Citrate serves as a signal molecule to modulate carbon metabolism and iron homeostasis in Staphylococcus aureus.

Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate.

The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa.

Bacterial cyclic-di-GMP (c-di-GMP) production is associated with biofilm development and the switch from acute to chronic infections. In Pseudomonas aeruginosa, the diguanylate cyclase (DGC) SiaD and phosphatase SiaA, which are co-transcribed as part of a siaABCD operon, are essential for cellular aggregation. However, the detailed functions of this operon and the relationships among its constituent genes are unknown. Here, we demonstrate that the siaABCD operon encodes for a signaling network that regulates SiaD enzymatic activity to control biofilm and aggregates formation. Through protein-protein interaction, SiaC promotes SiaD diguanylate cyclase activity. Biochemical and structural data revealed that SiaB is an unusual protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD and regulate c-di-GMP levels and subsequent virulence phenotypes. Collectively, our data provide insights into the molecular mechanisms underlying the modulation of DGC activity associated with chronic infections, which may facilitate the development of antimicrobial drugs.

Regulation of uric acid and glyoxylate metabolism by UgmR protein in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa has evolved several systems to adapt to complex environments. The GntR family proteins play important roles in the regulation of metabolic processes and bacterial pathogenesis. In this study, we uncovered that the gene clusters of PA1513-PA1518 and PA1498-PA1502 in P. aeruginosa are required for uric acid and glyoxylate metabolism respectively. We also identified a GntR family regulator UgmR that is involved in regulation of uric acid and glyoxylate metabolism. Promoter activity measurement and biochemical assays revealed that the UgmR directly represses the transcriptional activity of PA1513-PA1518 and PA1498-PA1502, and this inhibition was relieved by the addition of uric acid. Importantly, further experiments showed that UgmR also participates in the glyoxylate shunt. Collectively, these findings contribute to a better understanding of the UgmR factor involved in uric acid and glyoxylate metabolism, which provide insights into the complex metabolic pathways in P. aeruginosa.

The Pyocin Regulator PrtR Regulates Virulence Expression of Pseudomonas aeruginosa by Modulation of Gac/Rsm System and c-di-GMP Signaling Pathway.

In Pseudomonas aeruginosa, the second messenger cyclic-di-GMP and Gac/Rsm signaling pathways are associated with the transition from acute to chronic infection. Therefore, identification of the molecular mechanisms that govern lifestyle choice in bacteria is very important. Here, we identified a novel cyclic-di-GMP modulator, PrtR, which was shown to repress pyocin production by inhibition of PrtN and activate the type III secretion system (T3SS) through PtrB. Compared to a wild-type strain or a prtN mutant, the prtR prtN double mutant exhibited a wrinkly colony and hyperbiofilm phenotype, as well as an increase in intracellular c-di-GMP levels. Interestingly, a diguanylate cyclase (DGC) gene, siaD, was repressed by PrtR. Further experiments revealed that PrtR directly interacts with SiaD and facilitates the accumulation of c-di-GMP in cells. We also demonstrated that PrtR regulates the activity of the Gac/Rsm system, thus affecting expression of the T3SS and type VI secretion system (T6SS) and the formation of biofilm. Taken together, the present findings indicate that PrtR, as a c-di-GMP modulator, plays key roles in the adaptation to opportunistic infection of P. aeruginosa Additionally, this study revealed a novel mechanism for PrtR-mediated regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks.

Alteration of protein homeostasis mediates the interaction of Pseudomonas aeruginosa with Staphylococcus aureus.

Intracellular protein degradation is essential for the survival of all organisms, but its role in interspecies interaction is unknown. Here, we show that the ClpXP protease of Pseudomonas aeruginosa suppresses its antimicrobial activity against Staphylococcus aureus, a common pathogen co-isolated with P. aeruginosa from polymicrobial human infections. Using proteomic, biochemical, and molecular genetic approaches, we found that this effect is due to the inhibitory effects of ClpXP on the quorum sensing (QS) of P. aeruginosa, mainly by degrading proteins (e.g., PhnA, PhnB, PqsR, and RhlI) which are critical for the production of QS signal molecules PQS and C4-HSL. We provide evidence that co-culturing with S. aureus induces a decrease in the activity of ClpXP in P. aeruginosa, an effect which was also achieved by the treatment of P. aeruginosa with N-acetylglucosamine (GlcNAc), a widespread chemical present on the surface of diverse cell types from bacteria to humans. These findings extend the range of biological events governed by proteolytic machinery to microbial community structure, thus also suggesting that a chemical-induced alteration of protein homeostasis is a mechanism for interspecies interactions.

A type VI secretion system delivers a cell wall amidase to target bacterial competitors.

The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell wall-derived muropeptides. AmpD is cytoplasmic and plays a role in the recycling of cell wall muropeptides, with a link to antibiotic resistance. AmpDh2 is a periplasmic soluble enzyme with the former anchored to the inner leaflet of the outer membrane. We document, herein, that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa delivers AmpDh3 (but not AmpD or AmpDh2) to the periplasm of a prey bacterium upon contact. AmpDh3 hydrolyzes the cell wall peptidoglycan of the prey bacterium, which leads to its killing, thereby providing a growth advantage for P. aeruginosa in bacterial competition. We also document that the periplasmic protein PA0808, heretofore of unknown function, affords self-protection from lysis by AmpDh3. Cognates of the AmpDh3-PA0808 pair are widely distributed across Gram-negative bacteria. Taken together, these findings underscore the importance of their function as an evolutionary advantage and that of the H2-T6SS as the means for the manifestation of the effect.

Coordinated regulation of anthranilate metabolism and bacterial virulence by the GntR family regulator MpaR in Pseudomonas aeruginosa.

The GntR family regulators are widely distributed in bacteria and play critical roles in metabolic processes and bacterial pathogenicity. In this study, we describe a GntR family protein encoded by PA4132 that we named MpaR (MvfR-mediated PQS and anthranilate regulator) for its regulation of Pseudomonas quinolone signal (PQS) production and anthranilate metabolism in Pseudomonas aeruginosa. The deletion of mpaR increased biofilm formation and reduced pyocyanin production. RNA sequencing analysis revealed that the mRNA levels of antABC encoding enzymes for the synthesis of catechol from anthranilate, a precursor of the PQS, were most affected by mpaR deletion. Data showed that MpaR directly activates the expression of mvfR, a master regulator of pqs system, and subsequently promotes PQS production. Accordingly, deletion of mpaR activates the expression of antABC genes, and thus, increases catechol production. We also demonstrated that MpaR represses the rhl quorum-sensing (QS) system, which has been shown to control antABC activity. These results suggested that MpaR function is integrated into the QS regulatory network. Moreover, mutation of mpaR promotes bacterial survival in a mouse model of acute pneumonia infection. Collectively, this study identified a novel regulator of pqs system, which coordinately controls anthranilate metabolism and bacterial virulence in P. aeruginosa.

A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition.

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.

Molecular insights into the master regulator CysB-mediated bacterial virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major pathogen that causes serious acute and chronic infections in humans. The type III secretion system (T3SS) is an important virulence factor that plays essential roles in acute infections. However, the regulatory mechanisms of T3SS are not fully understood. In this study, we found that the deletion of cysB reduced the T3SS gene expression and swarming motility but enhanced biofilm formation. In a mouse acute pneumonia model, mutation of cysB decreased the average bacterial load compared to that of the wild-type strain. Further experiments demonstrated that CysB contributed to the reduced T3SS gene expression and bacterial pathogenesis by directly regulating the sensor kinase RetS. We also performed crystallographic studies of PaCysB. The overall fold of PaCysB NTD domain is similar to other LysR superfamily proteins and structural superposition revealed one possible DNA-binding model for PaCysB. Structural comparison also revealed great flexibility of the PaCysB RD domain, which may play an important role in bending and transcriptional regulation of target DNA. Taken together, these results expand our current understanding of the complex regulatory networks of T3SS and RetS pathways. The crystal structure of CysB provides new insights for studying the function of its homologs in other bacterial species.

Iron facilitates the RetS-Gac-Rsm cascade to inversely regulate protease IV (piv) expression via the sigma factor PvdS in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces several proteases, such as an elastase (LasB protease), a LasA protease, and protease IV (PIV), which are thought as significant virulence factors during infection. Regulators of LasA and LasB expression have been identified and well characterized; however, the molecular details of this regulation of protease IV (PIV) remained largely unknown. Here, we describe the interaction between protease IV and the RetS/Rsm signalling pathway, which plays a central role in controlling the production of multiple virulence factors and the switch from planktonic to biofilm lifestyle. We show that the expression of piv was reduced in ΔretS or ΔrsmA strain grown under restrictive conditions but was induced in ΔretS or ΔrsmA mutant grown under rich conditions as compared with wild-type parent. We compare the expression of piv under various conditions and found that iron facilitates RetS/Rsm system to lead this inverse regulation. Moreover, we reveal that the RetS/Rsm pathway regulates PIV production dependent on the alternative sigma factor PvdS. Collectively, this study extends the understanding of the RetS/Rsm regulatory cascade in response to environmental signals and provides insights into how P. aeruginosa adapts to the complex conditions.

Structural and Molecular Mechanism of CdpR Involved in Quorum-Sensing and Bacterial Virulence in Pseudomonas aeruginosa.

Although quorum-sensing (QS) systems are important regulators of virulence gene expression in the opportunistic human pathogen Pseudomonas aeruginosa, their detailed regulatory mechanisms have not been fully characterized. Here, we show that deletion of PA2588 resulted in increased production of pyocyanin and biofilm, as well as enhanced pathogenicity in a mouse model. To gain insights into the function of PA2588, we performed a ChIP-seq assay and identified 28 targets of PA2588, including the intergenic region between PA2588 and pqsH, which encodes the key synthase of Pseudomonas quinolone signal (PQS). Though the C-terminal domain was similar to DNA-binding regions of other AraC family members, structural studies revealed that PA2588 has a novel fold at the N-terminal region (NTR), and its C-terminal HTH (helix-turn-helix) domain is also unique in DNA recognition. We also demonstrated that the adaptor protein ClpS, an essential regulator of ATP-dependent protease ClpAP, directly interacted with PA2588 before delivering CdpR to ClpAP for degradation. We named PA2588 as CdpR (ClpAP-degradation and pathogenicity Regulator). Moreover, deletion of clpP or clpS/clpA promotes bacterial survival in a mouse model of acute pneumonia infection. Taken together, this study uncovered that CdpR is an important QS regulator, which can interact with the ClpAS-P system to regulate the expression of virulence factors and pathogenicity.

Atg7 Deficiency Intensifies Inflammasome Activation and Pyroptosis in Pseudomonas Sepsis.

Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure, with high morbidity and mortality. The literature implies that autophagy is a crucial regulator of inflammation in sepsis. In this article, we report that autophagy-related protein 7 (Atg7) is involved in inflammasome activation in Pseudomonas aeruginosa abdominal infection. Following i.p. challenge with P. aeruginosa, atg7fl/fl mice showed impaired pathogen clearance, decreased survival, and widespread dissemination of bacteria into the blood and lung tissue compared with wild-type mice. The septic atg7fl/fl mice also exhibited elevated neutrophil infiltration and severe lung injury. Loss of Atg7 resulted in increased production of IL-1ß and pyroptosis, consistent with enhanced inflammasome activation. Furthermore, we demonstrated that P. aeruginosa flagellin is a chief trigger of inflammasome activation in the sepsis model. Collectively, our results provide insight into innate immunity and inflammasome activation in sepsis.

Crystal structure of Pseudomonas aeruginosa RsaL bound to promoter DNA reaffirms its role as a global regulator involved in quorum-sensing.

Pseudomonas aeruginosa possesses at least three well-defined quorum-sensing (QS) (las, rhl and pqs) systems that control a variety of important functions including virulence. RsaL is a QS repressor that reduces QS signal production and ensures homeostasis by functioning in opposition to LasR. However, its regulatory role in signal homeostasis remains elusive. Here, we conducted a ChIP-seq assay and revealed that RsaL bound to two new targets, the intergenic regions of PA2228/PA2229 and pqsH/cdpR, which are required for PQS synthesis. Deletion of rsaL reduced transcription of pqsH and cdpR, thus decreasing PQS signal production. The ΔrsaL strain exhibited increased pyocyanin production and reduced biofilm formation, which are dependent on CdpR or PqsH activity. In addition, we solved the structure of the RsaL-DNA complex at a 2.4 Å resolution. Although the overall sequence similarity is quite low, RsaL folds into a HTH-like structure, which is conserved among many transcriptional regulators. Complementation results of the rsaL knockout cells with different rsaL mutants further confirmed the critical role of the DNA-binding residues (including Arg20, Gln27, Gln38, Gly35, Ser37 and Ser42) that are essential for DNA binding. Our findings reveal new targets of RsaL and provide insight into the detailed characterization of the RsaL-DNA interaction.

RpoN-Dependent Direct Regulation of Quorum Sensing and the Type VI Secretion System in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes in P. aeruginosa To gain further insights into the direct targets of RpoN in vivo, the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS (lasI, rhlI, and pqsR) and the T6SS (hcpA and hcpB). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), lux reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ΔrpoN::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in the ΔrpoN::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS.IMPORTANCE As a global regulator, RpoN controls a wide range of biological pathways, including virulence in P. aeruginosa PAO1. This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness. ChIP-seq provided a useful database to characterize additional functions and targets of RpoN in the future. The functional characterization of RpoN-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence in P. aeruginosa and other bacteria.

HrpS Is a Global Regulator on Type III Secretion System (T3SS) and Non-T3SS Genes in Pseudomonas savastanoi pv. phaseolicola.

The type III secretion system (T3SS) is the main machinery for Pseudomonas savastanoi and other gram-negative bacteria to invade plant cells. HrpR and HrpS form a hetero-hexamer, which activates the expression of HrpL, which induces all T3SS genes by binding to a 'hrp box' in promoters. However, the individual molecular mechanism of HrpR or HrpS has not been fully understood. Through chromatin immunoprecipitation coupled to high-throughput DNA sequencing, we found that HrpR, HrpS, and HrpL had four, 47, and 31 targets on the genome, respectively. HrpS directly bound to the promoter regions of a group of T3SS genes and non-T3SS genes. HrpS independently regulated these genes in a hrpL deletion strain. Additionally, a HrpS-binding motif (GTGCCAAA) was identified, which was verified by electrophoretic mobility shift assay and lux-reporter assay. HrpS also regulated motility and biofilm formation in P. savastanoi. The present study strongly suggests that HrpS alone can work as a global regulator on both T3SS and non-T3SS genes in P. savastanoi. [Formula: see text] Copyright © 2018 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

ChIP-seq reveals the global regulator AlgR mediating cyclic di-GMP synthesis in Pseudomonas aeruginosa.

AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa. However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR, which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A ΔalgR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A ΔalgRΔmucR double mutant produced lesser biofilm than did the single ΔalgR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR. In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation.

Molecular mechanisms of master regulator VqsM mediating quorum-sensing and antibiotic resistance in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although the AraC-family transcription factor VqsM has been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we report that VqsM directly binds to the lasI promoter region, and thus regulates its expression. To identify additional targets of VqsM in P. aeruginosa PAO1, we performed chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) and detected 48 enriched loci harboring VqsM-binding peaks in the P. aeruginosa genome. The direct regulation of these genes by VqsM has been confirmed by electrophoretic mobility shift assays and quantitative real-time polymerase chain reactions. A VqsM-binding motif was identified by using the MEME suite and verified by footprint assays in vitro. In addition, VqsM directly bound to the promoter regions of the antibiotic resistance regulator NfxB and the master type III secretion system (T3SS) regulator ExsA. Notably, the vqsM mutant displayed more resistance to two types of antibiotics and promoted bacterial survival in a mouse model, compared to wild-type PAO1. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems, T3SS, and antibiotic resistance.

Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae.

Pseudomonas syringae uses the two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanisms and the regulons of RhpRS have yet to be fully elucidated. Here, we show that RhpS functions as a kinase and a phosphatase on RhpR and as an autokinase upon itself. RhpR is phosphorylated by the small phosphodonor acetyl phosphate. A specific RhpR-binding site containing the inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by a DNase I footprint analysis. Electrophoretic mobility shift assay indicated that P-RhpR has a higher binding affinity to the IR motif than RhpR. To identify additional RhpR targets in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) and detected 167 enriched loci including the hrpR promoter, suggesting the direct regulation of T3SS cascade genes by RhpR. A genome-wide microarray analysis showed that, in addition to the T3SS cascade genes, RhpR differentially regulates a large set of genes with various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes by coordinating T3SS expression and many other biological processes.

Molecular mechanism of quinone signaling mediated through S-quinonization of a YodB family repressor QsrR.

Quinone molecules are intracellular electron-transport carriers, as well as critical intra- and extracellular signals. However, transcriptional regulation of quinone signaling and its molecular basis are poorly understood. Here, we identify a thiol-stress-sensing regulator YodB family transcriptional regulator as a central component of quinone stress response of Staphylococcus aureus, which we have termed the quinone-sensing and response repressor (QsrR). We also identify and confirm an unprecedented quinone-sensing mechanism based on the S-quinonization of the essential residue Cys-5. Structural characterizations of the QsrR-DNA and QsrR-menadione complexes further reveal that the covalent association of menadione directly leads to the release of QsrR from operator DNA following a 10° rigid-body rotation as well as a 9-Å elongation between the dimeric subunits. The molecular level characterization of this quinone-sensing transcriptional regulator provides critical insights into quinone-mediated gene regulation in human pathogens.