i.e., ExoS, ExoT, ExoU, and ExoY ([@B20]). However, majority of *P. aeruginosa* isolates do not encode all of the four effectors ([@B16]). For example, strain PAK expresses ExoS, ExoT, and ExoY, while strain PA14 expresses ExoU, ExoT, and ExoY.

In *P. aeruginosa*, the T3SS is induced in response to a variety of environmental conditions, such as direct contact with host cell, calcium depletion and the presence of serum ([@B33]; [@B23]). The expression of T3SS is activated by ExsA, an AraC-type DNA binding protein, which recognizes and binds to two adjacent highly conserved consensus sequences in the promoter region of the T3SS genes ([@B26]; [@B3]). The ExsA activity and transcriptional regulation on T3SS are intimately coupled to secretion by a partner-switching model involving three other proteins: ExsC, ExsD, and ExsE ([@B4]). Under non-inducing condition, the secretable repressor ExsE is kept inside bacterial cytosol and binds to ExsC, and ExsD binds to and inactivates ExsA. Whereas under inducing environment, ExsE is secreted by the T3SS machinery, which releases ExsC to sequester its low affinity partner ExsD, resulting in free ExsA that activates the transcription of whole T3SS gene cluster ([@B40]; [@B9]; [@B44]; [@B58], [@B57]; [@B54]; [@B11]).

The virulence factor regulator Vfr is a cAMP-dependent transcriptional regulator. It was originally identified as an activator of extracellular protease and exotoxin A expression. Now it is appreciated as a global regulator of virulence gene expression, including T3SS and pili biosynthesis genes ([@B61]; [@B63]; [@B39]). Intracellular cAMP is generated by adenylate cyclases CyaA and CyaB and hydrolyzed by the phosphodiesterase CpdA in *P. aeruginosa* ([@B63]; [@B17]). Besides, other regulators and proteins are also known to affect expression of the T3SS, such as the *rhl* quorum sensing system ([@B25]), stationary-phase sigma factor RpoS ([@B25]), transcriptional activator PsrA ([@B48]), global regulator MexT ([@B31]), alginate biosynthesis protein MucA ([@B64]), RNA-binding proteins RsmA and Crc ([@B41]; [@B12]), small proteins PtrA, PtrB, and PtrC ([@B19]; [@B65]; [@B31]), tryptophan synthase TrpA ([@B47]), tryptophan dioxygenase KynA ([@B47]), pseudouridinase enzyme TruA ([@B1]), nitrite reductase NirS ([@B60]), magnesium transporter MgtE ([@B2]), two-component system AlgZR ([@B30]), DNA binding protein Fis ([@B10]), multiple virulence regulator SuhB and RNA helicase DeaD ([@B34]; [@B29]). However, the molecular mechanism by which most of these proteins regulate T3SS is not fully elucidated yet. All of these suggest that expression of T3SS is controlled under a complicate regulatory network. Therefore, we aimed to identify novel regulators of T3SS and their regulatory mechanisms.

In the present study, we identified that NrtR (PA4916) is required for the expression of T3SS in *P. aeruginosa*. *In vivo* studies suggest that NrtR plays an important role in the pathogenesis of *P. aeruginosa*. Further studies demonstrated that NrtR affects expression of the T3SS through cAMP/Vfr signaling system that lies upstream of the ExsA. We demonstrated that NadD2 is involved in the NrtR mediated regulation of the T3SS by inhibiting the CyaB enzymatic activity and subsequent reducing intracellular cAMP level.

Materials and Methods {#s1}
=====================

Ethics Statement
----------------

All animal studies complied with National and Nankai University guidelines regarding the use of animals in research. All animal experiment protocols have been approved by the institutional animal care and use committee of the College of Life Sciences of Nankai University (permit number NK-04-2012).

Bacterial Strains, Plasmids, and Growth Conditions
--------------------------------------------------

Bacterial strains and plasmids used in this study are listed in [Table 1](#T1){ref-type="table"}. Both *E. coli* and *P. aeruginosa* were grown in Luria-Bertani broth (LB) medium ([@B10]) or on LB agar ([@B10]) plates at 37°C. Whenever needed, antibiotics were used at following concentrations (μg/ml): for *E. coli*, ampicillin 100, tetracycline 10, spectinomycin 50, streptomycin 25, gentamicin 10; for *P. aeruginosa*, carbenicillin 150, tetracycline 50, spectinomycin 200, streptomycin 200, gentamicin 100. When needed, 1 mM IPTG (isopropyl β-[D]{.smallcaps}-1-thiogalactopyranoside) or 50 mM cAMP were added to culture medium. Primers used to make various constructs and in RT-qPCR are listed in [Table 2](#T2){ref-type="table"}.

###### 

Bacterial strains and plasmids used in this study.

  Strain or plasmid                                  Description                                                                                                          Source
  -------------------------------------------------- -------------------------------------------------------------------------------------------------------------------- ---------------
  ***E. coli* strains**                                                                                                                                                   
  DH5α                                               F^-^ ϕ 80d*lacZ*ΔM15 *endA1 recA1 hsdR17*(r~K~^-^ m~K~^+^) *supE44 thi-1 relA1* Δ(*lacZYA-argF*)*U169 gyrA96 deoR*   TransGen
  S17-1                                              RP4-2 Tc::Mu Km::Tn*7* Tp^r^ Sm^r^ Pro Res^-^ Mod^+^                                                                 Dr. Ramphal
  HB101                                              Source for wild type *lac*P1 promoter sequence                                                                       TransGen
  ***P. aeruginosa* strains**                                                                                                                                             
  PA14                                               Wild type *P. aeruginosa* strain                                                                                     [@B36]
  PAK                                                Wild type *P. aeruginosa* strain                                                                                     David Bradley
  *exsA*::Tn                                         PA14 with *exsA* disrupted by insertion of Tn                                                                        [@B36]
  PA4336::Tn                                         PA14 with PA4336 inserted with Tn                                                                                    [@B36]
  PA4916::Tn                                         PA14 with PA4916 inserted with Tn                                                                                    [@B36]
  PA0020::Tn                                         PA14 with PA0020 inserted with Tn                                                                                    [@B36]
  PA4753::Tn                                         PA14 with PA4753 inserted with Tn                                                                                    [@B36]
  *exsA*::Ω                                          PAK with *exsA* disrupted by insertion of cassette Ω; Sp^r^, Sm^r^                                                   [@B34]
  Δ*nadD2*                                           PAK with *nadD2* deleted                                                                                             This study
  Δ*nadD2-nrtR*                                      PAK with *nadD2-nrtR* operon deleted                                                                                 This study
  ΔPA4916                                            PAK with PA4916 deleted                                                                                              This study
  ΔPA4916*/att7*::PA4916                             ΔPA4916 with PA4916 inserted into the chromosome with mini-Tn7 insertion                                             This study
  Δ*vfr*                                             PAK with *vfr* gene deleted                                                                                          This study
  Δ*nrtR*Δ*vfr*                                      PAK with both *nrtR* and *vfr* gene deleted                                                                          This study
  **Plasmids**                                                                                                                                                            
  pDN19                                              Shuttle vector between *E. coli* and *P. aeruginosa*; Tc^r^                                                          [@B34]
  pMMB67EH                                           Shuttle vector between *E. coli* and *P. aeruginosa*; Amp^r^                                                         [@B35]
  pE1553                                             Promoterless pUCP20; Amp^r^                                                                                          [@B35]
  pE1553a                                            *cyaA*-flag with own promoter in pE1553; Amp^r^                                                                      This study
  pE1553b                                            *cyaB*-flag with own promoter in pE1553; Amp^r^                                                                      This study
  pE1553-*cpdA*                                      *cpdA*-flag with own promoter in pE1553; Amp^r^                                                                      This study
  pMMB67-*cyaA*                                      *cyaA*-flag cloned into pMMB67EH driven by tac promoter; Amp^r^                                                      This study
  pMMB67-*cyaB*                                      *cyaB*-flag cloned into pMMB67EH driven by tac promoter; Amp^r^                                                      This study
  pMMB67-*nadD2*                                     *nadD2* gene of PAK on pMMB67EH driven by tac promoter; Amp^r^                                                       This study
  pMMB67-4918-20                                     PA4918-20 gene of PAK on pMMB67EH driven by tac promoter; Amp^r^                                                     This study
  pMMB67EH-*cyaB*~217-463~-His                       CyaB~217-463~-His cloned into pMMB67EH driven by tac promoter; Amp^r^                                                This study
  pMMB67EH-*cyaA*-His                                CyaA-His cloned into pMMB67EH driven by tac promoter; Amp^r^                                                         This study
  pUCP24                                             Shuttle vector between *E. coli* and *P. aeruginosa*; Gm^r^                                                          [@B50]
  pUCP24-*nadD2*-Flag                                *nadD2*-Flag cloned into pUCP24; Gm^r^                                                                               This study
                                                                                                                                                                          
  *exsA*                                             *exsA* gene of PAK on pDN19 driven by *lac* promoter; Tc^r^                                                          [@B34]
  *lac*P1                                            *Lac*P1 promoter of *E. coli* fused to promoterless *lacZ* on pDN19*lacZ*Ω; Sp^r^, Sm^r^, Tc^r^                      This study
  pUC18T-mini-Tn7T-Gm                                mini-Tn7 base vector insertion into chromosome attTn7 site, Gm^r^                                                    [@B34]
  pUC18T-mini-Tn7T-P*~exsC~*-*exsCEBA*-Flag-*ExsD*   *exsCEBAD* gene with *exsA*-Flag tagged on pUC18T-mini-Tn7T driven by *exsC* promoter, Gm^r^                         This study
  pTNS3                                              Helper plasmid encoding Tn7 site-specific transposition pathway; Amp^r^                                              [@B10]
  pEX18Tc                                            Gene knockout vector; Tc^r^                                                                                          [@B10]
  pZF01                                              PA4916 gene deletion on pEX18Tc; Tc^r^                                                                               This study
  pEX18Tc-Δ*nadD2*                                   *nadD2* gene deletion on pEX18Tc; Tc^r^                                                                              This study
  pEX18Tc-Δ*nadD2-nrtR*                              *nadD2-nrtR* operon deletion on pEX18Tc; Tc^r^                                                                       This study
  pEX18Tc-Δ*vfr*                                     *vfr* gene deletion on pEX18Tc; Tc^r^                                                                                This study
  pEX18Tc-Δ*cyaA*                                    *cyaA* gene deletion on pEX18Tc; Tc^r^                                                                               This study
  pEX18Tc-Δ*cyaB*                                    *cyaB* gene deletion on pEX18Tc; Tc^r^                                                                               This study
  pZF02                                              PA4916 gene on pUC18T-mini-Tn7T-Tc; Tc^r^                                                                            This study
  pET16b                                             Expression vector, Kan^r^                                                                                            Novagen
  pET16b-*nadD2*                                     *nadD2* gene of PAK cloned into pET16b                                                                               This study
  pET28a                                             Expression vector, Kan^r^                                                                                            Novagen
  pET28a-*cyaB*~217-463~                             *cyaB* gene encoding amino acid positions 217--463 cloned into pET28a                                                This study

###### 

Primers used in this study.

  Primer                      Sequence 5^′^--3^′b^                                                         Source
  --------------------------- ---------------------------------------------------------------------------- ------------
  PA4916UF^a^                 CTCG[GAATTC]{.ul}TTCCAGACGAAGAAGTCGTAG                                       This study
  PA4916UR                    CTGC[TCTAGA]{.ul}CGTCACTCCTCTCTTCAGCCC                                       This study
  PA4916DF                    GAGC[TCTAGA]{.ul}CCTGCCGCGCTTGCTAGACG                                        This study
  PA4916DR                    GCCC[AAGCTT]{.ul}AAATCATCGAGTCGCTGGTCCCC                                     This study
  *nadD2*UF                   G[GAATTC]{.ul}GAAGACCTCCACCTCCAGTGTCG                                        This study
  *nadD2*UR                   GC[TCTAGA]{.ul}AGAGAGGAGTGACGATGAGTTCAG                                      This study
  *nadD2*DF                   GC[TCTAGA]{.ul}AGAAAATACCTTCCACTGCG                                          This study
  *nadD2*DR                   CCC[AAGCTT]{.ul}GAGCAGGTTCTGCACAATGC                                         This study
  *cyaA*UF                    G[GAATTC]{.ul}CGGCATCCGTTGTTCCGCGCGGAGATCCAG                                 This study
  *cyaA*UR                    GC[TCTAGA]{.ul}GGGCGTCCGGGCACAGGCAAGGCCAGGCG                                 This study
  *cyaA*DF                    GC[TCTAGA]{.ul}CCCAGCGCCGCACCGCGCGGGGCTCGAC                                  This study
  *cyaA*DR                    CCC[AAGCTT]{.ul}CGCCGGCGAAGGCAAGGTCTCGATCCTC                                 This study
  *cyaB*UF                    G[GAATTC]{.ul}GGAAAGTCAGGTCGGACGCTTCCGCGATG                                  This study
  *cyaB*UR                    GC[TCTAGA]{.ul}GCGCTGGAGAGGATCCCTGTGTATTTTCG                                 This study
  *cyaB*DF                    GC[TCTAGA]{.ul}GTTCGTCGAACGCCGCCGGCAGTTCGTCGCGCC                             This study
  *cyaB*DR                    CCC[AAGCTT]{.ul}CCGCTCGGCTGGGCCGCGCGGCGCTGGC                                 This study
  *vfr*UF                     CTCG[GAATTC]{.ul}GTAGCAGATGTCGTAGATGTTG                                      This study
  *vfr*UR                     CTGG[GGTACC]{.ul}CGAGTCCCGAAAGAATAAAG                                        This study
  *vfr*DF                     GAGG[GGTACC]{.ul}TGGTGCATGTGAAAGGAAAGAC                                      This study
  *vfr*DR                     GCCC[AAGCTT]{.ul}GCGACCAGCCTGCACGAG                                          This study
  PA4916PF                    GC[GAGCTC]{.ul}TCCTTGCTGCCCAGGCGCAGC                                         This study
  PA4916PR                    AA[CTGCAG]{.ul}TCACTTGCCGAAGGCGTGGCGGTGG                                     This study
  PA4916F                     AA[CTGCAG]{.ul}GGCGGTCTGAAGAGAGGAGTGACG                                      This study
  PA4916R                     CCC[AAGCTT]{.ul}TGCCGCACCCGTTTGTCAGG                                         This study
  *cyaA*Fown                  GG[GGTACC]{.ul}GCAGCGCATCCTCGCCAGCGGCGAG                                     This study
  *cyaA*Ftac                  GG[GGTACC]{.ul}CTGGCCTTGCCTGTGCCCGGACGCCC                                    This study
  *cyaA*R                     CCC[AAGCTT]{.ul}TCACTTGTCGTCATCGTCCTTGTAGTCTTGTTCCAGCAGCGCCTGGTTCAGCGCCG     This study
  *cyaB*Fown                  GG[GGTACC]{.ul}TCGCCGAGTTCTACCCCTACTACCTGCAG                                 This study
  *cyaB*Ftac                  GG[GGTACC]{.ul}ATACACAGGGATCCTCTCCAGCGCATG                                   This study
  *cyaB*R                     CCC[AAGCTT]{.ul}TCACTTGTCGTCATCGTCCTTGTAGTCGAGGATGACCTTGTCGCGCAGGCGTTCGG     This study
  *nadD2*OF                   G[GAATTC]{.ul}CAAGCACTTGTACTACAAAATTTCGCAG                                   This study
  *nadD2*ORHis                CCC[AAGCTT]{.ul}TCAATGATGATGATGATGATG GCCCGCCTGGCGTTCGCCGCCATAGCAGTG         This study
  *nadD2*ORFlag               CCC[AAGCTT]{.ul}TCACTTGTCGTCATCGTCCTTGTAGTC GCCCGCCTGGCGTTCGCCGCCATAGCAGTG   This study
  PA4918-20OF                 GC[TCTAGA]{.ul}CAAGCGGAGGCTTCCATGAATCGCCCCAGC                                This study
  PA4918-20OR                 CCC[AAGCTT]{.ul}TCAGGGCGCCTTCGGCAGTTCGCGCTTGTG                               This study
  *cpdA*Fown                  GG[GGTACC]{.ul}CGCAGGCCTCGCGCCGGGTCGCGCTGAGCG                                This study
  *cpdA*R                     CCC[AAGCTT]{.ul}TCACTTGTCGTCATCGTCCTTGTAGTCGTATCCGGCGGTGTCGTAGTCCACTTC       This study
  pET16b-*nadD2*F             TTTTCT[CCATGG]{.ul}GCCGTGATGAAATAAGTTCCCGGATTCGCCGA                          This study
  pET16b-*nadD2*R             CCG[CTCGAG]{.ul}GCCCGCCTGGCGTTCGCCGCCATAGCAG                                 This study
  pET28a-*cyaB*~217-463~F     CATG[CCATGG]{.ul}GCAAGAGCGTGCGCCTGGAAACCCAGC                                 This study
  pET28a-*cyaB*~217-463~R     CCG[CTCGAG]{.ul}GAGGATGACCTTGTCGCGCAGGCGTTCGG                                This study
  pMMB67EH-*cyaB*~217-463~F   GG[GGTACC]{.ul}AAGAGCGTGCGCCTGGAAACCCAGC                                     This study
  pMMB67EH-*cyaB*~217-463~R   CCC[AAGCTT]{.ul}TCAATGATGATGATGATGATG GAGGATGACCTTGTCGCGCAGGCGTTCGG          This study
  pMMB67EH-*cyaA*-hisR        CCC[AAGCTT]{.ul}TCAATGATGATGATGATGATG TTGTTCCAGCAGCGCCTGGTTCAGCGCCG          This study
  *cyaB*-SDF                  [AATTC]{.ul}ATACACAGGGATCCTCTCCAGCGCATG[GGTAC]{.ul}                          This study
  *cyaB*-SDR                  [C]{.ul}CATGCGCTGGAGAGGATCCCTGTGTAT[G]{.ul}                                  This study
  *lac*P1F                    [GAATTC]{.ul}GCCCAATACGCAAACCGC                                              This study
  *lac*P1R                    [GGATCC]{.ul}TCAGGCGAAAGGGGGATGTGCTG                                         This study
  p~Tn7L~                     ATTAGCTTACGACGCTACACCC                                                       This study
  p~glmS-up~                  CTGTGCGACTGCTGGAGCTGA                                                        This study
  qPCR primer                                                                                              
  q*cyaA*F                    CTTCAAGGAGCAGGTATTC                                                          This study
  q*cyaA*R                    TTCGAGATGGCGATAGAC                                                           This study
  q*cyaB*F                    GACCTGCTCAACAACTACC                                                          This study
  q*cyaB*R                    GACGAACTTGTCGATGGT                                                           This study
  q*cpdA*F                    GCGGATCGACCTGATTCTC                                                          This study
  q*cpdA*R                    CTGCGGAAGCGTGTGTAG                                                           This study
  qPA4918F                    GTCATCGAATACCTGAGG                                                           This study
  qPA4918R                    GTTCTTCACGCAGTAGTC                                                           This study
  q*nadD2*F                   GGTGTATTGCGAACCGATC                                                          This study
  q*nadD2*R                   GCTTCCAGCAGGTCGATG                                                           This study
  q*exsA*F                    CACGTCGGATAATCCTGATT                                                         This study
  q*exsA*R                    TAGCGGAGAGGCATGAATA                                                          This study
  q*prpL*F                    TATCGTATTTCGCCGACTCCC                                                        This study
  q*prpL*R                    GCGAGTTGCCGTTGTTCAG                                                          This study
  q*toxA*F                    CGAGATGGGCGACGAGTTG                                                          This study
  q*toxA*R                    TGATGACCGTGGGCTTGATGT                                                        This study
  q*rpsL*F                    CAAGCGCATGGTCGACAAGAG                                                        This study
  q*rpsL*R                    ACCTTACGCAGTGCCGAGTTC                                                        This study

a

F, forward; R, reverse; U, upstream of specific gene; D, downstream of specific gene; P, promoter of specific gene; own, native promoter of specific gene; tac, tac promoter; O, for over-expression plasmid construction; SD, Shine-Dalgarno sequence.

b

The underlines are the sites of restriction enzymes.

For the deletion of the *nrtR* gene, a 1,058 bp fragment immediately upstream of the *nrtR* start codon and a 1,308 bp fragment downstream of the *nrtR* stop codon were PCR amplified, digested with *Xba*I-*Eco*RI and *Hin*dIII-*Xba*I, respectively. The two fragments were then ligated into pEX18Tc that was digested with *Eco*RI and *Hin*dIII, resulting in pZF01. Similar manipulation was used to construct the *vfr*, *cyaA*, *cyaB*, *nadD2* and *nadD2*-*nrtR* deletion plasmids (detailed descriptions in [Supplementary Text](#SM1){ref-type="supplementary-material"}).

For the *nrtR* complementation plasmid, a 735 bp *nrtR*-containing fragment and a 465 bp fragment containing the promoter of *nadD2-nrtR* operon was amplified by PCR using PAK genomic DNA as template (primers shown in [Table 2](#T2){ref-type="table"}). The 735 bp and 465 bp PCR products were digested with *Pst*I/*Hin*dIII and *Pst*I/*Sac*I, respectively, and then ligated into the vector pUC18T-mini-Tn7T digested with *Hin*dIII and *Sac*I, resulting in pZF02. The plasmid was introduced into the Δ*nrtR* mutant by electroporation, along with the helper plasmid pTNS3 ([@B6]). Insertion of the *nrtR* gene into the chromosome was confirmed by PCR with primers P~Tn7L~ and P~glmS-up~ (primers shown in [Table 2](#T2){ref-type="table"}).

For overexpression of *nadD2*, a 643 bp *nadD2*-containing fragment with its putative Shine-Dalgarno (SD) sequence was PCR amplified using PAK genomic DNA as the template (primers shown in [Table 2](#T2){ref-type="table"}). The PCR product was digested with *Hin*dIII and *Eco*RI, and then ligated into a shuttle vector pMMB67EH which was digested with the same restriction enzymes, resulting in pMMB67-*nadD2*. pMMB67-*cyaA*, pMMB67-*cyaB*, pMMB67-4918-20 (encoding PA4918-4920), pMMB67EH-*cyaA*-His and pUCP24-*nadD2*-Flag were constructed with the similar manipulation. For the translational fusion of CyaB-Flag, *cyaB* with c-terminal Flag-tag and its upstream 500 bp region was PCR amplified using PAK genomic DNA as template (primers shown in [Table 2](#T2){ref-type="table"}), digested with *Hin*dIII and *Kpn*I, and then ligated into a promoterless pUCP20 ([@B35]), resulting in pE1553b. Similar manipulation was used to construct the CyaA and CpdA translational fusion plasmids pE1553a and pE1553-*cpdA*. For expression of catalytic domain of CyaB, open reading frame from amino acids 217 to 463, which corresponds to the catalytic domain of CyaB ([@B18]), was PCR amplified and digested with *Kpn*I-*Hin*dIII. This digested fragment, together with the annealed native SD region of *cyaB* (primers shown in [Table 2](#T2){ref-type="table"}), was cloned into the pMMB67EH, resulting in pMMB67EH-*cyaB*~217-463~-His. The *lac*P1 reporter was created as described by [@B18], except that the *lac*P1 promoter was cloned into the vector pDN19*lacZ*Ω.

Western Blot Assay
------------------

Overnight bacterial cultures were diluted 50-fold into fresh LB with or without 5 mM EGTA. The *P. aeruginosa* strains were then cultured to an OD~600~ of 1.0 in a shaking incubator. Then the bacterial pellet and supernatant were separated by centrifugation at 13,000 ×*g* for 2 min. Supernatant and pellet samples from equivalent number of bacterial cells were mixed with SDS-PAGE loading buffer, boiled for 10 min at 99°C, separated on 12% SDS-PAGE (15% SDS-PAGE for ExsA-Flag), transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore), and probed with a rabbit polyclonal antibody against ExoS, the RNA polymerase beta subunit (RNAP, Abcam) or a mouse monoclonal antibody against Flag (Sigma). Signals were detected with the ECL-plus kit (Millipore).

Cytotoxicity Assay
------------------

Bacterial cytotoxicity was determined by measuring detachment of mammalian cells after *P. aeruginosa* infection. 1.4 × 10^5^ HeLa cells ([@B10]) were seeded into each well of a 24-well plate and cultured in Dulbecco's modified Eagle's medium (DMEM) ([@B10]) containing 10% fetal bovine serum (FBS) ([@B10]), penicillin (100 μg/ml) and streptomycin (50 μg/ml) at 37°C with 5% CO~2~ the night before infection. Three hours before infection, cells were washed twice with phosphate-buffered saline (PBS) ([@B10]) and incubated in DMEM with 10% FBS. Log phase bacteria were used to infect HeLa cells at a multiplicity of infection (MOI) of 50. 50 mM cAMP was added into DMEM medium at the start of infection as indicated. Three hours post infection, the culture medium was removed from each well, and cells remaining attached were washed twice with PBS and stained with 500 μl 0.1% crystal violet in 10% ethanol for 15 min at 37°C. After discarding the staining solution, each well was washed twice with 1 ml distilled water and dried in air. A 200 μl volume of 95% ethanol was added into each well and incubated at room temperature for 30 min with gentle shaking. The dissolved crystal violet was subjected to measurement of absorbance at a wavelength of 590 nm.

Murine Acute Pneumonia Model
----------------------------

Bacterial overnight culture was inoculated into fresh LB medium with 50-fold dilution and grown to an OD~600~ of 1.0. The bacteria were collected by centrifugation and adjusted to 1 × 10^9^ CFU/ml in PBS. The exact number of bacteria for each inoculum was further determined by serial dilution and plating. Six- to eight-week-old female BALB/c mice were anesthetized with 100 μl chloral hydrate (7.5%) by intraperitoneal injection, and then intranasally inoculated with 10 μl of bacterial suspension in each nostril, giving a total infection bacterial number of approximately 2 × 10^7^ per mouse. After 12 h, the mice were sacrificed and the lungs were dissected and homogenized in 1% protease peptone. The bacterial loads were determined by serial dilution and plating. The experimental results were analyzed with the GraphPad Prism software.

Histology
---------

Twelve hours post infection with *P. aeruginosa* strains or sterile PBS, mouse lungs were removed and fixed with 10% paraformaldehyde. Fixed tissues were dehydrated in grades of ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Images were taken with a 20× objective lens.

Total RNA Isolation and RT-qPCR
-------------------------------

Bacterial overnight culture was inoculated into fresh LB medium with 50-fold dilution and grown to an OD~600~ of 1.0 under T3SS inducing and non-inducing conditions. Total RNA was isolated using an RNA prep Pure cell/Bacteria Kit (Tiangen Biotech). cDNA was synthesized with a PrimeScript Reverse Transcriptase and random primers (Takara). The cDNA was mixed with indicated primers (shown in [Table 2](#T2){ref-type="table"}) and SYBR premix Ex Taq II (Takara). The 30S ribosomal protein encoding gene *rpsL* was used as an internal control.

cAMP Assay
----------

Intracellular cAMP concentration was measured as previously described ([@B18]). Overnight bacteria were subcultured with 50-fold dilution into LB and grown to an OD~600~ of 1.0. 1.5 ml of the bacteria were harvested by centrifugation at 13,000 ×*g* for 2 min at 4°C and washed twice with cold 0.9 M NaCl. Pellets were resuspended in 100 μl of 0.1 M HCl and incubated on ice for 10 min with occasional vortex to lyse bacteria. Cellular debris were removed by centrifugation at 13,000 ×*g* for 5 min at 4°C and the supernatant was used to measure intracellular cAMP using an enzyme-linked immunosorbant assay (ELISA kit, Cayman Chemical) following the manufacturer's protocol for sample acetylation. For protein concentration determination, duplicate bacterial pellets were resuspended in 100 μl PBS and lysed by three freeze/thaw cycles followed by centrifugation at 13,000 ×*g* for 5 min at 4°C. The protein concentration of the supernatant was measured by the BCA protein assay (Beyotime Biotechnology). Assay values for cAMP levels were converted to intracellular concentrations per mg of protein.

Co-immunoprecipitation Assay
----------------------------

The co-immunoprecipitation assay was performed as previously described with minor modifications ([@B50]). Δ*nadD2* containing pUCP24-*nadD2*-Flag and pMMB67EH-*cyaB*~217-463~-His, pMMB67EH-*cyaA*-His or the empty vector pMMB67EH were grown overnight and diluted 50-fold into fresh LB medium. When the OD~600~ reached 0.6, 1 mM IPTG was added to induce the expression of CyaB~217-463~-His or CyaA-His at 16°C for 18 h. Bacteria were harvested by centrifugation at 5,000 *g* for 10 min, resuspended in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 0.5% NP-40, pH 8.0) and lysed by sonication. Supernatants were collected by centrifugation and incubated with Ni-NTA agarose beads for 1 h at 4°C. The beads were washed five times with the lysis buffer and boiled in the SDS-PAGE loading buffer. Samples were separated by 15% SDS-PAGE (CyaB~217--463~-His) or 12% SDS-PAGE (CyaA-His) and probed with an anti-Flag (Sigma) or anti-His antibody (Millipore).

Expression and Purification of CyaB~217-463~ or NadD2 Protein
-------------------------------------------------------------

The full-length *nadD2* or catalytic domain of the *cyaB* (amino acid positions 217--463) was PCR amplified from PAK chromosomal DNA with primers shown in [Table 2](#T2){ref-type="table"} and cloned into pET16b or pET28a, resulting in pET16b-*nadD2* or pET28a-*cyaB*~217-463~, respectively. Overnight culture of the *E. coli* strain BL21 (DE3) carrying pET16b-*nadD2* or pET28a-*cyaB*~217-463~ was subcultured with 50-fold dilution into 500 ml fresh LB medium at 37°C. When the OD~600~ reached 0.6, 1 mM IPTG was added to induce the protein expression at 16°C for 16 h. The bacteria were collected by centrifugation at 4°C, 5,000 ×*g*, for 20 min and resuspended in the lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 3 mM β-mercaptoethanol, 10 mM imidazole, 0.5% NP-40, pH 8.0), followed by sonication. The bacterial lysate was centrifuged at 15,000 rpm for 20 min at 4°C and the supernatant was applied to a Ni-NTA column (Qiagen). After the Ni-NTA column was washed four times with the lysis buffer containing 20 mM imidazole, the protein was eluted with 400 mM imidazole prepared in lysis buffer, followed by dialysis against enzymatic reaction buffer (100 mM NaCl, 20 mM Tris-HCl, 10 mM MgCl~2~). The purified protein was examined by SDS-PAGE, and quantified by BCA protein assay (Beyotime Biotechnology). Expression and purification of the LasR protein was described in a previous report ([@B15]).

Adenylate Cyclase Activity Assays
---------------------------------

The adenylyl cyclase activity assays were performed as previously described with minor modifications ([@B55]). Briefly, the reaction was performed in a 50 μl volume of 100 mM NaCl, 20 mM Tris-HCl (pH 7.0), 10 mM MgCl~2~, 5 mM ATP, and 2.0--2.25 μg purified CyaB protein, with or without 4.5--5.5 μg NadD2 or LasR protein. Samples were incubated at 30°C for 30 min, and then heated at 95°C for 4 min. The reaction mixtures were centrifuged at 15,000 rpm for 1 min, followed by filtration with a 0.22 μm filter, and then the cAMP concentrations were determined using an ELISA kit (Cayman Chemical) according to the manufacturers' instructions.

Other Methods
-------------

The *nrtR* gene knock out was generated by homologous recombination as described previously ([@B24]). β-galactosidase activity assay was conducted to determine the *lac*P1 promoter transcriptional activity as described before ([@B64]). The measurement of ATP concentration was carried out following the manufacturer's instruction (Beyotime Biotechnology). DNA manipulations were performed according to Molecular Cloning ([@B46]).

Statistical Analysis
--------------------

GraphPad Prism software was used to perform the statistical analyses. Results were analyzed by Mann--Whitney test or the Student's *t*-test (two-tailed).

Results
=======

The PA4916 Mutant Is Defective in T3SS-Dependent Cytotoxicity
-------------------------------------------------------------

In our previous screen for T3SS related genes, we found 23 genes that affect T3SS ([@B34]). Among them, PA0020, PA3202, PA4336, PA4630, PA4916, and PA4753 encoded products are annotated as hypothetical proteins^[1](#fn01){ref-type="fn"}^ ([@B62]) with unknown biological functions. To confirm their relationships with T3SS as well as exclude strain specific phenotype, mutants with Transposon (Tn) insertions in the PA0020, PA4336, PA4916, and PA4753 from the PA14 Non-Redundant Transposon Insertion Mutant Set (PA14NR Set) were selected for further tests ([@B36]), however, mutant of the PA3202 or PA4630 is not available in the PA14NR Set. As T3SS plays a major role in cytotoxicity ([@B22]), we infected HeLa cells with those mutants. Detached cells due to cytotoxicity were washed away, and the remaining cells were observed and quantified by crystal violet staining. Similar to wild type PA14, mutant strains of PA0020, PA4336 and PA4753 detached most of the HeLa cell within 3 h, whereas the ΔPA4916 mutant caused minimal detachment ([Figure 1A](#F1){ref-type="fig"}), indicating a defective cytotoxicity.

![Cytotoxicity of indicated strains and the role of PA4916 in the expression and secretion of ExoS. **(A,B)** HeLa cells were infected with indicated strains at a MOI of 50. Three hours post infection, cells attached to the 24-well plate were washed with PBS and stained with crystal violet. The cell associated crystal violet was dissolved in ethanol and quantified by measuring OD~590~. HeLa cells with no bacterial infection served as a control. **(C)** Bacteria were cultured to an OD~600~ of 1.0 in LB with or without 5 mM EGTA. Proteins in supernatants and pellets from equivalent bacterial cells were loaded onto SDS-PAGE gels and probed with an antibody against ExoS or RNA polymerase beta subunit. S, supernatant; P, pellet.](fmicb-10-00085-g001){#F1}

To further confirm the role of PA4916, the whole open reading frame of PA4916 was deleted from the PAK chromosome via DNA recombination, resulting in ΔPA4916. As shown in [Figure 1B](#F1){ref-type="fig"}, this mutant also displayed a reduced cytotoxicity, which was restored nearly to that of wild type by complementation with an intact PA4916 gene.

PA4916 Is Required for ExoS Expression and Involved in Pathogenesis of *P. aeruginosa*
--------------------------------------------------------------------------------------

To verify whether the reduced cytotoxicity is due to a defective T3SS, wild type PAK and the ΔPA4916 mutant were grown under T3SS inducing and non-inducing conditions (in the presence and absence of 5 mM EGTA), and the expression and secretion of ExoS were examined by Western blot. Under T3SS inducing condition, the expression and secretion of ExoS were highly induced in the wild type PAK, however, faint ExoS was observed in the pellet and not detected in the supernatant of ΔPA4916. Complementation with an intact PA4916 gene restored the expression and secretion of ExoS in the ΔPA4916 mutant background ([Figure 1C](#F1){ref-type="fig"}).

The T3SS plays an important role in acute infections ([@B45]). The functional connection between PA4916 and the T3SS promoted us to examine its role in the pathogenesis of a mouse acute pneumonia model. Six- to eight-week-old female BALB/c mice were infected intranasally with 2 × 10^7^ CFU of wild type PAK or the ΔPA4916 mutant. Twelve hours post infection, lungs were isolated and homogenized. Bacterial loads were determined by serial dilution and plating. Compared to the wild type PAK strain, the number of ΔPA4916 mutant isolated from the lungs was significantly lower ([Figure 2A](#F2){ref-type="fig"}), suggesting a defective virulence. Reduced growth rate in the ΔPA4916 mutant might lead to a reduced virulence; however, the deletion mutant ΔPA4916 showed a rate of growth indistinguishable from that of wild type PAK strain when cultured in LB medium ([Supplementary Figure S1](#SM2){ref-type="supplementary-material"}).

![Role of PA4916 in the mouse acute pneumonia model. **(A)** Bacteria were grown to an OD~600~ of 1.0. Female BALB/c mice (6- to 8-week-old) were inoculated intranasally with 2 × 10^7^ CFU of wild-type PAK or its isogenic ΔPA4916 mutant. After 12 h, the mice were sacrificed, and the lungs were dissected and homogenized. The bacteria load in each lung was determined by serial dilution and plating. ^∗∗∗^*P* \< 0.001, by Mann--Whitney test. **(B--D)** Pathology sections of lungs infected with indicated strains. Mice were infected with 2 × 10^7^ CFU of wild type PAK **(B)**, ΔPA4916 mutant **(C)** or sterile PBS **(D)**. Lungs from infected mice were removed, fixed, sectioned, and stained with hematoxylin and eosin. Images were taken with a 20× objective lens.](fmicb-10-00085-g002){#F2}

To further validate the role of PA4916 in the bacterial virulence, the lungs infected by wild type or ΔPA4916 mutant were observed following histological section and staining. Lungs from mice infected with PAK for 12 h had significant neutrophil infiltration, edema and tissue damage ([Figure 2B](#F2){ref-type="fig"}). Most of the airways in the lungs of these mice were completely occluded with neutrophil and pyocyte infiltration. In contrast, infections with the ΔPA4916 mutant showed significantly reduced inflammatory characteristics ([Figure 2C](#F2){ref-type="fig"}), with substantially fewer neutrophils present in the alveolar spaces, compared to the infections with PAK, though the inflammation was more intense compared to the PBS instilled control ([Figure 2D](#F2){ref-type="fig"}). The extent of inflammation caused by the ΔPA4916 mutant correlated with the ability of this strain to colonize the lungs of infected mice. A recent study has also shown that PA4916 plasposon mutagenesis abrogated virulence of a robust mucoid *P. aeruginosa* cystic fibrosis airway isolate and named PA4916 as NrtR ([@B43]). So we further explored the regulation mechanism of PA4916 on the T3SS and referred PA4916 as NrtR hereafter.

Plasmid-Expressed *exsA* Restores the T3SS in Δ*nrtR* Mutant
------------------------------------------------------------

ExsA is a central regulator of T3SS ([@B26]). To investigate whether NrtR regulates T3SS through ExsA, total RNA was isolated and the mRNA levels of *exsA* were compared between PAK and the Δ*nrtR* mutant. As shown in [Figure 3A](#F3){ref-type="fig"}, under both T3SS inducing and non-inducing conditions, the *exsA* mRNA level was significantly decreased in the Δ*nrtR* mutant, which was restored by complementation with a *nrtR* gene. To further confirm the role of *exsA* in the NrtR mediated regulation of T3SS, a plasmid carrying an *exsA* gene driven by a *lac* promoter ([@B56]; [@B34]) was introduced into the Δ*nrtR* or an *exsA*::Ω mutant. Plasmid-expressed ExsA restored the expression of ExoS in both the *exsA*::Ω and Δ*nrtR* mutants ([Figure 3B](#F3){ref-type="fig"}). Although the expression of ExoS was similar in the *exsA*::Ω/*exsA* and Δ*nrtR/exsA* mutants, the ExsA amount in the Δ*nrtR/exsA* mutant was much less than that in the *exsA*::Ω/*exsA* mutant ([Figure 3C](#F3){ref-type="fig"}). Considering the fact that *lac* promoter is controlled by catabolite repression and T3SS is regulated by the cAMP-Vfr signaling pathway, the decreased ExsA amount may indicate a reduced cAMP level in the Δ*nrtR* mutant strain.

![Plasmid mediated expression of *exsA* restored T3SS in the Δ*nrtR* mutant. **(A)** The relative *exsA* mRNA levels in PAK, the Δ*nrtR* mutant and Δ*nrtR*/*att7*:: *nrtR* strain. Total RNA was isolated under T3SS inducing and non-inducing conditions and the *exsA* mRNA levels were determined by real-time PCR using *rpsL* as the internal control. ^∗^*P* \< 0.05, ^∗∗^*P* \< 0.01, by Student's *t*-test. **(B)** Bacteria harboring an *exsA*-Flag driven by a *lac* promoter or the empty vector pDN19 were grown to an OD~600~ of 1.0 in LB with or without EGTA. Proteins from equivalent number of bacterial cells of indicated strains were separated on SDS-PAGE and probed with an anti-ExoS antibody or an anti-RNA polymerase beta subunit antibody. Expression levels of the ExsA-Flag were determined with an anti-FLAG antibody **(C)**.](fmicb-10-00085-g003){#F3}

The cAMP-Vfr Signaling Pathway Is Involved in NrtR Mediated Regulation of T3SS
------------------------------------------------------------------------------

To investigate if the cAMP-Vfr signaling pathway is involved in the NrtR mediated T3SS regulation, we initially compared the cAMP contents between PAK and Δ*nrtR* mutant with a transcriptional fusion of the *lac*P1 promoter and a *lacZ* gene (*lac*P1-*lacZ*), whose expression has been shown to correlate to intracellular cAMP levels ([@B18]). A Δ*vfr* mutant was included as a control of Δ*nrtR* mutant ([@B18]). As expected, the LacZ levels in the Δ*nrtR* mutant under both T3SS inducing and non-inducing conditions were lower than those of the PAK strain, which were restored by complementation with a *nrtR* gene ([Figure 4A](#F4){ref-type="fig"}). This result was further confirmed by direct measurement of intracellular cAMP levels with a cAMP ELISA detection kit ([Figure 4B](#F4){ref-type="fig"}). A previous study has shown that exogenous addition of 50 mM cAMP restored the phenotypes of an adenylate cyclase mutant of *P. aeruginosa* ([@B18]). Therefore, we constructed a c-terminus Flag-tagged ExsA driven by its native promoter and examined the effect of exogenous addition of 50 mM cAMP on the T3SS in the Δ*nrtR* mutant. As shown in [Figure 4C,D](#F4){ref-type="fig"}, exogenous addition of cAMP restored the *exsA* expression levels in the Δ*nrtR* mutant at both transcriptional and protein levels, but not in the Δ*vfr* mutant. Furthermore, the expression and secretion of ExoS and cytotoxicity of Δ*nrtR* were restored to wild type level by exogenous addition of 50 mM cAMP ([Figure 4E,F](#F4){ref-type="fig"}). As expected, exogenous addition of 50 mM cAMP did not affect the expression and secretion of ExoS and cytotoxicity of the Δ*vfr* mutant ([Figure 4E,F](#F4){ref-type="fig"}). In addition, Δ*nrtR*Δ*vfr* double mutant strain was constructed. The *exsA* transcriptional level, as well as the expression and secretion of ExoS of Δ*nrtR*Δ*vfr* were compared with Δ*nrtR* and Δ*vfr* mutant. The results showed that, like Δ*vfr* mutant, exogenous cAMP addition did not affect *exsA* transcriptional level, as well as the expression and secretion of T3SS in Δ*nrtR*Δ*vfr* double mutant strain ([Figure 4C,E](#F4){ref-type="fig"}). These results demonstrate that NrtR regulates T3SS through the cAMP-Vfr signaling pathway in *P. aeruginosa*, likely by altering the intracellular cAMP level. Furthermore, twitching motility and the expression levels of *toxA* and *prpL* which were demonstrated to be affected by cAMP were examined in the Δ*nrtR* mutant ([@B63]). As expected, the expression levels of both *toxA* and *prpL* were decreased significantly in Δ*nrtR* mutant ([Supplementary Figure S2](#SM3){ref-type="supplementary-material"}). However, the twitching motility of the Δ*nrtR* mutant showed no detectable change compared to the wild type PAK strain ([Supplementary Figure S2](#SM3){ref-type="supplementary-material"}).

![Decreased cAMP contributed to the T3SS defect in the Δ*nrtR* mutant. **(A,B)** cAMP level was decreased in Δ*nrtR* mutant and Δ*vfr* mutant. β-galactosidase assay was used to examine the transcriptional activity of *lac*P1 promoter fused to a *lacZ* gene in indicated strains under T3SS inducing and non-inducing conditions **(A)**. **(B)** Intracellular cAMP levels were measured using an ELISA kit. Error bars represent standard deviations. ^∗^*P* \< 0.05, ^∗∗^*P* \< 0.01, ^∗∗∗^*P* \< 0.001 by Student's *t-*test. **(C--F)** Exogenous addition of cAMP recovers the expression of ExsA, ExoS and cytotoxicity of the Δ*nrtR* mutant, while not of or Δ*nrtR*Δ*vfr* mutant. **(C)** Relative mRNA levels of *exsA* in indicated strains with or without cAMP addition at the beginning of subculture in the presence or absence of 5 mM EGTA, with *rpsL* as an internal control. ns, not significant, ^∗^*P* \< 0.05, ^∗∗∗^*P* \< 0.001 by Student's *t-*test. **(D)** Indicated strains containing an *exsA*-Flag driven by its native promoter were grown at 37°C with or without 5 mM EGTA and 50 mM cAMP as indicated. Protein samples from equal number of bacteria were separated by SDS-PAGE and probed with an anti-Flag antibody or an anti-RNA polymerase beta subunit antibody. **(E)** Expression of ExoS in indicated strains were grown with or without 5 mM EGTA and 50 mM cAMP. The protein levels were detected with an antibody against ExoS or RNA polymerase beta subunit. S, supernatant; P, pellet. **(F)** Cytotoxicity of indicated strains in the presence or absence of 50 mM cAMP. HeLa cells were infected with indicated strains at a MOI of 50. 50 mM final concentration of cAMP was added to DMEM medium as indicated. Three hours post infection, cells attached to the 24-well plate were washed with PBS and stained with crystal violet. The cell associated crystal violet was dissolved in ethanol and quantified by measuring OD~590~. HeLa cells with no bacterial infection (blank and blank+50 mM cAMP) served as a control. ^∗∗^*P* \< 0.01, by Student's *t-*test.](fmicb-10-00085-g004){#F4}

Decreased Intracellular cAMP Is Not Caused by Altered Expression of CyaA or CyaB in the Δ*nrtR* Mutant
------------------------------------------------------------------------------------------------------

In *P. aeruginosa*, cAMP is synthesized by the adenylate cyclases CyaA and CyaB ([@B63]; [@B39]). To explore if the reduced cAMP level is caused by decreased expression of the adenylate cyclases in Δ*nrtR* mutant, we determined the expression levels of the two genes by RT-qPCR. As shown in [Figure 5A](#F5){ref-type="fig"}, the mRNA level of *cyaA* was lower in the Δ*nrtR* mutant than those in both wild type PAK and the complemented strain, while the *cyaB* was similar among these three strains. To confirm this observation, C-terminal Flag-tagged CyaA or CyaB driven by their respective native promoters, were transformed into PAK and the Δ*nrtR* mutant, and their protein expression levels were examined by Western blot assay. Consistent with the RT-qPCR result, similar level of CyaB-Flag, while slightly lower level of CyaA-Flag protein were observed in the Δ*nrtR* mutant compared to that in PAK ([Figure 5B](#F5){ref-type="fig"}). To further understand if the observed slight reduction of the adenylate cyclase is the cause of the decreased cAMP in Δ*nrtR* mutant, the functional C-terminal Flag tagged CyaA or CyaB was driven by a *tac* promoter and transformed into the Δ*nrtR* mutant. Similar levels of expression and secretion of ExoS were observed by Western blot assay between Δ*nrtR*/pMMB67EH and Δ*nrtR*/pMMB67-*cyaA* or Δ*nrtR*/pMMB67-*cyaB* ([Figure 5C,D](#F5){ref-type="fig"}). These results suggest that the reduced cAMP level in the Δ*nrtR* strain is not due to alteration of adenylate cyclases expression.

![The decrease of cAMP is not caused by changed expression of CyaA, CyaB or CpdA. **(A)** Relative mRNA levels of *cyaA*, *cyaB*, and *cpdA*. Total RNA of indicated strains was isolated and the mRNA levels of these genes were determined by real time PCR with *rpsL* serving as an internal control. ns, not significant, ^∗^*p* \< 0.05 by Student's *t*-test. **(B)** Indicated strains containing a *cyaA*-Flag (*cyaB*-Flag or *cpdA*-Flag) driven by its native promoter were grown at 37°C with or without 5 mM EGTA until OD~600~ of 1.0. Samples from equal number of bacteria were separated by SDS-PAGE and probed with an anti-Flag antibody or an anti-RNA polymerase beta subunit antibody. **(C,D)** Indicated strains containing a *cyaA*-Flag or *cyaB*-Flag driven by a *tac* promoter or the empty vector pMMB67EH were grown to an OD~600~ of 1.0 in LB with 1 mM IPTG with or without 5 mM EGTA. Proteins from equivalent bacterial cells of indicated strains were separated by SDS-PAGE and probed with an anti-ExoS antibody, an anti-Flag antibody or an anti-RNA polymerase beta subunit antibody. S, supernatant; P, pellet.](fmicb-10-00085-g005){#F5}

Decreased Intracellular cAMP Is Not Caused by Altered Expression of CpdA in the Δ*nrtR* Mutant
----------------------------------------------------------------------------------------------

Since we did not get evidence for an effect of altered expression of adenylate cyclases on the T3SS in the Δ*nrtR* mutant, we next wanted to investigate if the degradation of cAMP was affected in the Δ*nrtR* mutant. To date, CpdA is the only known phosphodiesterase degrading cAMP in *P. aeruginosa* ([@B17]). One possibility for the observed decrease in cAMP levels and subsequent T3SS in the Δ*nrtR* mutant could be that CpdA expression is upregulated in the Δ*nrtR* mutant. This would in turn result in an increased degradation of cAMP and a decrease in T3SS. To test this possibility, total RNA was isolated and mRNA levels of *cpdA* were compared among wild type PAK, Δ*nrtR* mutant and Δ*nrtR* complemented strain by RT-qPCR. As shown in [Figure 5A](#F5){ref-type="fig"}, the mRNA level of *cpdA* was lower, rather than higher, in the Δ*nrtR* mutant than those in both wild type PAK and complemented strain. To confirm this observation, C-terminal Flag-tagged CpdA driven by its native promoter, was transformed into PAK and the Δ*nrtR* mutant, and their protein expression levels were examined by Western blot assay. Consistent with the RT-qPCR result, lower CpdA-Flag protein level were observed in the Δ*nrtR* mutant than that in PAK ([Figure 5B](#F5){ref-type="fig"}). These results thus suggest that decreased cAMP and subsequent T3SS does not occur through increased CpdA-mediated cAMP degradation.

Increased NadD2 Level Contributes to the Decreased cAMP and T3SS in the Δ*nrtR* Mutant
--------------------------------------------------------------------------------------

*NrtR* encodes a transcriptional regulator which binds to the DNA intergenic region between the *nadD2*- *nrtR* and PA4918-4920 operons to repress their expression ([@B43]). RT-qPCR assay showed that the mRNA levels of *nadD2* and PA4918 in the Δ*nrtR* mutant were much higher than those in PAK ([Figure 6A](#F6){ref-type="fig"} and [Supplementary Figure S3A](#SM4){ref-type="supplementary-material"}). Thus, we examined whether NadD2 or PA4918-4920 operon was involved in the NrtR mediated regulation of T3SS. Overexpression of *nadD2* in wild type PAK reduced the expression and secretion of the ExoS, as well as the intracellular cAMP level ([Figure 6B,C](#F6){ref-type="fig"}). However, overexpression of PA4918-4920 operon in PAK showed no inhibitory effect on the expression and secretion of T3SS ([Supplementary Figure S3B](#SM4){ref-type="supplementary-material"}). In addition, deletion of *nadD2* in wild type PAK did not affect the expression and secretion of ExoS, whereas deletion of *nadD2* restored the expression and secretion of ExoS in the Δ*nrtR* mutant ([Figure 6D](#F6){ref-type="fig"}). These results indicate that the increased NadD2 level might be responsible for the decreased cAMP and T3SS in the Δ*nrtR* mutant. NadD2 is an ATP consuming enzyme ([@B43]). Adenylate cyclases catalyzed the synthesis of cAMP from ATP. Reduced ATP availability due to increased NadD2 level might result in the decreased cAMP level in PAK/pMMB67-*nadD2*. However, the PAK/pMMB67-*nadD2* strain displayed a higher ATP level than the strain with an empty vector (PAK/pMMB67EH) ([Figure 6E](#F6){ref-type="fig"}).

![Increased expression of NadD2 contributes to the decreased intracellular cAMP and expression of T3SS in the Δ*nrtR* mutant. **(A)** Relative mRNA levels of *nadD2*. Total RNA of indicated strains was isolated and mRNA levels of *nadD2* were determined by real time PCR with *rpsL* serving as an internal control. Data represents the mean ± standard deviation. ^∗^*p* \< 0.05 by Student's *t*-test. **(B)** PAK containing *nadD2* driven by a *tac* promoter or the empty vector pMMB67EH were grown to an OD~600~ of 1.0 in LB containing 1 mM IPTG with or without 5 mM EGTA. Proteins from equivalent bacterial cells of indicated strains were separated by SDS-PAGE and probed with an antibody against ExoS or RNA polymerase beta subunit. S, supernatant; P, pellet. **(C)** The cAMP levels were decreased in the PAK/pMMB67-*nadD2* strain. β-Galactosidase assay was used to examine the LacZ level driven by the *lac*P1 promoter in indicated strains with 1 mM IPTG induction. Error bars represent standard deviations. ^∗∗∗^*P* \< 0.001 by Student's *t-*test. **(D)** Indicated bacteria were cultured to an OD~600~ of 1.0 in LB with or without 5 mM EGTA. Proteins in supernatants and pellets from equivalent bacterial cells were loaded onto SDS-PAGE gels and probed with an antibody against ExoS or RNA polymerase beta subunit. S, supernatant; P, pellet. **(E)** ATP levels in indicated strains. ^∗∗∗^*P* \< 0.001 by Student's *t-*test. **(F)** Interaction between NadD2 and CyaB~217-463~ or CyaA. Δ*nadD2* carrying pUCP24-*nadD2*-Flag with pMMB67EH-*cyaB*~217-463~-His, pMMB67EH-*cyaA*-His or pMMB67EH were grown to an OD~600~ of 0.6 and incubated with 1 mM IPTG for 18 h at 16°C. Bacteria were lysed and subjected to chromatography with Ni-NTA beads. His-tagged CyaB~217--463~ or CyaA and FLAG-tagged NadD2 were detected by Western blot assay. **(G)** Inhibition of CyaB adenylyl cyclase activity by NadD2. 2.0--2.25 μg CyaB was incubated with 5 mM ATP. 4.5--5.5 μg NadD2 or LasR was added as indicated. After 30 min at 30°C, the cAMP levels were measured using an ELISA kit. Error bars represent standard deviations. ^∗∗^*P* \< 0.01 by Student's *t-*test.](fmicb-10-00085-g006){#F6}

Since the decreased cAMP level is not caused by the changed expression of the adenylate cyclases in the Δ*nrtR* mutant, it is possible that their enzymatic activities might be affected by NadD2. To test this possibility, a CyaA-His or a CyaB~217-463~-His fusion protein (the catalytic domain of CyaB without the transmembrane region), was constructed and overexpressed in Δ*nadD2* carrying a NadD2-Flag fusion protein. The His-Tagged CyaA and CyaB~217-463~ were purified with Ni-affinity chromatography. As shown in [Figure 6F](#F6){ref-type="fig"}, NadD2-Flag was co-purified with the CyaB~217-463~-His, but not with CyaA-His.

Next, we examined whether NadD2 directly represses the adenylyl cyclase activity of CyaB. CyaB~217-463~ and NadD2 were expressed in *E. coli* and purified ([Supplementary Figure S4A](#SM5){ref-type="supplementary-material"}). The purified NadD2 was not contaminated by ATPase or phosphodiesterase ([Supplementary Figures S4B,C](#SM5){ref-type="supplementary-material"}). The purified catalytic domain of CyaB (CyaB~217-463~) was incubated with ATP with or without NadD2 and the cAMP level was determined with a cAMP ELISA kit (Cayman Chemical). An unrelated protein LasR was used as a negative control. As shown in [Figure 6G](#F6){ref-type="fig"}, the cAMP level was reduced by the presence of NadD2 but not LasR, indicating a repression of the adenylyl cyclase activity by the NadD2. These results suggest that NadD2 might suppress the enzymatic activity of CyaB.

Discussion
==========

In the present study, we identified that NrtR is required for the T3SS and involved in pathogenesis of *P. aeruginosa* in a murine acute pneumonia model. Further experimental results demonstrated that NrtR regulates expression of T3SS through the cAMP/Vfr signaling system. *NadD2*, which is in the same operon of *nrtR* and repressed by NrtR, is involved in the NrtR mediated regulation of the T3SS by inhibition of adenylyl cyclase activity of CyaB in *P. aeruginos*a.

In our previous study, we identified PA0020, PA4336, PA4916 and PA4753 as T3SS related genes by screening Tn insertion mutant library of PAK with an ELISA assay ([@B34]). While in this study, only the *nrtR* mutant in PA14 background displayed a significant change in T3SS related cytotoxicity. The previous screening by ELISA was performed with EGTA as the T3SS inducing condition, whereas in the cytotoxicity assay, contact with host cell is the inducing signal. The different results in the two tests indicate that PA0020, PA4336 and PA4753 might play different roles in bacterial response to the two signals. In addition, the differences of the Tn insertion sites in each of the genes might cause different effects on the gene function, thus leading to different phenotypes. Further studies are required to fully understand the functions of those genes.

*NrtR* encodes a putative ADP-ribose pyrophosphatase with a Nudix hydrolase domain. Nudix protein specifically hydrolyzes varieties of substrates with a common structure of a [Nu]{.ul}cleoside [di]{.ul}phosphates linked to moiety, [X]{.ul}, such as (d) NTPs, coenzymes and capped RNAs ([@B42]; [@B5]; [@B52]). It has been reported that some of these proteins may play important regulatory roles in response to stress, invasion to host cell and in pathogenesis ([@B13]; [@B59]; [@B37]). A recent study reported that *nrtR* of *P. aeruginosa* PA14 encodes a transcriptional repressor, which has no ADP-ribose pyrophosphatase activity and can bind to the DNA intergenic region between *nadD2-nrtR* and PA4918-4920 operons to repress their expression ([@B43]). Consistent with this, our study demonstrated that the transcriptional levels of *nadD2* and PA4918 increased 11- and 46-fold, respectively, in the Δ*nrtR* mutant ([Figure 6A](#F6){ref-type="fig"} and [Supplementary Figure S3A](#SM4){ref-type="supplementary-material"}). Overexpression of NadD2 repressed the expression of T3SS in PAK strain, whereas overexpression of PA4918-4920 in PAK did not show any inhibitory effect on the expression of T3SS ([Supplementary Figure S3B](#SM4){ref-type="supplementary-material"}), thus NrtR positively controls the expression of T3SS in *P. aeruginosa* through repression of *nadD2* specifically. *nadD2* deletion restored the expression and secretion of T3SS in Δ*nrtR* mutant, but had no influence on the expression and secretion of T3SS in wild type PAK strain, indicating that the altered expression level of NadD2 is not sufficient to repress the expression of T3SS in wild type PAK strain.

*NadD2*, encoding a nicotinate mononucleotide adenylyltransferase, is located downstream of the transcriptional repressor NrtR and upstream of the PA4918-4920 (*pncA*-*pncB1*-*nadE*) operon. The interaction between NadD2 and other proteins were predicted using the STRING database^[2](#fn02){ref-type="fn"}^), a pre-computed database to predict both physical and functional interactions. The high confidence score (\>0.7) exist between NadD2 and proteins encoded by its neighboring genes on the chromosome. It has been demonstrated that *pncA*, *pncB1*, and *nadE* encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase, respectively ([@B43]). Therefore, NrtR negatively regulates the salvage pathway I of the NAD biosynthesis. In addition, the co-immunoprecipitation assay in the present study suggests that NadD2 interacts with CyaB, but not with CyaA, indicating that NadD2 influences the cAMP production through inhibition of the adenylyl cyclase activity of CyaB. However, the inability of functional CyaA to complement the expression of T3SS in Δ*nrtR* mutant suggests that NrtR may also influence the adenylyl cyclase activity of CyaA indirectly.

cAMP, as an important second messenger, has been shown to regulate the T3SS, exotoxin A, protease IV and type IV pili biosynthesis ([@B63]). As the cAMP was decreased in the Δ*nrtR* mutant, we also tested the twitching motility and the expression levels of *toxA* and *prpL* in the Δ*nrtR* mutant. As expected, the expression levels of both *toxA* and *prpL* were decreased significantly in Δ*nrtR* mutant. However, the twitching motility of the Δ*nrtR* mutant showed no detectable change compared to the wild type PAK strain ([Supplementary Figure S2](#SM3){ref-type="supplementary-material"}). It might be possible that the T3SS is more sensitive to changes in the cAMP level than twitching motility. The generation of cAMP in *P. aeruginosa* relies on CyaA and CyaB, while its degradation relies on phosphodiesterase CpdA ([@B17]). Expression of CpdA is lower in the Δ*nrtR* mutant, which may be caused by decreased levels of intracellular cAMP, as the *cpdA* can be directly activated by Vfr in response to intracellular cAMP as a feedback loop ([@B17]).

The intracellular cAMP levels are modulated by calcium concentration ([@B28]), and the EGTA-induced calcium depletion has been shown to increase intracellular cAMP levels in *P. aeruginosa* ([@B63]). Consistent with these results, our study demonstrated that intracellular cAMP levels were increased in both wild type PAK and Δ*nrtR* mutant under EGTA inducing condition, even in the Δ*vfr* mutant. Considering that EGTA addition did not affect the expression of adenylate cyclases in both PAK and Δ*nrtR* mutant, this increase suggests that EGTA might increase the activity of adenylate cyclases. The fact that the activation of *lac*P1 was Vfr-dependent in *P. aeruginosa* might result in no obvious increase of reporter activity in Δ*vfr* mutant under EGTA inducing condition ([@B18]). In contrast to the almost eliminated reporter activity, the Δ*vfr* mutant displayed an approximately 50% reduction in intracellular cAMP compared to the PAK strain, which is consistent with previous report ([@B18]).

The T3SS of *P. aeruginosa* can be induced by EGTA addition (calcium depletion) ([@B63]). Previous studies reported that most secretion apparatus component and effector genes were regulated by calcium depletion except for the ExsA, whose expression was relatively unaffected ([@B63]). Inconsistent with this finding, in our study, the mRNA levels of *exsA* showed a significant increase under EGTA inducing condition. This may be caused by the different experimental methods used in these two studies (transcriptomic analysis vs. RT-qPCR). Furthermore, the ETGA-dependent increase of ExsA protein levels was demonstrated previously ([@B30]; [@B27]). However, in contrast to the previous reports and our transcriptional levels of *exsA*, in this study, the protein levels of ExsA were not affected by EGTA addition both in PAK and the Δ*nrtR* mutant. In the previous studies, *P. aeruginosa* was grown in Trypticase soy broth, while we cultured bacteria in L-broth medium. The difference between the transcriptional levels and protein levels of *exsA* might be due to the sensitivity of RT-qPCR or some unknown post-transcriptional regulatory mechanism.

Recently, [@B43] demonstrated that NrtR regulates nicotinamide adenine dinucleotide (NAD) biosynthesis and is involved in the virulence of a *P. aeruginosa* clinical isolate. Our study revealed novel functions of NrtR and NadD2 in the cAMP biosynthesis in *P. aeruginosa*.

Author Contributions
====================

YJ, WW, and SJ conceived and designed the experiments, and wrote the paper. YJ, MZ, FZ, QP, YW, QZ, and CL performed the experiments. YJ, WW, FB, ZC, and SJ analyzed the data.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This work was supported by National Science Foundation of China (31600110 and 31670130), Science and Technology Committee of Tianjin (17JCQNJC09200, 15JCZDJC33000, and 15JCYBJC53900), Program of International S&T Cooperation (2015DFG32500), and the State Key Laboratory of Medicinal Chemical Biology (2017005).

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<https://string-db.org/>

Supplementary Material
======================

The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2019.00085/full#supplementary-material>

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Growth curve of PAK and the ΔPA4916 mutant.

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Role of NrtR in twitching motility or expression of *prpL* and *toxA* in *P. aeruginosa*. **(A)** Twitching motilities of the indicated strains were examined on 1% LB agar with a Δ*vfr* mutant serving as a control. The twitching zones were visualized with 0.1% crystal violet staining. **(B)** Relative mRNA levels of *prpL* and *toxA*. Total RNA of indicated strains was isolated and mRNA levels of *prpL* and *toxA* were determined by real time PCR with *rpsL* serving as an internal control. Data represents the mean ± standard deviation. ^∗∗^*p* \< 0.01, ^∗∗∗^*p* \< 0.001 by Student's *t*-test.

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PA4918 is not involved in the regulation of T3SS. **(A)** Relative mRNA levels of PA4918. Total RNA of indicated strains was isolated and mRNA levels of PA4918 were determined by real time PCR with *rpsL* serving as an internal control. Data represents the mean ± standard deviation. ^∗∗∗^*p* \< 0.001 by Student's *t*-test. **(B)** PAK containing the PA4918-20 operon driven by a *tac* promoter or the empty vector pMMB67EH were grown to an OD~600~ of 1.0 in LB with 1 mM IPTG with or without 5 mM EGTA. Proteins from equivalent bacterial cells of indicated strains were separated by SDS-PAGE and probed with an anti-ExoS antibody or an anti-RNA polymerase beta subunit antibody. S, supernatant; P, pellet.

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Coomassie blue staining of purified CyaB~217-463~ and NadD2. **(B)** Purified NadD2 was not contaminated by ATPase. Equal amount ATP was incubated with or without 5 μg NadD2. After 30 min at 30°C, the ATP levels were measured using an ATP detection kit. Error bars represent standard deviations. ns, not significant, by Student's *t-*test. **(C)** Purified NadD2 was not contaminated by phosphodiesterase. Equal amount cAMP was incubated with or without 5 μg NadD2. After 30 min at 30°C, the cAMP levels were measured using an ELISA kit. Error bars represent standard deviations. ns, not significant, by Student's *t-*test.

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[^1]: Edited by: Yuji Morita, Meiji Pharmaceutical University, Japan

[^2]: Reviewed by: Tim Yahr, The University of Iowa, United States; Sylvie Elsen, Center for the National Scientific Research (CNRS), France; Kangmin Duan, University of Manitoba, Canada

[^3]: ^†^These authors have contributed equally to this work

[^4]: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
