Thomas Danhorn

Phenazine-1-Carboxylic Acid Promotes Bacterial Biofilm Development via Ferrous Iron Acquisition

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels of iron in excess of what is required for planktonic growth have been shown to promote biofilm formation, and therapies that interfere with ferric iron [Fe(III)] uptake combined with antibiotics may help treat P. aeruginosa infections. However, use of these therapies presumes that iron is in the Fe(III) state in the context of infection. Here we report the ability of phenazine-1-carboxylic acid (PCA), a common phenazine made by all phenazine-producing pseudomonads, to help P. aeruginosa alleviate Fe(III) limitation by reducing Fe(III) to ferrous iron [Fe(II)]. In the presence of PCA, a P. aeruginosa mutant lacking the ability to produce the siderophores pyoverdine and pyochelin can still develop into a biofilm. As has been previously reported (P. K. Singh, M. R. Parsek, E. P. Greenberg, and M. J. Welsh, Nature 417:552-555, 2002), biofilm formation by the wild type is blocked by subinhibitory concentrations of the Fe(III)-binding innate-immunity protein conalbumin, but here we show that this blockage can be rescued by PCA. FeoB, an Fe(II) uptake protein, is required for PCA to enable this rescue. Unlike PCA, the phenazine pyocyanin (PYO) can facilitate biofilm formation via an iron-independent pathway. While siderophore-mediated Fe(III) uptake is undoubtedly important at early stages of infection, these results suggest that at later stages of infection, PCA present in infected tissues may shift the redox equilibrium between Fe(III) and Fe(II), thereby making iron more bioavailable.

Motility and Chemotaxis in Agrobacterium tumefaciens Surface Attachment and Biofilm Formation

Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen Agrobacterium tumefaciens drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in A. tumefaciens. Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic A. tumefaciens derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.

Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system.

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions. Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results. The increase in surface interactions under phosphate limitation was observed in both static culture and continuous-culture flow cells. Statistical analysis of confocal micrographs obtained from the flow cell biofilms revealed that phosphate limitation increased both the overall attached biomass and the surface coverage, whereas the maximum thickness of the biofilm was not affected. Functions encoded on the two large plasmids of A. tumefaciens C58, pTiC58 and pAtC58, were not required for the observed phosphate effect. The phosphate concentration at which increased attachment was observed triggered the phosphate limitation response, controlled in many bacteria by the two-component regulatory system PhoR-PhoB. The A. tumefaciens phoB and phoR orthologues could only be disrupted in the presence of plasmid-borne copies of the genes, suggesting that this regulatory system might be essential. Expression of the A. tumefaciens phoB gene from a tightly regulated inducible promoter, however, correlated with the amount of biofilm under both phosphate-limiting and nonlimiting conditions, demonstrating that components of the Pho regulon influence A. tumefaciens surface interactions.

Phosphorus limitation increases attachment in Agrobacterium tumefaciens and reveals a conditional functional redundancy in adhesin biosynthesis.

Bacterial responses to phosphorus limitation, commonly inorganic phosphate (P(i)), are important survival mechanisms in a variety of environments. The two-component sensor kinase PhoR and its cognate response regulator PhoB are central to the P(i) limitation response of many bacteria and control the large Pho regulon. Limitation for P(i) significantly increased attachment and biofilm formation by the plant pathogen Agrobacterium tumefaciens, and this was driven by PhoB. Surprisingly, it was also found that both phoR and phoB were essential in A. tumefaciens. Expression of a plasmid-borne copy of the low affinity P(i) transporter (pit) from Sinorhizobium meliloti in A. tumefaciens abolished the phoB and phoR essentiality in A. tumefaciens and allowed direct demonstration of the requirement for this regulatory system in the biofilm response. Increased attachment under P(i) limitation required a unipolar polysaccharide (UPP) adhesin. Mutation of a polyisoprenylphosphate hexose-1-phosphate transferase (PHPT) called uppE abolished UPP production and prevented surface attachment under P(i)-replete conditions, but this was rescued under P(i) limitation, and this rescue required phoB. In low P(i) conditions, either uppE or a paralogous gene Atu0102 is functionally redundant, but only uppE functions in UPP synthesis and attachment when P(i) is replete. This conditional functional redundancy illustrates the influence of phosphorus availability on A. tumefaciens surface colonization.

Three Unique Interstitial Macrophages in the Murine Lung at Steady State

&NA; The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal‐derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal‐derived macrophages. In this study, we demonstrate in the steady‐state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX3CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto‐oncogene, tyrosine kinase, CD64, CD11b, and CX3CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve‐1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner

Virtual memory cells (VM) are an antigen-specific, memory phenotype CD8 T-cell subset found in lymphoreplete, unchallenged mice. Previous studies indicated that VM cells were the result of homeostatic proliferation (HP) resembling the proliferation observed in a lymphopenic environment. Here we demonstrate that HP is ongoing in lymphoreplete mice, the degree of which is dictated by the number of naive CD8 T cells with a sufficiently high affinity for self-antigen interacting with peripheral IL-15. VM cell transcriptional profiles suggest a capacity to mediate protective immunity via antigen non-specific bystander killing, a function we show is dependent on IL-15. Finally, we show a VM-like population of human cells that accumulate with age and traffic to the liver, displaying phenotypic and functional attributes consistent with the bystander protective functions of VM cells identified in the mouse. These data identify developmental and functional attributes of VM cells, including their likely role in protective immunity.

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation’ DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Cell Origin Dictates Programming of Resident versus Recruited Macrophages during Acute Lung Injury

&NA; Two populations of alveolar macrophages (AMs) coexist in the inflamed lung: resident AMs that arise during embryogenesis, and recruited AMs that originate postnatally from circulating monocytes. The objective of this study was to determine whether origin or environment dictates the transcriptional, metabolic, and functional programming of these two ontologically distinct populations over the time course of acute inflammation. RNA sequencing demonstrated marked transcriptional differences between resident and recruited AMs affecting three main areas: proliferation, inflammatory signaling, and metabolism. Functional assays and metabolomic studies confirmed these differences and demonstrated that resident AMs proliferate locally and are governed by increased tricarboxylic acid cycle and amino acid metabolism. Conversely, recruited AMs produce inflammatory cytokines in association with increased glycolytic and arginine metabolism. Collectively, the data show that even though they coexist in the same environment, inflammatory macrophage subsets have distinct immunometabolic programs and perform specialized functions during inflammation that are associated with their cellular origin.

ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980

The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC. ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence. ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9.13. The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage lambda Int family. Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

Deletion of c-FLIP from CD11bhi Macrophages Prevents Development of Bleomycin-induced Lung Fibrosis

&NA; Idiopathic pulmonary fibrosis is a progressive lung disease with complex pathophysiology and fatal prognosis. Macrophages (M&PHgr;) contribute to the development of lung fibrosis; however, the underlying mechanisms and specific M&PHgr; subsets involved remain unclear. During lung injury, two subsets of lung M&PHgr; coexist: Siglec‐Fhi resident alveolar M&PHgr; and a mixed population of CD11bhi M&PHgr; that primarily mature from immigrating monocytes. Using a novel inducible transgenic system driven by a fragment of the human CD68 promoter, we targeted deletion of the antiapoptotic protein cellular FADD‐like IL‐1&bgr;‐converting enzyme‐inhibitory protein (c‐FLIP) to CD11bhi M&PHgr;. Upon loss of c‐FLIP, CD11bhi M&PHgr; became susceptible to cell death. Using this system, we were able to show that eliminating CD11bhi M&PHgr; present 7‐14 days after bleomycin injury was sufficient to protect mice from fibrosis. RNA‐seq analysis of lung M&PHgr; present during this time showed that CD11bhi M&PHgr;, but not Siglec‐Fhi M&PHgr;, expressed high levels of profibrotic chemokines and growth factors. Human M&PHgr; from patients with idiopathic pulmonary fibrosis expressed many of the same profibrotic chemokines identified in murine CD11bhi M&PHgr;. Elimination of monocyte‐derived M&PHgr; may help in the treatment of fibrosis. We identify c‐FLIP and the associated extrinsic cell death program as a potential pathway through which these profibrotic M&PHgr; may be pharmacologically targeted.