Elevated exopolysaccharide levels in Pseudomonas aeruginosa flagellar mutants have implications for biofilm growth and chronic infections.

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.

Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600.

The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here, we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus.

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3',5'-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose.

Correction: The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus.

[This corrects the article DOI: 10.1371/journal.ppat.1005192.].

Explorative gene analysis of antibiotic tolerance-related genes in adherent and biofilm cells of Pseudomonas aeruginosa.

BACKGROUND: Antibiotic tolerance has attracted worldwide attention, as it leads to chronic, refractory, and persistent infections that are difficult to control. Bacterial biofilms are well known to be more tolerant to antibiotics compared to planktonic bacteria. We previously revealed that adherent bacteria on a solid surface also exhibited tolerance to antibiotics before forming a biofilm. However, little is known about the mechanisms of antibiotic tolerance for adherent or biofilm cells. OBJECTIVES: We investigated the mechanisms of antibiotic tolerance in the biofilm life cycle using adherent and biofilm cells, and evaluated the possibility that common mechanisms operate at each stage. METHODS: We constructed transposon mutants of Pseudomonas aeruginosa PAO1 and screened for low-tolerant mutants with two different methods, using adherent cells and biofilm cells. RESULTS: Fourteen and nine mutants exhibiting low antibiotic tolerance were detected in the adherent cells and biofilm cells, and 14 and 7 candidate genes linked to this tolerance were identified by sequencing, respectively. Eight of the 14 genes related to the antibiotic tolerance of the adherent cells were involved in biofilm formation. Two of the seven genes related to the antibiotic tolerance of biofilm cells participated in the antibiotic tolerance of adherent cells. CONCLUSIONS: The antibiotic tolerance of adherent cells and biofilm formation appear to be under the same regulation mechanism to promote survival in the presence of antibiotics. Antibiotic tolerance shows a complex regulation mechanism at each stage of biofilm formation.

ChIP-Seq and RNA-Seq reveal an AmrZ-mediated mechanism for cyclic di-GMP synthesis and biofilm development by Pseudomonas aeruginosa.

The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.

Self-produced exopolysaccharide is a signal that stimulates biofilm formation in Pseudomonas aeruginosa.

Bacteria have a tendency to attach to surfaces and grow as structured communities called biofilms. Chronic biofilm infections are a problem because they tend to resist antibiotic treatment and are difficult to eradicate. Bacterial biofilms have an extracellular matrix that is usually composed of a mixture of polysaccharides, proteins, and nucleic acids. This matrix has long been assumed to play a passive structural and protective role for resident biofilm cells. Here we show that this view is an oversimplification and that the biofilm matrix can play an active role in stimulating its own synthesis. Working with the model biofilm bacterium Pseudomonas aeruginosa, we found that Psl, a major biofilm matrix polysaccharide for this species, acts as a signal to stimulate two diguanylate cyclases, SiaD and SadC, to produce the intracellular secondary messenger molecule c-di-GMP. Elevated intracellular concentrations of c-di-GMP then lead to the increased production of Psl and other components of the biofilm. This mechanism represents a unique positive feedback regulatory circuit, where the expression of an extracellular polysaccharide promotes biofilm growth in a manner analogous to autocrine signaling in eukaryotes.

Single-cell control of initial spatial structure in biofilm development using laser trapping.

Biofilms are sessile communities of microbes that are spatially structured by an embedding matrix. Biofilm infections are notoriously intractable. This arises, in part, from changes in the bacterial phenotype that result from spatial structure. Understanding these interactions requires methods to control the spatial structure of biofilms. We present a method for growing biofilms from initiating cells whose positions are controlled with single-cell precision using laser trapping. The native growth, motility, and surface adhesion of positioned microbes are preserved, as we show for model organisms Pseudomonas aeruginosa and Staphylococcus aureus. We demonstrate that laser-trapping and placing bacteria on surfaces can reveal the effects of spatial structure on bacterial growth in early biofilm development.

The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix.

Extracellular polysaccharides comprise a major component of the biofilm matrix. Many species that are adept at biofilm formation have the capacity to produce multiple types of polysaccharides. Pseudomonas aeruginosa produces at least three extracellular polysaccharides, alginate, Pel and Psl, that have been implicated in biofilm development. Non-mucoid strains can use either Pel or Psl as the primary matrix structural polysaccharide. In this study, we evaluated a range of clinical and environmental P.aeruginosa isolates for their dependence on Pel and Psl for biofilm development. Mutational analysis demonstrates that Psl plays an important role in surface attachment for most isolates. However, there was significant strain-to-strain variability in the contribution of Pel and Psl to mature biofilm structure. This analysis led us to propose four classes of strains based upon their Pel and Psl functional and expression profiles. Our data also suggest that Pel and Psl can serve redundant functions as structural scaffolds in mature biofilms. We propose that redundancy could help preserve the capacity to produce a biofilm when exopolysaccharide genes are subjected to mutation. To test this, we used PAO1, a common lab strain that primarily utilizes Psl in the matrix. As expected, a psl mutant strain initially produced a poor biofilm. After extended cultivation, we demonstrate that this strain acquired mutations that upregulated expression of the Pel polysaccharide, demonstrating the utility of having a redundant scaffold exopolysaccharide. Collectively, our studies revealed both unique and redundant roles for two distinct biofilm exopolysaccharides.

Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa.

The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.

Black Queen Hypothesis, partial privatization, and quorum sensing evolution.

Microorganisms produce costly cooperative goods whose benefit is partially shared with nonproducers, called 'mixed' goods. The Black Queen Hypothesis predicts that partial privatization has two major evolutionary implications. First, to favor strains producing several types of mixed goods over nonproducing strains. Second, to favor the maintenance of cooperative traits through different strains instead of having all cooperative traits present in a single strain (metabolic specialization). Despite the importance of quorum sensing regulation of mixed goods, it is unclear how partial privatization affects quorum sensing evolution. Here, we studied the influence of partial privatization on the evolution of quorum sensing. We developed a mathematical population genetics model of an unstructured microbial population considering four strains that differ in their ability to produce an autoinducer (quorum sensing signaling molecule) and a mixed good. Our model assumes that the production of the autoinducers and the mixed goods is constitutive and/or depends on quorum sensing. Our results suggest that, unless autoinducers are costless, partial privatization cannot favor quorum sensing. This result occurs because with costly autoinducers: (1) a strain that produces both autoinducer and goods (fully producing strain) cannot persist in the population; (2) the strain only producing the autoinducer and the strain producing mixed goods in response to the autoinducers cannot coexist, i.e., metabolic specialization cannot be favored. Together, partial privatization might have been crucial to favor a primordial form of quorum sensing-where autoinducers were thought to be a metabolic byproduct (costless)-but not the transition to nowadays costly autoinducers.

Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and post-transcriptionally by RsmA.

Extracellular polysaccharides are important components of biofilms. In non-mucoid Pseudomonas aeruginosa strains, the Pel and Psl polysaccharides are major structural components of the biofilm matrix. In this study, we demonstrate that the alternative σ-factor RpoS is a positive transcriptional regulator of psl gene expression. Furthermore, we show that psl mRNA has an extensive 5' untranslated region, to which the post-transcriptional regulator RsmA binds and represses psl translation. Our observations suggest that upon binding RsmA, the region spanning the ribosome binding site of psl mRNA folds into a secondary stem-loop structure that blocks the Shine-Dalgarno sequence, preventing ribosome access and protein translation. This constitutes a novel mechanism for translational repression by this family of regulators.

Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function.

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with KDs in the nM range. These HMW carbohydrates contain 74.9-80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewisx, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Hfq-Assisted RsmA Regulation Is Central to Pseudomonas aeruginosa Biofilm Polysaccharide PEL Expression.

To appropriately switch between sessile and motile lifestyles, bacteria control expression of biofilm-associated genes through multiple regulatory elements. In Pseudomonas aeruginosa, the post-transcriptional regulator RsmA has been implicated in the control of various genes including those related to biofilms, but much of the evidence for these links is limited to transcriptomic and phenotypic studies. RsmA binds to target mRNAs to modulate translation by affecting ribosomal access and/or mRNA stability. Here, we trace a global regulatory role of RsmA to inhibition of the expression of Vfr-a transcription factor that inhibits transcriptional regulator FleQ. FleQ directly controls biofilm-associated genes that encode the PEL polysaccharide biosynthesis machinery. Furthermore, we show that RsmA alone cannot bind vfr mRNA but requires the assistance of RNA chaperone protein Hfq. This is the first example where a RsmA protein family member requires another protein for binding to its target RNA.

Discovery and development of safe-in-man broad-spectrum antiviral agents.

Viral diseases are one of the leading causes of morbidity and mortality in the world. Virus-specific vaccines and antiviral drugs are the most powerful tools to combat viral diseases. However, broad-spectrum antiviral agents (BSAAs, i.e. compounds targeting viruses belonging to two or more viral families) could provide additional protection of the general population from emerging and re-emerging viral diseases, reinforcing the arsenal of available antiviral options. Here, we review discovery and development of BSAAs and summarize the information on 120 safe-in-man agents in a freely accessible database (https://drugvirus.info/). Future and ongoing pre-clinical and clinical studies will increase the number of BSAAs, expand the spectrum of their indications, and identify drug combinations for treatment of emerging and re-emerging viral infections as well as co-infections.

In vivo colonization profile study of Bordetella bronchiseptica in the nasal cavity.

Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvg(i)) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.

Cyclic di-GMP-Responsive Transcriptional Reporter Bioassays in Pseudomonas aeruginosa.

3',5'-cyclic diguanosine monophosphate (cyclic di-GMP) is a bacterial secondary messenger molecule that regulates many important cellular activities and behaviors, such as motility and biofilm formation. While mass spectrometry protocols for quantitative analyses of intracellular cyclic di-GMP concentrations have been developed, they are time intensive, expensive, low-throughput, and incapable of directly monitoring dynamic changes in vivo. In this protocol, we provide a Pseudomonas aeruginosa-specific detailed methodology to assay the intracellular levels of cyclic di-GMP using biological reporters.

Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production.

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances, largely polysaccharides. Multiple types of extracellular polymeric substances can be produced by a single bacterial strain. The distinct polymer components of biofilms are known to provide chemical protection, but little is known about how distinct extracellular polysaccharides may also protect biofilms against mechanical stresses such as shear or phagocytic engulfment. Decades-long infections of Pseudomonas. aeruginosa biofilms in the lungs of cystic fibrosis patients are natural models for studies of biofilm fitness under pressure from antibiotics and the immune system. In cystic fibrosis infections, production of the extracellular polysaccharide alginate has long been known to increase with time and to chemically protect biofilms. More recently, it is being recognized that chronic cystic fibrosis infections also evolve to increase production of another extracellular polysaccharide, Psl; much less is known about Psl's protective benefits to biofilms. We use oscillatory bulk rheology, on biofilms grown from longitudinal clinical isolates and from genetically-manipulated lab strains, to show that increased Psl stiffens biofilms and increases biofilm toughness, which is the energy cost to cause the biofilm to yield mechanically. Further, atomic force microscopy measurements reveal greater intercellular cohesion for higher Psl expression. Of the three types of extracellular polysaccharides produced by P. aeruginosa, only Psl increases the stiffness. Stiffening by Psl requires CdrA, a protein that binds to mannose groups on Psl and is a likely cross-linker for the Psl components of the biofilm matrix. We compare the elastic moduli of biofilms to the estimated stresses exerted by neutrophils during phagocytosis, and infer that increased Psl could confer a mechanical protection against phagocytic clearance.

The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms.

Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level).IMPORTANCE Many studies have shown that bacterial traits, such as siderophores and quorum sensing, are social in nature. This has led to an impression that secreted traits act as public goods, which are costly to produce but benefit both the producing cell and its surrounding neighbors. Theories and subsequent experiments have shown that such traits are exploitable by asocial cheats, but we show here that this does not always hold true. We demonstrate that the Pseudomonas aeruginosa exopolysaccharide PSL provides social benefits to populations but that it is nonexploitable, because most of the fitness benefits accrue to PSL-producing cells. Our work builds on an increasing body of work showing that secreted traits can have both private and public benefits to cells.

A Pseudomonas aeruginosa Quorum-Sensing autoinducer analog enhances the activity of antibiotics against resistant strains.

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1ΔoprD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1ΔoprD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem-resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria. J. Med. Invest. 64: 101-109, February, 2017.