Modulate Biofilm Formation Research Articles

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

Identification of a cellular role of hemolysin co-regulatory protein (Hcp) in Vibrio alginolyticus modulating substrate metabolism and biofilm formation by cAMP-CRP

Cyclic AMP (cAMP) and cAMP receptor protein (CRP) system controls catabolic enzyme expression based on metabolite concentrations in bacteria. Hemolysin co-regulatory protein (Hcp) is well known as a molecular chaperone for virulence factor secretion of the type VI secretion system (T6SS). However, the intracellular role of Hcp involving in bacterial physiological processes remains unknown. To clarify that, we constructed a single hcp mutant strain and analyzed their effects on the physiological processes of Vibrio alginolyticus. The omics results revealed the extensive involvement of Hcp in the catabolic metabolism in bacteria. Simultaneously, Hcp1 and Hcp2 played opposing regulatory roles on the bacterial growth, biofilm formation, and intracellular cAMP-CRP levels during cultivation in a glucose medium. Furthermore, the interacting protein screening and co-immunoprecipitation (Co-IP) assays confirmed that the glucose-specific phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) enzyme IIA component (EIIAglc) was a key interacting partner with Hcp proteins as well as class I adenylyl cyclase (AC-I) in Vibrio alginolyticus. These results indicated that, to achieve cellular homeostasis, Hcp1 and Hcp2 might exert antagonistic and synergistic effects, respectively, on the interaction between EIIAglc and AC thus cooperatively regulating intracellular cAMP-CRP production.

N-Acyl Homoserine Lactone Production by the Marine Isolate, Dasania marina.

Dasania marina (isolate SD1D, with 98.5% sequence similarity to Dasania marina DMS 21967 KOPRI 20902) is a marine bacterium that was isolated from ballast tank fluids as part of a biofilm study in 2014. Our previous work indicated that although this strain produced no detectable biofilm, it was the only isolate to produce N-acyl homoserine lactones (AHLs) in assays using the broad-range reporter strain, Agrobacterium tumefaciens KYC55. The goal of the current study was to determine the types of AHL molecules produced by the D. marina isolate using gas chromatography-mass spectroscopy (GCMS) and C4- to C14-AHL as standards. A time course assay indicated that the D. marina strain produced the highest level of AHLs at 20 h of growth. When extracts were subjected to GCMS, detectable levels of C8- and C10-AHL and higher levels of C12-AHL were observed. Interestingly, several biofilm-forming isolates obtained from the same source also produced detectable amounts of several AHLs. Of the isolates tested, a strain designated SD5, with 99.83% sequence similarity to Alteromonas tagae BCRC 17571, produced unstable biofilms, yet detectable levels of C6-, C8-, C10- and C12-AHL, and isolate SD8, an Alteromonas oceani S35 strain (98.85% sequence similarity), produced robust and stable biofilms accompanied by detectable levels of C8- and C12-AHL. All isolates tested produced C12-AHL at higher levels than the other AHLs. Results from this study suggest that quorum sensing and biofilm formation are uncoupled in D. marina. Whether the suite of AHLs produced by this isolate could modulate biofilm formation in other strains requires further study.

Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1- mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1- mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1- mutant. In the sloughing assay, nuc1- mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.

Proteomics and metabolomics analyses of Streptococcus agalactiae isolates from human and animal sources

Streptococcus agalactiae (S. agalactiae), group B Streptococcus (GBS), a major cause of infection in a wide variety of diseases, have been compared in different human and animal sources. We aimed to compare the bacterial proteome and metabolome profiles of human and animal S. agalactiae strains to delineate biological interactions relevant to infection. With the innovative advancement in mass spectrometry, a comparative result between both strains provided a solid impression of different responses to the host. For instance, stress-related proteins (Asp23/Gls24 family envelope stress response protein and heat shock protein 70), which play a role in the survival of GBS under extreme environmental conditions or during treatment, are highly expressed in human and animal strains. One human strain contains ꞵ-lactamase (serine hydrolase) and biofilm regulatory protein (lytR), which are important virulence regulators and potential targets for the design of novel antimicrobials. Another human strain contains the aminoglycosides-resistance bifunctional AAC/APH (A0A0U2QMQ5) protein, which confers resistance to almost all clinically used aminoglycosides. Fifteen different metabolites were annotated between the two groups. L-aspartic acid, ureidopropionic acid, adenosine monophosphate, L-tryptophan, and guanosine monophosphate were annotated at higher levels in human strains. Butyric acid, fumaric acid, isoleucine, leucine, and hippuric acid have been found in both human and animal strains. Certain metabolites were uniquely expressed in animal strains, with fold changes greater than 2. For example, putrescine modulates biofilm formation. Overall, this study provides biological insights into the substantial possible bacterial response reflected in its macromolecular production, either at the proteomic or metabolomic level.

Genomic Island-Encoded Diguanylate Cyclase from Vibrio alginolyticus Regulates Biofilm Formation and Motility in Pseudoalteromonas.

Many bacteria use the second messenger c-di-GMP to regulate exopolysaccharide production, biofilm formation, motility, virulence, and other phenotypes. The c-di-GMP level is controlled by the complex network of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that synthesize and degrade c-di-GMP. In addition to chromosomally encoded DGCs, increasing numbers of DGCs were found to be located on mobile genetic elements. Whether these mobile genetic element-encoded DGCs can modulate the physiological phenotypes in recipient bacteria after horizontal gene transfer should be investigated. In our previous study, a genomic island encoding three DGC proteins (Dgc137, Dgc139, and Dgc140) was characterized in Vibrio alginolyticus isolated from the gastric cavity of the coral Galaxea fascicularis. Here, the effect of the three DGCs in four Pseudoalteromonas strains isolated from coral Galaxea fascicularis and other marine environments was explored. The results showed that when dgc137 is present rather than the three DGC genes, it obviously modulates biofilm formation and bacterial motility in these Pseudoalteromonas strains. Our findings implied that mobile genetic element-encoded DGC could regulate the physiological status of neighboring bacteria in a microbial community by modulating the c-di-GMP level after horizontal gene transfer.

Identification of VP0143 gene which modulates biofilm formation of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic foodborne pathogen associated with seafood consumption and can cause acute inflammatory gastroenteritis. The biofilm formed by V. parahaemolyticus facilitates its colonization and persistence in the food production chain, posing a continuous threat to food safety. In this study, through transposon mutants screening, we identified a novel gene, VP0143, which positively regulated biofilm formation of V. parahaemolyticus. The VP0143 deletion mutant (ΔVP0143) exhibited less biofilm formation as determined by crystal violet staining assay and showed decreased viability of biofilm cells by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay compared to the wild type strain. The ΔVP0143 strain also showed weakened flagella-mediated motility and surface hydrophobicity. Moreover, VP0143 deletion significantly affected the production of autoinducer-2 and exopolysaccharides in biofilm, and reduced Raman peak intensity of biofilm components. Microscopic analysis confirmed that biofilm formed by ΔVP0143 strain showed loosened structure. In addition, ΔVP0143 strain exhibited compromised ability to form biofilm on silicon wafers, glass and stainless-steel plates, as well as on shrimp and crab surfaces. All these defects were reversed in the complementation strain ΔVP0143C. RT-qPCR further indicated that expression of genes associated with biofilm formation was decreased after VP0143 deletion. Therefore, our data suggest that VP0143 is involved in regulating biofilm formation of V. parahaemolyticus and extend our understanding of genetic determinants of V. parahaemolyticus biofilm formation.

Reciprocal regulation of NagC and quorum sensing systems and their roles in hmsHFRS expression and biofilm formation in Yersinia pseudotuberculosis.

Biofilm formation by Yersinia pseudotuberculosis is regulated by quorum sensing (QS) and dependent on the haemin storage locus hms, required for the extracellular polysaccharide poly-N-acetylglucosamine (poly-GlcNAc) production. In Escherichia coli NagC regulates both GlcNAc biosynthesis and metabolism with GlcNAc acting as a signal that co-ordinates these and other activities. However, the contribution of NagC and GlcNAc to biofilm development in Y. pseudotuberculosis is not known. Here we show that a Y. pseudotuberculosis nagC mutant is impaired for biofilm production on abiotic (glass) and biotic (Caenorhabitis elegans) surfaces. Genetic complementation restored poly-GlcNAc production and biofilm formation on C. elegans. Using lux-based promoter fusions, hmsHFRS expression was found to be nagC dependent. Given that NagC and QS both regulate aggregation and biofilm formation, we investigated their regulatory relationship using lux-based promoter fusions. These revealed that (i) nagC is negatively autoregulated, but expression can be partially restored in the nagC mutant by exogenous GlcNAc, (ii) NagC negatively regulates the ytbI and ypsI QS genes and (iii) nagC expression is reduced in the ytbI, ypsI and ypsR mutants but not the ytbR mutant. These data establish the existence of a reciprocal regulatory relationship between NagC and QS, which in the case of the luxRI pair ytbRI, is also GlcNAc-dependent. NagC and GlcNAc are therefore components of a regulatory system involving QS that modulates biofilm formation and aggregation.

Modulating biofilm can potentiate activity of novel plastic-degrading enzymes

Plastic pollution is an increasing global issue desperately requiring a solution. Only 9% of all plastic waste has been recycled, and whilst recycling gives a second life to plastic, it is costly and there are limited downstream uses of recycled plastic, therefore an alternative is urgently needed. Biodegradation of plastic by microorganisms is a developing field of interest with the potential for bioreactors to be used alongside recycling to degrade plastic that may otherwise be sent to landfill. Here, we have identified two novel polyethylene terephthalate (PET) degrading enzymes through genomic mining and characterised their activity, including their ability to degrade PET. One of the main roadblocks facing the development of microbial enzymes as a plastic biodegradation solution, is that their efficiency is too low to facilitate development as bioremediation tools. In an innovative approach to tackle this roadblock, we hypothesised that enhancing a bacteria’s ability to attach to and form a biofilm on plastic could maximise the local concentration of the enzyme around the target substrate, therefore increasing the overall rate of plastic degradation. We found that increasing biofilm levels, by manipulating the levels of the second messenger, Cyclic-di-GMP, led to increased levels of polyester degradation in cells expressing novel and well characterised polyester-degrading enzymes. This indicates that modulating biofilm formation is a viable mechanism to fast track the development of bacterial plastic bioremediation solutions.

The regulator FleQ both transcriptionally and post-transcriptionally regulates the level of RTX adhesins of Pseudomonas fluorescens.

Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a ΔfleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the ΔfleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the ΔfleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens. IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens, biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens. Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.

Impacts of Biofilm Formation on the Physicochemical Properties and Toxicity of Microplastics: A Concise Review

Microplastics (MPs) are of global concern due to their slow degradation in the environment and the potential of inducing adverse effects on organisms. In aquatic ecosystems, microbes routinely colonize MPs and develop biofilms on their surfaces. Biofilms are assemblages of surface-associated microbial cells that are enclosed in an extracellular polymeric substance. Emerging evidence has suggested that the development of biofilm can alter the physicochemical properties and the pollutant adsorption capability on MPs. In this article, we review the impacts of biofilm formation on MP properties, ecotoxicity, and fate. First, we summarize the environmental factors that modulate biofilm formation, as well as the unique components of biofilm on MP. Next, we review current understanding on the influence of biofilm formation on the physical and chemical properties of MPs and discuss how these changes affect their pollutant adsorption capacity. Finally, we discuss how biofilm formation on MPs affects their ingestion by organisms and nutrient cycling in aquatic environments.

The Sinorhizobium meliloti NspS-MbaA system affects biofilm formation, exopolysaccharide production and motility in response to specific polyamines.

We previously showed that specific polyamines (PAs) present in the extracellular environment markedly affect extracellular polysaccharide (EPS) production, biofilm formation and motility in Sinorhizobium meliloti Rm8530. We hypothesized that extracellular PA signals were sensed and transduced by the NspS and MbaA proteins, respectively, which are homologs of the PA-sensing, c-di-GMP modulating NspS-MbaA proteins described in Vibrio cholerae. Here we show that the decrease in biofilm formation and EPS production in the quorum-sensing (QS)-deficient S. meliloti wild-type strain 1021 in cultures containing putrescine or spermine did not occur in a 1021 nspS mutant (1021 nspS). The transcriptional expression of nspS in strain 1021 was significantly increased in cultures containing either of these polyamines, but not by exogenous cadaverine, 1,3-diaminopropane (DAP), spermidine (Spd) or norspermidine (NSpd). Cell aggregation in liquid cultures did not differ markedly between strain 1021 and 1021 nspS in the presence or absence of PAs. The S. meliloti QS-proficient Rm8530 wild-type and nspS mutant (Rm8530 nspS) produced similar levels of biofilm under control conditions and 3.2- and 2.2-fold more biofilm, respectively, in cultures with NSpd, but these changes did not correlate with EPS production. Cells of Rm8530 nspS aggregated from two- to several-fold more than the wild-type in cultures without PAs or in those containing Spm. NSpd, Spd and DAP differently affected swimming and swarming motility in strains 1021 and Rm8530 and their respective nspS mutants. nspS transcription in strain Rm8530 was greatly reduced by exogenous Spm. Bioinformatic analysis revealed similar secondary structures and functional domains in the MbaA proteins of S. meliloti and V. cholerae, while their NspS proteins differed in some residues implicated in polyamine recognition in the latter species. NspS-MbaA homologs occur in a small subset of soil and aquatic bacterial species that commonly interact with eukaryotes. We speculate that the S. meliloti NspS-MbaA system modulates biofilm formation, EPS production and motility in response to environmental or host plant-produced PAs.

The phosphodiesterase RmcA contributes to the adaptation of Pseudomonas putida to l-arginine.

Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon, and even ATP supply and to regulate multicellular behaviors such as biofilm formation. Arginine modulates the intracellular levels of 3'-5'cyclic diguanylic acid (c-di-GMP), a second messenger that controls biofilm formation, maintenance and dispersion. In Pseudomonas putida, KT2440, a versatile microorganism with wide biotechnological applications, modulation of c-di-GMP levels by arginine requires the transcriptional regulator ArgR, but the connections between arginine metabolism and c-di-GMP are not fully characterized. It has been recently demonstrated that arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa through the transducer RmcA protein (Redox regulator of c-di-GMP), which can directly decrease c-di-GMP levels and possibly affect biofilm architecture. A RmcA homolog is present in P. putida, but its function and involvement in arginine perceiving or biofilm life cycle had not been studied. Here, we present a preliminary characterization of the RmcA-dependent response to arginine in P. putida in modulating biofilm formation, c-di-GMP levels, and energy metabolism. This work contributes to further understanding the molecular mechanisms linking biofilm homeostasis and environmental adaptation.

Mutation of gdpS gene induces a viable but non-culturable state in Staphylococcus epidermidis and changes in the global transcriptional profile

BackgroundIn the genome of staphylococci, only the gdpS gene encodes the conserved GGDEF domain, which is the characteristic of diguanylate cyclases. In our previous study, we have demonstrated that the gdpS gene can modulate biofilm formation by positively regulating the expression of ica operon in Staphylococcus epidermidis. Moreover, this regulation seems to be independent of the c-di-GMP signaling pathway and the protein-coding function of this gene. Therefore, the biological function of the gdpS gene remains to be further investigated.ResultsIn the present study, it was observed that mutation of the gdpS gene induced S. epidermidis to enter into a presumed viable but nonculturable state (VBNC) after cryopreservation with glycerol. Similarly, when moved from liquid to solid culture medium, the gdpS mutant strain also exhibited a VBNC state. Compared with the wild-type strain, the gdpS mutant strain autolyzed more quickly during storage at 4℃, indicating its increased susceptibility to low temperature. Transcriptional profiling analysis showed that the gdpS mutation affected the transcription of 188 genes (92 genes were upregulated and 96 genes were downregulated). Specifically, genes responsible for glycerol metabolism were most markedly upregulated and most of the altered genes in the mutant strain are those involved in nitrogen metabolism. In addition, the most significantly downregulated genes included the betB gene, whose product catalyzes the synthesis of glycine betaine and confers tolerance to cold.ConclusionThe preliminary results suggest that the gdpS gene may participate in VBNC formation of S. epidermidis in face of adverse environmental factors, which is probably achieved by regulating expression of energy metabolism genes. Besides, the gdpS gene is critical for S. epidermidis to survive low temperature, and the underlying mechanism may be partly explained by its influence on expression of betB gene.

Quorum Sensing Orchestrates Antibiotic Drug Resistance, Biofilm Formation, and Motility in Escherichia coli and Quorum Quenching Activities of Plant-derived Natural Products: A Review

Quorum sensing (QS) is a type of cell-to-cell communication that is influenced by an increase in signaling molecules known as autoinducers, which is correlated to the increase in the density of microbial communities. In this review, we aim to discuss and provide updates on the different signaling molecules used by Escherichia coli, such as acyl-homoserine lactone (AHL), autoinducer-2 (AI-2), and indole to influence key phenotypes such as antibiotic drug resistance, biofilm formation, and motility during quorum sensing. Based on the literature, E. coli signaling molecules have different functions during cell-to-cell communication such that the increase in AHL and indole was found to cause the modulation of antibiotic resistance and inhibition of biofilm formation and motility. Meanwhile, AI-2 is known to modulate biofilm formation, antibiotic resistance, and motility. On the other hand, in the existing literature, we found that various plants possess phytochemicals that can be used to alter QS and its downstream key phenotypes such as biofilm formation, swimming and swarming motility, and genes related to motility, curli and AI-2 production. However, the exact physiological and molecular mechanisms of these natural compounds are still understudied. Understanding the mechanisms of those phytochemicals during QS are therefore highly recommended to conduct as a necessary step for future scholars to develop drugs that target the actions of QS-signaling molecules and receptors linked to antibiotic resistance, biofilm formation, and motility without putting bacteria under stress, thereby preventing the development of drug resistance.

Anthranilate Acts as a Signal to Modulate Biofilm Formation, Virulence, and Antibiotic Tolerance of Pseudomonas aeruginosa and Surrounding Bacteria.

ABSTRACTAnthranilate is a diffusible molecule produced by Pseudomonas aeruginosa and accumulates as P. aeruginosa grows. Anthranilate is an important intermediate for the synthesis of tryptophan and the Pseudomonas quinolone signal (PQS), as well as metabolized by the anthranilate dioxygenase complex (antABC operon products). Here we demonstrate that anthranilate is a key factor that modulates the pathogenicity-related phenotypes of P. aeruginosa and other surrounding bacteria in the environment, such as biofilm formation, antibiotic tolerance, and virulence. We found that the anthranilate levels in P. aeruginosa cultures rapidly increased in the stationary phase and then decreased again, forming an anthranilate peak. Biofilm formation, antibiotic susceptibility, and virulence of P. aeruginosa were significantly altered before and after this anthranilate peak. In addition, these phenotypes were all modified by the mutation of antABC and exogenous addition of anthranilate. Anthranilate also increased the antibiotic susceptibility of other species of bacteria, such as Escherichia coli, Salmonella enterica, Bacillus subtilis, and Staphylococcus aureus. Before the anthranilate peak, the low intracellular anthranilate level was maintained through degradation from the antABC function, in which induction of antABC was also limited to a small extent. The premature degradation of anthranilate, due to its high levels, and antABC expression early in the growth phase, appears to be toxic to the cells. From these results, we propose that by generating an anthranilate peak as a signal, P. aeruginosa may induce some sort of physiological change in surrounding cells.IMPORTANCE Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an “anthranilate peak”. This peak acts as a signal that induces physiological changes in surrounding cells, decreasing their antibiotic tolerance and biofilm formation. This study is important in that it provides a new insight into how microbial signaling substances can induce changes in the pathogenicity-related phenotypes of cells in the environment. In addition, this study shows that anthranilate can be used as an adjuvant to antibiotics.

Understanding the Role of the Antioxidant Drug Erdosteine and Its Active Metabolite on Staphylococcus aureus Methicillin Resistant Biofilm Formation.

Increasing numbers of researches have suggested that some drugs with reactive oxygen species (ROS)-mediated mechanisms of action modulate biofilm formation of some pathogenic strains. However, the full contribution of ROS to biofilm development is still an open question. In this paper, the correlations between the antioxidant drug Erdosteine (Er) and its active Metabolite I (Met I), ROS and biofilm development of two strains of methicillin resistant Staphylococcus aureus are presented. Experiments revealed that Er and Met I at 2 and 5 mg/L increased up to three orders of magnitude the number of biofilm-dwelling cells, while the content of ROS within the biofilms was reduced above the 87%, with a major effect of Met I in comparison to Er. Comparative proteomics showed that, 5 mg/L Met I modified the expression of 30% and 65% of total proteins in the two strains respectively. Some proteins involved in cell replication were upregulated, and a nitric oxide-based mechanism is assumed to modulate the biofilm development by changing quorum sensitive pathways. Additionally, several proteins involved in virulence were downregulated in the presence of Met I, suggesting that treated cells, despite being greater in number, might have lost part of their virulence.

The Vancomycin Resistance-Associated Regulatory System VraSR Modulates Biofilm Formation of Staphylococcus epidermidis in an ica-Dependent Manner.

ABSTRACTThe two-component system VraSR responds to the cell wall-active antibiotic stress in Staphylococcus epidermidis. To study its regulatory function in biofilm formation, a vraSR deletion mutant (ΔvraSR) was constructed using S. epidermidis strain 1457 (SE1457) as the parent strain. Compared to SE1457, the ΔvraSR mutant showed impaired biofilm formation both in vitro and in vivo with a higher ratio of dead cells within the biofilm. Consistently, the ΔvraSR mutant produced much less polysaccharide intercellular adhesin (PIA). The ΔvraSR mutant also showed increased susceptibility to the cell wall inhibitor and SDS, and its cell wall observed under a transmission electron microscope (TEM) appeared to be thinner and interrupted, which is in accordance with higher susceptibility to the stress. Complementation of vraSR in the ΔvraSR mutant restored the biofilm formation and the cell wall thickness to wild-type levels. Transcriptome sequencing (RNA-Seq) showed that the vraSR deletion affected the transcription levels of 73 genes, including genes involved in biofilm formation, bacterial programmed cell death (CidA-LrgAB system), glycolysis/gluconeogenesis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, etc. The results of RNA-Seq were confirmed by quantitative real-time reverse transcription-PCR (qRT-PCR). In the ΔvraSR mutant, the expression of icaA and lrgAB was downregulated and the expression of icaR and cidA was upregulated, in comparison to that of SE1457. The transcriptional levels of antibiotic-resistant genes (pbp2, serp1412, murAA, etc.) had no significant changes. An electrophoretic mobility shift assay further revealed that phosphorylated VraR bound to the promoter regions of the ica operon, as well as its own promoter region. This study demonstrates that in S. epidermidis, VraSR is an autoregulator and directly regulates biofilm formation in an ica-dependent manner. Upon cell wall stress, it indirectly regulates cell death and drug resistance in association with alterations to multiple metabolism pathways.IMPORTANCE S. epidermidis is a leading cause of hospital-acquired catheter-related infections, and its pathogenicity depends mostly on its ability to form biofilms on implants. The biofilm formation is a complex procedure that involves multiple regulating factors. Here, we show that a vancomycin resistance-associated two-component regulatory system, VraSR, plays an important role in modulating S. epidermidis biofilm formation and tolerance to stress. We demonstrate that S. epidermidis VraSR is an autoregulated system that selectively responds to stress targeting cell wall synthesis. Besides, phosphorylated VraR can bind to the promoter region of the ica operon and directly regulates polysaccharide intercellular adhesin production and biofilm formation in S. epidermidis. Furthermore, VraSR may indirectly modulate bacterial cell death and extracellular DNA (eDNA) release in biofilms through the CidA-LrgAB system. This work provides a new molecular insight into the mechanisms of VraSR-mediated modulation of the biofilm formation and cell death of S. epidermidis.

Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

Bidirectional Cell-Cell Communication via Indole and Cyclo(Pro-Tyr) Modulates Interspecies Biofilm Formation.

The extracellular signaling molecule indole plays a pivotal role in biofilm formation by the enteric gammaproteobacterium Escherichia coli; this process is particularly correlated with the extracellular indole concentration. Using the indole-biodegrading betaproteobacterium Burkholderia unamae, we examined the mechanism by which these two bacteria modulate biofilm formation in an indole-dependent manner. We quantified the spatial organization of cocultured microbial communities at the micrometer scale through computational image analysis, ultimately identifying how bidirectional cell-to-cell communication modulated the physical relationships between them. Further analysis allowed us to determine the mechanism by which the B. unamae-derived signaling diketopiperazine cyclo(Pro-Tyr) considerably upregulated indole biosynthesis and enhanced E. coli biofilm formation. We also determined that the presence of unmetabolized indole enhanced the production of cyclo(Pro-Tyr). Thus, bidirectional cell-to-cell communication that occurred via interspecies signaling molecules modulated the formation of a mixed-species biofilm between indole-producing and indole-consuming species. IMPORTANCE Indole is a relatively stable N-heterocyclic aromatic compound that is widely found in nature. To date, the correlations between indole-related bidirectional cell-to-cell communications and interspecies communal organization remain poorly understood. In this study, we used an experimental model, which consisted of indole-producing and indole-degrading bacteria, to evaluate how bidirectional cell-to-cell communication modulated interspecies biofilm formation via intrinsic and environmental cues. We identified a unique spatial patterning of indole-producing and indole-degrading bacteria within mixed-species biofilms. This spatial patterning was an active process mediated by bidirectional physicochemical interactions. Our findings represent an important step in gaining a more thorough understanding of the process of polymicrobial biofilm formation and advance the possibility of using indole-degrading bacteria to address biofilm-related health and industry issues.