Microcolony formation by the opportunistic pathogen Pseudomonas aeruginosa requires pyruvate and pyruvate fermentation.

A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that microcolony formation is associated with stressful, oxygen-limiting but electron-rich conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation, while addition of pyruvate partly restored microcolony formation in mifR mutant biofilms. In contrast, expression of ldhA in mifR::Mar fully restored microcolony formation by this mutant strain. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation of the P. aeruginosa biofilm environment.

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN.

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.

The novel Pseudomonas aeruginosa two-component regulator BfmR controls bacteriophage-mediated lysis and DNA release during biofilm development through PhdA.

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of polysaccharides, proteins, and extracellular (e)DNA, with eDNA required for biofilm formation and integrity. Here we demonstrate that eDNA release is controlled by BfmR, a regulator essential for Pseudomonas aeruginosa biofilm development. Expression of bfmR coincided with localized cell death and DNA release, and could be stimulated by conditions resulting in membrane perturbation and cell lysis. ΔbfmR mutant biofilms demonstrated increased cell lysis and eDNA release suggesting BfmR to suppress, but not eliminate, these processes. Genome-wide transcriptional profiling indicated that BfmR was required for repression of genes associated with bacteriophage assembly and bacteriophage-mediated lysis. Chromatin immunoprecipitation analysis of direct BfmR targets identified the promoter of PA0691, termed here phdA, encoding a previously undescribed homologue of the prevent-host-death (Phd) family of proteins. Lack of phdA expression coincided with impaired biofilm development and increased cell death, a phenotype comparable to ΔbfmR. Expression of phdA in ΔbfmR restored eDNA release, cell lysis and biofilm formation to wild-type levels, with phdA overexpression promoting resistance to the superinfective bacteriophage Pf4, detected only in biofilms. Therefore, we propose that BfmR regulates biofilm development by limiting bacteriophage-mediated lysis and thus, eDNA release, via PhdA.

Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner.

AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide production, and type IV pilus-mediated twitching motility. In this study, the role of AlgR in biofilms was examined in continuous-flow and static biofilm assays. Strain PSL317 (DeltaalgR) produced one-third the biofilm biomass of wild-type strain PAO1. Complementation with algR, but not fimTU-pilVWXY1Y2E, restored PSL317 to the wild-type biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAO1 and PSL317 revealed that a number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a biofilm, but not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine lactone were detected in the biofilm effluent but not the planktonic supernatants of the algR mutant. Additionally, AlgR specifically bound to the rhlA and rhlI promoters in mobility shift assays. Moreover, PAO1 containing a chromosomal mutated AlgR binding site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that AlgR may control transcription in a contact-dependent or biofilm-specific manner.

Interleukin-17 receptor deficiency results in impaired synovial expression of interleukin-1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall-induced arthritis.

OBJECTIVE: To examine the role of interleukin-17 receptor (IL-17R) signaling in cartilage destruction and its interrelationship with synovial IL-1 expression during chronic reactivated streptococcal cell wall (SCW)-induced arthritis. METHODS: SCW arthritis was repeatedly induced in wild-type (WT) and IL-17R-deficient (IL-17R-/-) mice. At different time points, joint inflammation was assessed by using calipers to measure joint swelling. On day 42, mice were killed, and knee joints were removed for histologic analysis. Quantitative polymerase chain reaction (PCR) analyses for different proinflammatory mediators and matrix metalloproteinases (MMPs) were performed on inflamed synovium from WT and IL-17R-/- mice after 5 repeated injections of SCW fragments. RESULTS: IL-17R signaling did not play a significant role in acute joint swelling induced by a single injection of SCW fragments directly into the joint. However, repeated local injections of SCW fragments into the knee joints of IL-17R-/- mice resulted in fewer infiltrating cells in the joint compared with WT mice. Moreover, histologic analysis on day 42 revealed a significant suppression of the degree of chondrocyte death and an absence of cartilage surface erosion in IL-17R-/- mice. Quantitative PCR analysis revealed impaired synovial expression of IL-1, IL-6, cyclooxygenase 2, stromelysin (MMP-3), gelatinase B (MMP-9), and collagenase 3 (MMP-13) in IL-17R-/- mice. CONCLUSION: These data show a critical role of IL-17R signaling in driving the synovial expression of proinflammatory and catabolic mediators, such as IL-1 and different MMPs, during progression from an acute, macrophage-driven joint inflammation to a chronic, cartilage-destructive, T cell-mediated synovitis. Prevention of IL-17R signaling warrants consideration as a therapeutic target in chronic destructive arthritis.

Requirement of IL-17 receptor signaling in radiation-resistant cells in the joint for full progression of destructive synovitis.

IL-17 is a proinflammatory cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. In the present study, we report that IL-17R signaling is required in radiation-resistant cells in the joint for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (streptococcal cell wall) directly into the knee joint of naive IL-17R-deficient (IL-17R-/-) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Microarray analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, cytokines, and collagenase-3 in the synovium of IL-17R-/- mice. Bone marrow (BM) chimeric mice revealed the need for IL-17R expression on radiation-resistant joint cells for destructive inflammation. Chimeric mice of host wt and donor IL-17R-/- BM cells developed destructive synovitis in this chronic reactivated streptococcal cell wall arthritis model similar to wt-->wt chimeras. In contrast, chimeric mice of host IL-17R-/- and donor wt BM cells were protected from chronic destructive arthritis similar as IL-17R-/- -->IL-17R-/- chimeras. These data strongly indicate that IL-17R signaling in radiation-resistant cells in the joint is required for turning an acute macrophage-mediated inflammation into a chronic destructive synovitis.

Altered phenotype of dextran sulfate sodium colitis in interferon regulatory factor-1 knock-out mice.

BACKGROUND AND AIMS: Interferon regulatory factor-1 (IRF-1) is a transcription factor with antiviral, proinflammatory and tumor suppressor properties. We examined the role of IRF-1 in dextran sulfate sodium colitis, a murine model of inflammatory bowel disease, to determine if absence of the gene would protect against colitis. METHODS: C57BL/6J mice with a targeted disruption of IRF-1 and wild-type C57BL/6J controls received five 7-day cycles of 2% dextran sulfate sodium alternating with five 7-day cycles of water. Colonic tissue was formalin fixed for histological analysis and total RNA extracted for gene chip and SYBR green real-time polymerase chain reaction (PCR) analysis. RESULTS: Histological analysis revealed increased distortion of crypt architecture in the dextran sulfate sodium-treated, IRF-1 -/- animals as compared to dextran sulfate sodium-treated wild-type animals. Five of 15 dextran sulfate sodium-treated IRF-1 -/- mice, but only one of 14 dextran sulfate sodium-treated wild-type mice, developed colonic dysplasia. Microarray analysis comparing colonic gene expression in IRF-1 -/- and wild-type animals revealed decreased expression of caspases, genes involved in antigen presentation, and tumor suppressor genes in the IRF-1 -/- animals. Increased expression of genes involved in carcinogenesis and immunoglobulin and complement genes was also noted in the knock-out animals. CONCLUSIONS: Absence of IRF-1 is not protective in dextran sulfate sodium colitis.

Central role of toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria.

Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dissemination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4 signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection, C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that this subsequent gene expression determines survival of the host.

The transcriptional regulator AlgR controls cyanide production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis (CF) patients. One characteristic of P. aeruginosa CF isolates is the overproduction of the exopolysaccharide alginate, controlled by AlgR. Transcriptional profiling analyses comparing mucoid P. aeruginosa strains to their isogenic algR deletion strains showed that the transcription of cyanide-synthesizing genes (hcnAB) was approximately 3-fold lower in the algR mutants. S1 nuclease protection assays corroborated these findings, indicating that AlgR activates hcnA transcription in mucoid P. aeruginosa. Quantification of hydrogen cyanide (HCN) production from laboratory isolates revealed that mucoid laboratory strains made sevenfold more HCN than their nonmucoid parental strains. In addition, comparison of laboratory and clinically derived nonmucoid strains revealed that HCN was fivefold higher in the nonmucoid CF isolates. Moreover, the average amount of cyanide produced by mucoid clinical isolates was 4.7 +/- 0.85 micromol of HCN/mg of protein versus 2.4 +/- 0.40 micromol of HCN/mg of protein for nonmucoid strains from a survey conducted with 41 P. aeruginosa CF isolates from 24 patients. Our data indicate that (i) mucoid P. aeruginosa regardless of their origin (laboratory or clinically derived) produce more cyanide than their nonmucoid counterparts, (ii) AlgR regulates HCN production in P. aeruginosa, and (iii) P. aeruginosa CF isolates are more hypercyanogenic than nonmucoid laboratory strains. Taken together, cyanide production may be a relevant virulence factor in CF lung disease, the production of which is regulated, in part, by AlgR.

Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis.

The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression. In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P. aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P. aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid. Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes. Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2). An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1. Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P. aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain. AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557. Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant. This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb3-type cytochrome PA1557.

Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells.

The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology. Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.

Cutting edge: roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection.

Local production of IL-17 is a significant factor in effective host defense against Gram-negative bacteria. However, the proximal events mediating IL-17 elaboration by T cells remain unclear. In this study, we show in vivo that intact Toll-like receptor 4 signaling in the lung is required for induction of both the p19 transcript of IL-23 and IL-17 protein elaboration in response to Klebsiella pneumoniae. Although IL-17 is widely considered a CD4(+) T cell product, we also demonstrate significant in vitro IL-17 production by CD8(+) T cells after culture in medium from dendritic cells exposed to these bacteria. The dominant portion of this IL-17-inducing activity for both CD4(+) and CD8(+) T cells is IL-23. These data demonstrate the critical signaling pathway for IL-17 induction in the host response to Gram-negative pulmonary infection and suggest a direct role for IL-23 in CD8(+) T cell IL-17 production.

Acute alcohol inhibits TNF-alpha processing in human monocytes by inhibiting TNF/TNF-alpha-converting enzyme interactions in the cell membrane.

Alcohol abuse has long been known to adversely affect innate immune responses and predispose to infections. One cellular mechanism responsible for this effect is alcohol-induced suppression of TNF-alpha by mononuclear phagocytes. We undertook experiments to better understand the cellular mechanisms by which alcohol dose-dependently suppresses TNF elaboration by human monocytes. Here we show in human primary monocytes and cell lines that alcohol suppresses LPS-induced TNF secretion post-transcriptionally by inhibiting cellular processing by TNF-alpha-converting enzyme (TACE). Using fluorescent resonance energy transfer microscopy, physiological relevant levels of alcohol resulted in a reversible dose-dependent decrease in fluorescent resonance energy transfer efficiency between TNF and TACE. These data demonstrate that alcohol inhibits interactions between TNF and its converting enzyme, TACE, possibly by affecting membrane fluidity. These data in part explain the cellular mechanisms by which alcohol impairs monocyte function and may identify immunotherapeutic targets aimed at restoring immune function in this at-risk patient population.

c-Myc is required for the glucose-mediated induction of metabolic enzyme genes.

Glucose exerts powerful effects on hepatocyte gene transcription by mechanisms that are incompletely understood. c-Myc regulates hepatic glucose metabolism by increasing glycolytic enzyme gene transcription while concomitantly decreasing gluconeogenic and ketogenic enzyme gene expression. However, the molecular mechanisms by which c-Myc exerts these effects is not known. In this study, the glucose-mediated induction of L-type pyruvate kinase and glucose-6-phosphatase mRNA levels was diminished by maneuvers involving recombinant adenoviral vectors that interfere with (i) c-Myc protein levels by antisense expression or (ii) c-Myc function through a dominant-negative Max protein. These results were obtained using both HL1C rat hepatoma cells and primary rat hepatocytes. Furthermore, a decrease in c-Myc abundance reduced glucose production in HL1C cells, presumably by decreasing glucose-6-phosphatase activity. The repression of hormone-activated phosphoenolpyruvate carboxykinase gene transcription by glucose was not affected by a reduction in c-Myc levels. The basal mRNA levels for L-pyruvate kinase and glucose-6-phosphatase were not altered to any significant degree by adenoviral treatment. Furthermore, adenoviral overexpression of the c-Myc protein induced glucose-6-phosphatase mRNA in the absence of glucose stimulation. We conclude that multiple mechanisms exist to communicate the glucose-derived signal and that c-Myc has a key role in the hepatic glucose signaling pathway.