cis-DA-dependent dispersion by Pseudomonas aeruginosa biofilm and identification of cis-DA-sensory protein DspS.

IMPORTANCE: Dispersion is an essential stage of the biofilm life cycle resulting in the release of bacteria from a biofilm into the surrounding environment. Dispersion contributes to bacterial survival by relieving overcrowding within a biofilm and allowing dissemination of cells into new habitats for colonization. Thus, dispersion can contribute to biofilm survival as well as disease progression and transmission. Cells dispersed from a biofilm rapidly lose their recalcitrant antimicrobial-tolerant biofilm phenotype and transition to a state that is susceptible to antibiotics. However, much of what is known about this biofilm developmental stage has been inferred from exogenously induced dispersion. Our findings provide the first evidence that native dispersion is coincident with reduced cyclic dimeric guanosine monophosphate levels, while also relying on at least some of the same factors that are central to the environmentally induced dispersion response, namely, BdlA, DipA, RbdA, and AmrZ. Additionally, we demonstrate for the first time that cis-DA signaling to induce dispersion is attributed to the two-component sensor/response regulator DspS, a homolog of the DSF sensor RpfC. Our findings also provide a path toward manipulating the native dispersion response as a novel and highly promising therapeutic intervention.

Laboratory Grown Biofilms of Bacteria Associated with Human Atherosclerotic Carotid Arteries Release Collagenases and Gelatinases during Iron-Induced Dispersion.

The association of bacteria with arterial plaque lesions in patients with atherosclerosis has been widely reported. However, the role these bacteria play in the progression of atherosclerosis is still unclear. Previous work in our lab has demonstrated that bacteria exist in carotid artery plaques as biofilm deposits. Biofilms are communities of microorganisms enmeshed within a protective, self-produced extracellular matrix and have been shown to contribute to chronic infections in humans. Biofilm communities have the potential to impact surrounding tissues in an infection if they undergo a dispersion response, releasing bacteria into the surrounding environment by enzymatic degradation of the extracellular matrix. One concern relating to these enzymes is that they could cause collateral damage to host tissues. In this study, we present an in vitro multispecies biofilm culturing model used to investigate the potential role of bacterial biofilm dispersion in the progression of atherosclerosis. This work has demonstrated an increase in cell release from mixed-species biofilms formed by bacteria associated with human carotid arterial plaque deposits following treatment with iron or a combination of norepinephrine and transferrin. Greater extracellular lipase, protease, and collagenase/gelatinase activity was also associated with iron-treated biofilms. The results of this work suggest that bacteria in this model undergo iron-induced biofilm dispersion, as evidenced by the increased cell release and higher enzyme activity following treatment. This work demonstrates the potential for multispecies biofilm dispersion to contribute to arterial tissue degradation by bacteria and suggests that in atherosclerotic infections, biofilm dispersion may contribute to thrombogenesis, which can lead to heart attack or stroke. IMPORTANCE Atherosclerosis, or hardening of the arteries, is a leading cause of congestive heart failure, heart attack, and stroke in humans. Mounting evidence, in the literature and from our lab, points to the regular involvement of bacteria within arterial plaque deposits in patients with advanced atherosclerosis. Very little is known about the behavior of these bacteria and whether they may contribute to tissue damage in infected arteries. Tissue damage within the arterial plaque lesion can lead to rupture of the plaque contents into the bloodstream, where a clot may form, resulting in a potential heart attack or stroke. This study shows that plaque-associated bacteria, when cultured as mixed-species biofilms in the laboratory, can release degradative enzymes into their environment as the result of a dispersion response triggered by iron. These degradative enzymes can digest proteins and lipids which are associated with the tissues that separate the plaque lesion from the arterial lumen. Thus, this study demonstrates that if mixed species biofilms are induced to undergo dispersion in an infected atherosclerotic lesion when exposed to an elevated concentration of free iron, they have the potential to contribute to the weakening of arterial tissues, which may contribute to atherosclerotic plaque destabilization.

A review of microscopy-based evidence for the association of Propionibacterium acnes biofilms in degenerative disc disease and other diseased human tissue.

PURPOSE: Recent research shows an increasing recognition that organisms not traditionally considered infectious in nature contribute to disease processes. Propionibacterium acnes (P. acnes) is a gram-positive, aerotolerant anaerobe prevalent in the sebaceous gland-rich areas of the human skin. A ubiquitous slow-growing organism with the capacity to form biofilm, P. acnes, recognized for its role in acne vulgaris and medical device-related infections, is now also linked to a number of other human diseases. While bacterial culture and molecular techniques are used to investigate the involvement of P. acnes in such diseases, definitive demonstration of P. acnes infection requires a technique (or techniques) sensitive to the presence of biofilms and insensitive to the presence of potential contamination. Fortunately, there are imaging techniques meeting these criteria, in particular, fluorescence in situ hybridization and immunofluorescence coupled with confocal laser scanning microscopy, as well as immunohistochemistry. METHODS: Our literature review considers a range of microscopy-based studies that provides definitive evidence of P. acnes colonization within tissue from a number of human diseases (acne vulgaris, degenerative disc and prostate disease and atherosclerosis), some of which are currently not considered to have an infectious etiology. RESULTS/CONCLUSION: We conclude that P. acnes is an opportunistic pathogen with a likely underestimated role in the development of various human diseases associated with significant morbidity and, in some cases, mortality. As such, these findings offer the potential for new studies aimed at understanding the pathological mechanisms driving the observed disease associations, as well as novel diagnostic strategies and treatment strategies, particularly for degenerative disc disease. These slides can be retrieved under Electronic Supplementary Material.

Control of Biofilms with the Fatty Acid Signaling Molecule cis-2-Decenoic Acid.

Biofilms are complex communities of microorganisms in organized structures attached to surfaces. Importantly, biofilms are a major cause of bacterial infections in humans, and remain one of the most significant challenges to modern medical practice. Unfortunately, conventional therapies have shown to be inadequate in the treatment of most chronic biofilm infections based on the extraordinary innate tolerance of biofilms to antibiotics. Antagonists of quorum sensing signaling molecules have been used as means to control biofilms. QS and other cell-cell communication molecules are able to revert biofilm tolerance, prevent biofilm formation and disrupt fully developed biofilms, albeit with restricted effectiveness. Recently however, it has been demonstrated that Pseudomonas aeruginosa produces a small messenger molecule cis-2-decenoic acid (cis-DA) that shows significant promise as an effective adjunctive to antimicrobial treatment of biofilms. This molecule is responsible for induction of the native biofilm dispersion response in a range of Gram-negative and Gram-positive bacteria and in yeast, and has been shown to reverse persistence, increase microbial metabolic activity and significantly enhance the cidal effects of conventional antimicrobial agents. In this manuscript, the use of cis-2-decenoic acid as a novel agent for biofilm control is discussed. Stimulating the biofilm dispersion response as a novel antimicrobial strategy holds significant promise for enhanced treatment of infections and in the prevention of biofilm formation.

Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge In Vitro.

In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium Propionibacterium acnes and its potential association with biofilm structures within the arterial wall. The P. acnes 16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable P. acnes was one among 10 different bacterial species recoverable in culture. Fluorescence in situ hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with P. acnes detectable in 4 samples. We also demonstrated that laboratory-grown cultures of P. acnes biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by P. acnes were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced P. acnes VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that P. acnes can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least in vitro, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes.

BdlA, DipA and induced dispersion contribute to acute virulence and chronic persistence of Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing ΔbdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing ΔbdlA and ΔdipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections.

Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture.

UNLABELLED: Atherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin. IMPORTANCE: The association of bacteria with atherosclerosis has been only superficially studied, with little attention focused on the potential of bacteria to form biofilms within arterial plaques. In the current work, we show that bacteria form biofilm deposits within carotid arterial plaques, and we demonstrate that one species we have identified in plaques can be stimulated in vitro to undergo a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of transferrin. Biofilm dispersion is characterized by the release of bacterial enzymes into the surroundings of biofilm microcolonies, allowing bacteria to escape the biofilm matrix. We believe these enzymes may have the potential to damage surrounding tissues and facilitate plaque rupture if norepinephrine is able to stimulate biofilm dispersion in vivo. This research, therefore, suggests a potential mechanistic link between hormonal state and the potential for heart attack and stroke.

The putative enoyl-coenzyme A hydratase DspI is required for production of the Pseudomonas aeruginosa biofilm dispersion autoinducer cis-2-decenoic acid.

In the present study, we report the identification of a putative enoyl-coenzyme A (CoA) hydratase/isomerase that is required for synthesis of the biofilm dispersion autoinducer cis-2-decenoic acid in the human pathogen Pseudomonas aeruginosa. The protein is encoded by PA14_54640 (PA0745), named dspI for dispersion inducer. The gene sequence for this protein shows significant homology to RpfF in Xanthomonas campestris. Inactivation of dspI was shown to abolish biofilm dispersion autoinduction in continuous cultures of P. aeruginosa and resulted in biofilms that were significantly greater in thickness and biomass than those of the parental wild-type strain. Dispersion was shown to be inducible in dspI mutants by the exogenous addition of synthetic cis-2-decenoic acid or by complementation of ΔdspI in trans under the control of an arabinose-inducible promoter. Mutation of dspI was also shown to abolish cis-2-decenoic acid production, as revealed by gas chromatography-mass spectrometry (GC-MS) analysis of cell-free spent culture medium. The transcript abundance of dspI correlated with cell density, as determined by quantitative reverse transcriptase (RT) PCR. This regulation is consistent with the characterization of cis-2-decenoic acid as a cell-to-cell communication molecule that regulates biofilm dispersion in a cell density-dependent manner.

A fatty acid messenger is responsible for inducing dispersion in microbial biofilms.

It is well established that in nature, bacteria are found primarily as residents of surface-associated communities called biofilms. These structures form in a sequential process initiated by attachment of cells to a surface, followed by the formation of matrix-enmeshed microcolonies, and culminating in dispersion of the bacteria from the mature biofilm. In the present study, we have demonstrated that, during growth, Pseudomonas aeruginosa produces an organic compound we have identified as cis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development. When added exogenously to P. aeruginosa PAO1 biofilms at a native concentration of 2.5 nM, cis-2-decenoic acid was shown to induce the dispersion of biofilm microcolonies. This molecule was also shown to induce dispersion of biofilms, formed by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Bacillus subtilis, Staphylococcus aureus, and the yeast Candida albicans. Active at nanomolar concentrations, cis-2-decenoic acid appears to be functionally and structurally related to the class of short-chain fatty acid signaling molecules such as diffusible signal factor, which act as cell-to-cell communication molecules in bacteria and fungi.

Characterization of temporal protein production in Pseudomonas aeruginosa biofilms.

Phenotypic and genetic evidence supporting the notion of biofilm formation as a developmental process is growing. In the present work, we provide additional support for this hypothesis by identifying the onset of accumulation of biofilm-stage specific proteins during Pseudomonas aeruginosa biofilm maturation and by tracking the abundance of these proteins in planktonic and three biofilm developmental stages. The onset of protein production was found to correlate with the progression of biofilms in developmental stages. Protein identification revealed that proteins with similar function grouped within similar protein abundance patterns. Metabolic and housekeeping proteins were found to group within a pattern separate from virulence, antibiotic resistance, and quorum-sensing-related proteins. The latter were produced in a progressive manner, indicating that attendant features that are characteristic of biofilms such as antibiotic resistance and virulence may be part of the biofilm developmental process. Mutations in genes for selected proteins from several protein production patterns were made, and the impact of these mutations on biofilm development was evaluated. The proteins cytochrome c oxidase, a probable chemotaxis transducer, a two-component response regulator, and MexH were produced only in mature and late-stage biofilms. Mutations in the genes encoding these proteins did not confer defects in growth, initial attachment, early biofilm formation, or twitching motility but were observed to arrest biofilm development at the stage of cell cluster formation we call the maturation-1 stage. The results indicated that expression of theses genes was required for the progression of biofilms into three-dimensional structures on abiotic surfaces and the completion of the biofilm developmental cycle. Reverse transcription-PCR analysis confirmed the detectable change in expression of the respective genes ccoO, PA4101, and PA4208. We propose a possible mechanism for the role of these biofilm-specific proteins in biofilm formation.

Biosynthesis of the allene (-)-marasin in Marasmius ramealis.

[1-14C]-E-dehydromatricaria methyl ester and dimethyl [1-14C]-deca-4,6,8-triyne-1,10-dioate are incorporated into the allene (-)-marasin in Marasmius ramealis without scrambling of the 14C label. This and the levels of the incorporations (0.8% and 4.9% respectively) strongly suggests that the above esters, or close relatives, can be converted directly into (-)-marasin in M. ramealis, and that the diyne-allene moiety in this latter compound arises by the rearrangement, under enzymic control, of an alkyltriyne moiety.

Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm.

Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.