The N-linked glycosylations of TIGIT Asn32 and Asn101 facilitate PVR/TIGIT interaction.

Although the PVR/TIGIT immune checkpoint axis has been suggested as a promising target for cancer immunotherapy and multiple TIGIT-targeting therapies are undergoing clinical trials, the underlying regulatory mechanisms of PVR/TIGIT interaction remain inconclusive. Here we show that TIGIT N-glycosylations are critical for maintaining the interaction between TIGIT and PVR. TIGIT has two N-glycosylation residues, N32 and N101. N-glycosylation on N101 of TIGIT and, to less extent, on N32, play potent roles in PVR binding. Taken together, these findings suggest that the N-glycosylation sites on TIGIT, especially residue N101, may be potential targets for PVR/TIGIT immune checkpoint blockade.

Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin ß1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.

Zinc-dependent interaction between JAB1 and pre-S2 mutant large surface antigen of hepatitis B virus and its implications for viral hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The pre-S2 mutant large HBV surface protein (Δ2 LHBS), which contains an in-frame deletion of approximately 17 amino acids in LHBS, is highly associated with risks and prognoses of HBV-induced HCC. It was previously reported that Δ2 LHBS interacts with the Jun activation domain-binding protein 1 (JAB1), a zinc metalloprotease. This promotes the degradation of the cell cycle regulator p27(Kip1) and is believed to be the major mechanism for Δ2 LHBS-induced HCC. In this study, it was found that the interaction between JAB1 and Δ2 LHBS is facilitated by divalent metal Zn(2+) ions. The binding of JAB1 to Δ2 LHBS requires the JAB1/CSN5 MPN metalloenzyme (JAMM) motif and residue H138 that binds to Zn(2+) ions in JAB1. Isothermal titration calorimetry showed that Δ2 LHBS binds directly to Zn(2+) ions in a two-site binding mode. Residues H71 and H116 in Δ2 LHBS, which also contact Zn(2+) ions, are also indispensable for Δ2 LHBS-mediated p27(Kip1) degradation in human HuH7 cells. These results suggest that developing drugs that interrupt interactions between Δ2 LHBS and JAB1 can be used to mitigate Δ2 LHBS-associated risks for HCC.

Screening and characterization of myositis-related autoantibodies in COVID-19 patients.

An efficient host immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) appears to be crucial for controlling and resolving this viral infection. However, many studies have reported autoimmune characteristics in severe COVID-19 patients. Moreover, clinical observations have revealed that COVID-19-associated acute distress respiratory syndrome shares many features in common with inflammatory myopathy including interstitial lung disease (ILD), most particularly rapidly progressive (RP)-ILD. This study explored this phenomenon by seeking to identify and characterize myositis-specific and related autoantibodies in 25 COVID-19 patients with mild or severe symptoms. Line blot analysis with the EUROLINE Myopathies Ag kit identified 9 (36%) patients with COVID-19 with one or more autoantibodies against several myositis-related antigens (Jo-1, Ku, Mi-2ß, PL-7, PL-12, PM-Scl 75, PM-Scl 100, Ro-52, and SRP); no anti-MDA5 antibodies were detected. As the presence of antibodies identified by line blots was unrelated to disease severity, we further characterized the autoantibodies by radioimmunoassay, in which [35 S]methionine-labeled K562 cellular antigens were precipitated and visualized by gel electrophoresis. This result was confirmed by an immunoprecipitation assay and immunoblotting; 2 patients exhibited anti-Ku70 and anti-Ku80 antibodies. Our data suggest that it is necessary to use more than one method to characterize and evaluate autoantibodies in people recovered from COVID-19, in order to avoid misinterpreting those autoantibodies as diagnostic markers for autoimmune diseases.

Epidemiological observations on breaking COVID-19 transmission: from the experience of Taiwan.

With almost no community-transmitted cases and without any complete lockdown throughout 2020, Taiwan is one of very few countries worldwide that has recorded minimal impact from the COVID-19 pandemic attack. This is despite being only 130 km from China and having frequent business communications with that country, where COVID-19 first emerged. At the end of December 2020, Taiwan had recorded just 873 cases and 7 deaths, in a country of around 24 million people. How to determine the effectiveness of public health policies is an important issue that must be resolved, especially in those countries that have experienced few cases of community-transmitted COVID-19. Our analysis of epidemiological data in Taiwan relating to influenza-like illness (ILI), enterovirus and diarrhoea from the past 3 years reveals dramatic reductions in the incidence of ILI and enterovirus in 2020, compared with 2018 and 2019. These reductions occurred within 2 weeks of the government issuing public health policies for COVID-19 and indicate that such policies can effectively reduce infectious diseases overall. In contrast, no such reduction in ILI activity was observed in 2020 after the first COVID-19 case was reported in the USA. We suggest that infectious diseases data can be used to inform effective public health policies needed to break the transmission chain of COVID-19 and that ongoing monitoring of infectious diseases data can provide confidence about nationwide health.

Evans blue dye as an indicator of albumin permeability across a brain endothelial cell monolayer in vitro.

An increase in the brain endothelial (BEnd) cell permeability of blood albumin is often seen as an early sign of blood-brain barrier (BBB) disruption and can precede increases in the BEnd permeability of small molecules and other plasma proteins in the course of brain disease. Therefore, Evans blue dye (EBD), an albumin-binding fluorescent tracer that is simple to detect and quantify, has been widely utilized for studying BEnd permeability during BBB disruption. Here, we investigated whether EBD is a suitable indicator of albumin permeability across mouse BEnd cell monolayers, alone or cocultured with mouse cortical astrocytes, in an in-vitro permeability assay; given the strong affinity of EBD for albumin, we further asked whether EBD can affect albumin permeability and vice versa. Albumin and EBD readily crossed membrane cell culture inserts with pore diameters of no less than 1 µm in the absence of a cellular barrier, and their permeability was substantially reduced when the membranes were overlaid with a monolayer of BEnd cells. In line with albumin binding, the BEnd permeability of EBD was substantially reduced by the presence of albumin. While EBD at an EBD-to-albumin ratio similar to those typically used in in vivo BBB experiments had little effect on the BEnd permeability of albumin, a much higher concentration of EBD augmented the BEnd permeability of albumin. In conclusion, we investigated the use of EBD as an indicator of albumin permeability in vitro, explored some of its drawbacks and further demonstrated that EBD at the concentration used in vivo does not affect albumin permeability.

Interleukin-18 Is a Potential Biomarker to Discriminate Active Adult-Onset Still's Disease From COVID-19.

Background: Hyperinflammation with dysregulated production of galectins and cytokines may develop in COVID-19 or adult-onset Still's disease (AOSD). Given the similar clinical features in both diseases, it is necessary to identify biomarkers that can differentiate COVID-19 from AOSD. However, the related data remain scarce currently. Methods: In this cross-sectional study, plasma levels of galectin-3, galectin-9, and soluble TIM-3 (sTIM-3) were determined by ELISA in 55 COVID-19 patients (31 non-severe and 24 severe), 23 active AOSD patients, and 31 healthy controls (HC). The seropositivity for SARS-CoV-2 was examined using an immunochromatographic assay, and cytokine profiles were determined with the MULTIPLEX platform. Results: Significantly higher levels of galectin-3, galectin-9, IL-1ß, IL-1Ra, IL-10, IFN-α2, IL-6, IL-18, and TNF-α were observed in severe COVID-19 and active AOSD patients compared with HC (all p<0.001). AOSD, but not COVID-19, showed significantly higher IFN-γ and IL-17A compared with HC (both p<0.01). Moreover, active AOSD patients had 68-fold higher IL-18 levels and 5-fold higher ferritin levels than severe COVID-19 patients (both p<0.001). IL-18 levels at the cut-off value 190.5pg/mL had the highest discriminative power for active AOSD and severe COVID-19, with AUC 0.948, sensitivity 91.3%, specificity 95.8%, and accuracy of 91.5% (p<0.005). Multivariate regression analysis revealed IL-18 as a significant predictor of active AOSD (p<0.05). Conclusion: Active AOSD patients share features of hyperinflammation and cytokine storm with severe COVID-19 patients but possess a distinct cytokine profile, including elevated IL-18, IL-6, IFN-γ, and IL-17A. IL-18 is a potential discriminator between AOSD and COVID-19 and may significantly predict active AOSD.

Evaluations and Mechanistic Interrogation of Natural Products Isolated From Paeonia suffruticosa for the Treatment of Inflammatory Bowel Disease.

Mu Dan Pi (MDP), a traditional Chinese medicine derived from the root bark of Paeonia suffruticosa Andrews, is used to treat autoimmune diseases due to its anti-inflammatory properties. However, the impact of MDP on inflammatory bowel disease (IBD) and its principal active compounds that contribute to the anti-inflammatory properties are uncertain. Thus, this study systemically evaluated the anti-inflammatory effects of fractionated MDP, which has therapeutic potential for IBD. MDP fractions were prepared by multistep fractionation, among which the ethyl acetate-fraction MDP5 exhibited the highest potency, with anti-inflammatory activity screened by the Toll-like receptor (TLR)-2 agonist, Pam3CSK4, in a cell-based model. MDP5 (at 50 µg/ml, p < 0.001) significantly inhibited nuclear factor kappa-B (NF-κB) reporters triggered by Pam3CSK4, without significant cell toxicity. Moreover, MDP5 (at 10 µg/ml) alleviated proinflammatory signaling triggered by Pam3CSK4 in a dose-dependent manner and reduced downstream IL-6 and TNF-α production (p < 0.001) in primary macrophages. MDP5 also mitigated weight loss, clinical inflammation, colonic infiltration of immune cells and cytokine production in a murine colitis model. Index compounds including paeoniflorin derivatives (ranging from 0.1 to 3.4%), gallic acid (1.8%), and 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (1.1%) in MDP5 fractions were identified by LC-MS/MS and could be used as anti-inflammatory markers for MDP preparation. Collectively, these data suggest that MDP5 is a promising treatment for IBD patients.

Redox sensor NPGPx restrains ZAP70 activity and modulates T cell homeostasis.

Emerging evidences implicate the contribution of ROS to T cell activation and signaling. The tyrosine kinase, ζ-chain-associated protein of 70 kDa (ZAP70), is essential for T cell development and activation. However, it remains elusive whether a direct redox regulation affects ZAP70 activity upon TCR stimulation. Here, we show that deficiency of non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), a redox sensor, results in T cell hyperproliferation and elevated cytokine productions. T cell-specific NPGPx-knockout mice reveal enhanced T-dependent humoral responses and are susceptible to experimental autoimmune encephalomyelitis (EAE). Through proteomic approaches, ZAP70 is identified as the key interacting protein of NPGPx through disulfide bonding. NPGPx is activated by ROS generated from TCR stimulation, and modulates ZAP70 activity through redox switching to reduce ZAP70 recruitment to TCR/CD3 complex in membrane lipid raft, therefore subduing TCR responses. These results reveal a delicate redox mechanism that NPGPx serves as a modulator to curb ZAP70 functions in maintaining T cell homeostasis.

Effects of Non-insulin Anti-hyperglycemic Agents on Gut Microbiota: A Systematic Review on Human and Animal Studies.

Background: As growing evidence links gut microbiota with the therapeutic efficacy and side effects of anti-hyperglycemic drugs, this article aims to provide a systematic review of the reciprocal interactions between anti-hyperglycemic drugs and gut microbiota taxa, which underlie the effect of the gut microbiome on diabetic control via bug-host interactions. Method: We followed the PRISMA requirements to perform a systematic review on human vs. animal gut microbiota data in PubMed, SCOPUS, and EMBASE databases, and used Cochrane, ROBIN-I, and SYRCLE tools to assess potential bias risks. The outcomes of assessment were trends on gut microbiota taxa, diversity, and associations with metabolic control (e.g., glucose, lipid) following anti-hyperglycemic treatment. Results: Of 2,804 citations, 64 studies (17/humans; 47/mice) were included. In human studies, seven were randomized trials using metformin or acarbose in obese, pre-diabetes, and type 2 diabetes (T2D) patients. Treatment of pre-diabetes and newly diagnosed T2D patients with metformin or acarbose was associated with decreases in genus of Bacteroides, accompanied by increases in both Bifidobacterium and Lactobacillus. Additionally, T2D patients receiving metformin showed increases in various taxa of the order Enterobacteriales and the species Akkermansia muciniphila. Of seven studies with significant differences in beta-diversity, the incremental specific taxa were associated with the improvement of glucose and lipid profiles. In mice, the effects of metformin on A. muciniphila were similar, but an inverse association with Bacteroides was reported. Animal studies on other anti-hyperglycemic drugs, however, showed substantial variations in results. Conclusions: The changes in specific taxa and ß-diversity of gut microbiota were associated with metformin and acarbose in humans while pertinent information for other anti-hyperglycemic drugs could only be obtained in rodent studies. Further human studies on anti-hyperglycemic drugs other than metformin and acarbose are needed to explore gut microbiota's role in their therapeutic efficacies and side effects.

A systematic identification of anti-inflammatory active components derived from Mu Dan Pi and their applications in inflammatory bowel disease.

Mu Dan Pi (MDP), also known as Moutan Cortex Radicis, is a traditional Chinese medicine used to treat autoimmune diseases. However, the impact of MDP and its principal active compounds on inflammatory bowel disease (IBD) is uncertain. This study therefore systemically assessed the anti-inflammatory effects of MDP and its known active compounds in IBD. The anti-inflammatory activities of water extract and individual compounds were screened by NF-κB and interferon regulatory factor (IRF) reporter assays in THP-1 cells induced with either Toll-like receptor or retinoic acid inducible gene I/melanoma differentiation-associated gene 5 activators and further verified in bone marrow-derived macrophages. MDP water extract significantly inhibited the activation of NF-κB and IRF reporters, downstream signaling pathways and the production of IL-6 and TNF-α, in a dose-dependent manner. Among 5 known active components identified from MDP (1,2,3,4,6-penta-O-galloyl-ß-d-glucose [PGG], gallic acid, methyl gallate, paeoniflorin, and paeonol), PGG was the most efficient at inhibiting both reporters (with an IC50 of 5-10 µM) and downregulating IL-6 and TNF-α. Both MDP powder for clinical use and MDP water extract, but not PGG, reduced colitis and pathological changes in mice. MDP and its water extract show promise as a novel therapy for IBD patients.

Glutathione peroxidase 8 negatively regulates caspase-4/11 to protect against colitis.

Human caspase-4 and its mouse homolog caspase-11 are receptors for cytoplasmic lipopolysaccharide. Activation of the caspase-4/11-dependent NLRP3 inflammasome is required for innate defense and endotoxic shock, but how caspase-4/11 is modulated remains unclear. Here, we show that mice lacking the oxidative stress sensor glutathione peroxidase 8 (GPx8) are more susceptible to colitis and endotoxic shock, and exhibit reduced richness and diversity of the gut microbiome. C57BL/6 mice that underwent adoptive cell transfer of GPx8-deficient macrophages displayed a similar phenotype of enhanced colitis, indicating a critical role of GPx8 in macrophages. GPx8 binds covalently to caspase-4/11 via disulfide bonding between cysteine 79 of GPx8 and cysteine 118 of caspase-4 and thus restrains caspase-4/11 activation, while GPx8 deficiency leads to caspase-4/11-induced inflammation during colitis and septic shock. Inhibition of caspase-4/11 activation with small molecules reduces the severity of colitis in GPx8-deficient mice. Notably, colonic tissues from patients with ulcerative colitis display low levels of Gpx8 and high caspase-4 expression. In conclusion, these results suggest that GPx8 protects against colitis by negatively regulating caspase-4/11 activity.

The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43.

Many bacterial species utilize acetoin as a carbon source. In Klebsiella pneumoniae, the utilization of acetoin is catalyzed by an acetoin dehydrogenase complex encoded by the acoABCD operon, which is positively regulated in the presence of acetoin by the transcriptional factor AcoK. AcoK contains a LuxR type DNA-binding domain at the C-terminal region and putative Walker A and B nucleotide-binding motifs in the N-terminal region. The comprehensive deletion and mutation study performed here shows that mutations in the putative Walker A motif result in a significant reduction of ATP hydrolysis and trans-activation by AcoK of acoABCD expression, presumably due to a loss of ATP-binding ability. AcoK was shown to bind specifically to nucleotides -66 to -36 of the acoABCD promoter, though the DNA-binding ability was not affected by the Walker A motif mutation. Thus, this study provides an additional example of how a member of the signal transduction ATPases with numerous domains family activates its target gene expression.

Identification of an HptB-mediated multi-step phosphorelay in Pseudomonas aeruginosa PAO1.

We herein demonstrate that the hybrid sensor PA1611 carries out specific signal transduction, through HptB (PA3345), to the response regulator PA3346 in Pseudomonas aeruginosa PAO1. As assessed by phenotypic changes in the hptB deletion mutant, the pathway is likely to be involved in the regulation of flagellar activity, the chemotaxis response, twitching motility, and biofilm formation in the bacteria.

NPGPx (GPx7): a novel oxidative stress sensor/transmitter with multiple roles in redox homeostasis.

NPGPx (GPx7) is a member of the glutathione peroxidase (GPx) family without any GPx activity. GPx7 displays a unique function which serves as a stress sensor/transmitter to transfer the signal to its interacting proteins by shuttling disulfide bonds in response to various stresses. In this review, we focus on the exceptional structural and biochemical features of GPx7 compared to other 7 family members and described how GPx7 regulates the diverse signaling targets including GRP78, PDI, CPEB2, and XRN2, and their different roles in unfolded protein response, oxidative stress, and non-targeting siRNA stress response, respectively. The phenotypes associated with GPx7 deficiency in mouse or human including ROS accumulations, highly elevated cancer incidences, auto-immune disorders, and obesity are also revealed in this paper. Finally, we compare GPx8 with GPx7, which shares the highest structural similarity but different biological roles in stress response. These insights have thus provided a more comprehensive understanding of the role of GPx7 in the maintenance of redox homeostasis.

Essential role of ß-human 8-oxoguanine DNA glycosylase 1 in mitochondrial oxidative DNA repair.

8-Oxoguanine (8-OG) is the major mutagenic base lesion in DNA caused by reactive oxygen species (ROS) and accumulates in both nuclear and mitochondrial DNA (mtDNA). In humans, 8-OG is primarily removed by human 8-OG DNA glycosylase 1 (hOGG1) through the base excision repair (BER) pathway. There are two major hOGG1 isoforms, designated α- and ß-hOGG1, generated by alternative splicing, and they have distinct subcellular localization: cell nuclei and mitochondria, respectively. Using yeast two-hybrid screening assays, we found that ß- but not α-hOGG1 directly interacts with the mitochondrial protein NADH:ubiquinone oxidoreductase 1 beta subcomplex 10 (NDUFB10), an integral factor in Complex 1 on the mitochondrial inner membrane. Using coimmunoprecipitation and immunofluorescence studies, we found that this interaction was greatly increased by hydrogen peroxide-induced oxidative stress, suggesting that ß- but not α-hOGG1 is localized in the mitochondrial inner membrane. Analyses of nuclear and mtDNA damage showed that the ß- but not α- hogg1 knockdown (KD) cells were severely defective in mitochondrial BER, indicating an essential requirement of ß-hOGG1 for mtDNA repair. ß-hogg1 KD cells were also found to be mildly deficient in Complex I activity, suggesting that ß-hOGG1 is an accessory factor for the mitochondrial integral function for ATP synthesis. In summary, our findings define ß-hOGG1 as an important factor for mitochondrial BER and as an accessory factor in the mitochondrial Complex I function.

Genomic instability caused by hepatitis B virus: into the hepatoma inferno.

Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide, especially in Asia. HBV induces HCC through multiple oncogenic pathways. Hepatitis-induced hepatocyte inflammation and regeneration stimulates cell proliferation. The interplay between the viral and host factors activates oncogenic signaling pathways and triggers cell transformation. In this review, we summarize previous studies, which reported that HBV induces host genomic instability and that HBV-induced genomic instability is a significant factor that accelerates carcinogenesis. The various types of genomic changes in HBV-induced HCC--chromosomal instability, telomere attrition, and gene-level mutations--are reviewed. In addition, the two viral factors, HBx and the pre-S2 mutant large surface antigen, are discussed for their roles in promoting genomic instability as their main features as viral oncoproteins.

Surface rigidity change of Escherichia coli after filamentous bacteriophage infection.

In this study, the feasibility using atomic force microscopy (AFM) to study the interaction between bacteriophages (phages) and bacteria in situ was demonstrated here. Filamentous phage M13 specifically infects the male Escherichia coli, which expresses F-pili. After infection, E. coli become fragile and grows at a slower rate. AFM provides a powerful tool for investigating these changes in a near-physiological environment. Using high-resolution AFM in phosphate-buffered saline, the damage to the lipopolysaccharide (LPS) layer on the outer membrane of the M13 phage-infected E. coli was observed. The membrane became smoother and more featureless compared to those that were not infected. Besides, the force-distance (f-d) curves were measured to reveal the surface rigidity change in E. coli after M13 phage infection. The effective spring constant and Young's modulus of E. coli decreased after M13 phage infection. Furthermore, the AFM tip was pressed against E. coli to study the response of E. coli under load before and after M13 phage infection. The results showed that after infection E. coli became less rigid and the membrane was also damaged. However, the stiffness changes, including the spring constant and Young's modulus of E. coli, are negligible after M13 phage infection compared with those in previous reports, which may be one of the reasons that E. coli still can maintain its viability after filamentous phage infection.

Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1.

Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt proteins, a phosphorelay profiling assay was performed. Among the 12 recombinant orphan sensor kinases tested, 4 of these sensors (PA1611, PA1976, PA2824, and RetS) transferred the phosphoryl group to HptB (PA3345). The in vivo interaction between HptB and each of the sensors was also confirmed using the bacterial two-hybrid assay. Interestingly, the phosphoryl groups from these sensors all appeared to be transferred via HptB to PA3346, a novel phosphatase consisting of an N-terminal receiver domain and a eukaryotic type Ser/Thr phosphatase domain, and resulted in a significant increase of its phosphatase activity. The subsequent reverse transcription-PCR analysis revealed an operon structure of hptB-PA3346-PA3347, suggesting a coordinate expression of the three genes to carry out a signal transduction. The possibility was supported by the analysis showing PA3347 is able to be phosphorylated on Ser-56, and this phosphoryl group could be removed by PA3346 protein. Finally, analysis of PA3346 and PA3347 gene knock-out mutants revealed that these genes are associated with bacterial swarming activity and biofilm formation. Together, these results disclose a novel multistep phosphorelay system that is essential for P. aeruginosa to respond to a wide spectrum of environmental signals.