Disruption of c-di-GMP Signaling Networks Unlocks Cryptic Expression of Secondary Metabolites during Biofilm Growth in Burkholderia pseudomallei.

The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soilborne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at various temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypobiofilm- and hyperbiofilm-forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) 2, 11, 14 (syrbactin), and 15 (malleipeptin) in parental and ΔII2523 backgrounds also reveal the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under different environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions. IMPORTANCE Burkholderia pseudomallei is a saprophytic bacterium residing in the environment that switches to a pathogenic lifestyle during infection of a wide range of hosts. The environmental cues that serve as the stimulus to trigger this change are largely unknown. However, it is well established that the cellular level of c-di-GMP, a secondary signal messenger, controls the switch from growth as planktonic cells to growth as a biofilm. Disrupting the signaling mediated by c-di-GMP allows for a better understanding of the regulation and the contribution of the surface associated and secreted molecules that contribute to the various lifestyles of this organism. The genome of B. pseudomallei also encodes cryptic biosynthetic gene clusters predicted to encode small molecules that potentially contribute to growth as a biofilm, adaptation, and interactions with other organisms. A better understanding of the regulation of these molecules is crucial to understanding how this versatile pathogen alters its lifestyle.

2-Aminoimidazoles Inhibit Mycobacterium abscessus Biofilms in a Zinc-Dependent Manner.

Biofilm growth is thought to be a significant obstacle to the successful treatment of Mycobacterium abscessus infections. A search for agents capable of inhibiting M. abscessus biofilms led to our interest in 2-aminoimidazoles and related scaffolds, which have proven to display antibiofilm properties against a number of Gram-negative and Gram-positive bacteria, including Mycobacterium tuberculosis and Mycobacterium smegmatis. The screening of a library of 30 compounds led to the identification of a compound, AB-2-29, which inhibits the formation of M. abscessus biofilms with an IC50 (the concentration required to inhibit 50% of biofilm formation) in the range of 12.5 to 25 µM. Interestingly, AB-2-29 appears to chelate zinc, and its antibiofilm activity is potentiated by the addition of zinc to the culture medium. Preliminary mechanistic studies indicate that AB-2-29 acts through a distinct mechanism from those reported to date for 2-aminoimidazole compounds.

Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium.

Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms.

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae - Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum - and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis. The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

Model of Chronic Equine Endometritis Involving a Pseudomonas aeruginosa Biofilm.

Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux-engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent lux-labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria (P < 0.05). A greater incidence (P < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences (P > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased (P < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly (P < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that P. aeruginosa produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.

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.

Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B. pseudomallei biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to B. pseudomallei 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and B. pseudomallei has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire B. pseudomallei biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the B. pseudomallei complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in B. mallei, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of B. pseudomallei and improve diagnostics and therapeutic treatment strategies.

Thermoregulation of Biofilm Formation in Burkholderia pseudomallei Is Disrupted by Mutation of a Putative Diguanylate Cyclase.

Burkholderia pseudomallei, a tier 1 select agent and the etiological agent of melioidosis, transitions from soil and aquatic environments to infect a variety of vertebrate and invertebrate hosts. During the transition from an environmental saprophyte to a mammalian pathogen, B. pseudomallei encounters and responds to rapidly changing environmental conditions. Environmental sensing systems that control cellular levels of cyclic di-GMP promote pathogen survival in diverse environments. Cyclic di-GMP controls biofilm production, virulence factors, and motility in many bacteria. This study is an evaluation of cyclic di-GMP-associated genes that are predicted to metabolize and interact with cyclic di-GMP as identified from the annotated genome of B. pseudomallei 1026b. Mutants containing transposon disruptions in each of these genes were characterized for biofilm formation and motility at two temperatures that reflect conditions that the bacteria encounter in the environment and during the infection of a mammalian host. Mutants with transposon insertions in a known phosphodiesterase (cdpA) and a predicted hydrolase (Bp1026b_I2285) gene exhibited decreased motility regardless of temperature. In contrast, the phenotypes exhibited by mutants with transposon insertion mutations in a predicted diguanylate cyclase gene (Bp1026b_II2523) were strikingly influenced by temperature and were dependent on a conserved GG(D/E)EF motif. The transposon insertion mutant exhibited enhanced biofilm formation at 37°C but impaired biofilm formation at 30°C. These studies illustrate the importance of studying behaviors regulated by cyclic di-GMP under varied environmental conditions in order to better understand cyclic di-GMP signaling in bacterial pathogens.IMPORTANCE This report evaluates predicted cyclic di-GMP binding and metabolic proteins from Burkholderia pseudomallei 1026b, a tier 1 select agent and the etiologic agent of melioidosis. Transposon insertion mutants with disruptions in each of the genes encoding these predicted proteins were characterized in order to identify key components of the B. pseudomallei cyclic di-GMP-signaling network. A predicted hydrolase and a phosphodiesterase that modulate swimming motility were identified, in addition to a diguanylate cyclase that modulates biofilm formation and motility in response to temperature. These studies warrant further evaluation of the contribution of cyclic di-GMP to melioidosis in the context of pathogen acquisition from environmental reservoirs and subsequent colonization, dissemination, and persistence within the host.