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Quorum Sensing and Bacterial Biofilm Formation

Updated August 24, 2021
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Quorum Sensing and Bacterial Biofilm Formation essay

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Introduction

Biofilm is a type of microbial colony that secretes mucilaginous substances enabling microorganisms to anchor to surfaces and each other. There are many beneficial roles for biofilm such as breaking down contaminants in soil or assisting in the digestive tract (Ungvarsky, 2015). However, biofilm can also be harmful when the sticky, matrix-enclosed communities allow bacteria to evade the immune system, increase antibiotic resistance and shed planktonic cells to other locations in the body (Jackson et al., 2002). Biofilm formation is multifactorial and complex. Extracellular signals and quorum sensing play a role in cell-to-cell communication during biofilm development while nutrient load and intracellular carbon flux mediated by the RNA-binding global regulator protein CsrA (carbon storage regulator) may alter cellular expression allowing bacteria to adapt during the process (Jackson et al., 2002; Kjelleberg & Molin, 2002).

Background

Biofilm can form on a variety of surfaces such as living tissues, medical devices, water system piping and natural aquatic systems (Donlan, 2002). These sessile communities are unique because they possess properties, behaviors, and abilities that the individual cell does not exhibit (Ungvarsky, 2015). A dose of antibiotic may be enough to destroy a single cell but in the case of a biofilm colony, a treatment many hundreds of times stronger would be required (Donlan, 2002). As a public health concern, biofilm has been implicated in infectious diseases and chronic bacterial infections including Escherichia coli (E. coli) urinary catheter cystitis, biliary tract infections, and cystic fibrosis (Davies et al., 1998; Jackson et al., 2002).

In a paper by Davies et al., it was demonstrated that a correlation exists between cell-to-cell signaling and biofilm development in Pseudomonas aeruginosa. Further analysis of genomic functions revealed the performance and properties of biofilm leading to a consensus model among researchers (Kjelleberg & Molin, 2002). The first part of the model states that microbial communities develop on surfaces through processes of adhesion, growth, motility and ESP formation. Second, the development of biofilm involves quorum sensing (QS)-mediated control signals. Kjelleberg and Molin assessed the merit of the QS model in biofilm formation in their paper.

Quorum sensing is the method by which bacteria regulate gene expression in a density-dependent manner (M. B. Miller & Bassler, 2001). During quorum sensing, bacteria produce and release chemical signals called autoinducers that can freely diffuse across cell membranes and alter gene expressions (Kjelleberg & Molin, 2002; Vadakkan, Choudhury, Gunasekaran, Hemapriya, & Vijayanand, 2018). The positive feedback mechanisms of autoinducers allow a population of cells to express the appropriate phenotype necessary for survival or cellular differentiation purposes (Kjelleberg & Molin, 2002).

The gene, csrA, encodes for the small RNA-binding protein CsrA. Research has shown that disruption of this gene in E. coli causes adherence to culture tubes and formation of a coating resembling biofilm, but it was only observed under a single growth condition (Jackson et al., 2002; Romeo et al., 1993). In E. coli, CsrA acts on several metabolic pathways to mediate carbon flux. In the stationary phase of growth, CsrA suppresses glycogen biosynthesis, glycogen catabolism, and gluconeogenesis (Jackson et al., 2002; T. Romeo, Gong, Liu, & Brun-Zinkernagel, 1993; Sabnis, Yang, & Romeo, 1995). However, in glycolysis, acetate metabolism, and motility, CsrA has an activating role (Jackson et al., 2002; Sabnis et al., 1995).

During post-transcriptional regulation, CsrA can upregulate or downregulate gene expression by binding to mRNA transcripts (Jackson et al., 2002; M. Y. Liu & Romeo, 1997; Wei, Shin, LaPorte, Wolfe, & Romeo, 2000). Prior studies have shown that csrA homologs are present in Bacteria, Firmicutes, Proteobacteria, Thermotogales, and Spirochaetales but not eukaryotes, suggesting that CsrA affects the interaction between host and microbe (Tony Romeo, 2002). A study conducted by Jackson et al. sought to investigate the role of CsrA regulation in biofilm development further.

Methods

Bacterial cell line and media: Cultures used in biofilm assays grew at 26°C in Luria-Bertani (LB) media (Jackson et al., 2002; J. H. Miller, 1972). Colony-forming antigen (CFA) media and artificial urine media were used to carry out biofilm assays (Evans, Evans, & Tjoa, 1977; Jackson et al., 2002). Assessment of glycogen biosynthesis was performed using Kornberg agar media (Jackson et al., 2002; M. Y. Liu, Yang, & Romeo, 1995).

Quantitative biofilm assay: Inoculated cultures grew overnight in 96-well microtiter plate. Biofilm growth was assessed using crystal violet staining assay, and planktonic cell growth was evaluated by microtiter plate reader absorbance at 600 nm (Jackson et al., 2002; O’Toole & Kolter, 2002).

Molecular and genetic techniques: Expression studies required the use of a chromosomal csrA’-lacZ translational fusion transduced into E. coli K-12 strain CF7789 (Jackson et al., 2002; J. H. Miller, 1972).

β-Galactosidase, protein, and glycogen assays: After removing planktonic cells from cultures used for β-galactosidase assays, the remaining biofilm was pipetted in CFA media to break up aggregates. β-galactosidase activity was assessed following protocols from Romeo et al. (Jackson et al., 2002; Tony Romeo, Black, & Preiss, 1990). Protein assays were performed using the bicinchoninic acid method (Jackson et al., 2002; Smith et al., 1985). Glycogen assay looked at phenotypes by staining cells with iodine vapor (Jackson et al., 2002; Mu Ya Liu et al., 1997).

Results

Quorum sensing on biofilm formation: The acylhomoserine lactone (AHL)-based quorum sensing system observed in several gram-negative bacteria is suggested to regulate genes for colonization of eukaryotes in contrast to other QS systems (Kjelleberg & Molin, 2002). Kjelleberg and Molin review studies that have observed AHL-QS dependence governing biofilm formation on living surfaces such as P. aeruginosa and on inanimate surfaces in mixed- and monoculture species(Charlton et al., 2008; J. W. Costerton, Stewart, & Greenberg, 1999; McLean, Whiteley, Stickler, & Fuqua, 1997). Claims regarding AHL influence on biofilm heterogeneity, architecture, resistance to stress, maintenance and shedding were also noted. Kjelleberg and Molin state that when comparing WT and isogenic variants, research shows that a deficiency in QS signal molecules provides evidence for a relationship between QS regulation and biofilm formation. However, QS is not the only type of control exploited by bacteria. The biofilm community will utilize multiple mechanisms to survive and thrive under various conditions. For example, the global regulator protein CsrA has been shown to exert influence on biofilm formation through regulation of central carbon flux.

csrA mutant vs. wild-type (WT) biofilm formation: Researchers observed similar growth rates in WT E. coli K-12 strain MG1655 and the isogenic csrA mutant, including delayed biofilm development until the cultures reached the stationary phase of growth (Jackson et al., 2002). However, following the stationary phase of growth, csrA mutant biofilm formation was observed to be more prolific. The csrA mutant was found to exhibited features of mature biofilm such as pillars and channels used to facilitate nutrient exchange and elimination of waste (J. William Costerton, Lewandowski, Caldwell, Korber, & Lappin-Scott, 1995; Jackson et al., 2002). Researchers noted that disruption to the csrA gene stimulates increased adherence and development of biofilm in every growth medium tested in contrast to the single growth condition previously studied.

csrA overexpression in E. coli and clinical strains: In csrA overexpression studies, overexpression inhibited growth in WT E. coli K-12 and the csrA mutant. Biofilm formation in clinical strains that colonize urinary catheters, E. coli P18, and Citrobacter freundii P5, were also observed to be inhibited, while food-borne pathogens, E. coli 0157:h7 strain EF302 and S. enterica serovar Typhimurium ATCC 14028, showed moderate inhibition (Jackson et al., 2002).

CsrB antagonizes CsrA activity by sequestering roughly 18 of its subunits. Null mutations in csrB result in similar outcomes seen with csrA overexpression, while overexpression of csrB increases csrA disruption (Mu Ya Liu et al., 1997; Seshagirirao Gudapaty, Kazushi Suzuki, Xin Wang, Paul Babitzke, & Romeo, 2001). Jackson et al. experiments confirmed that biofilm formation was deficient in the csrB null mutant.

Effects of CsrA in strains defective for extracellular and/or surface molecules factors on the formation of biofilm: Attachment and adherence in E. coli involves curli fimbriae, type I pili, and flagella (Pratt & Kolter, 1999; Vidal et al., 1998). Researchers performed experiments to investigate biofilm formation in E. coli with strains lacking one or more of these extracellular and/or surface factors that participate in the development of biofilm. Experiments showed that isogenic csrA mutants grew considerably compared to the WT. Loss of type I pili in csrA mutant decreased the formation of biofilm and loss of curli fimbriae in mutants lacking colonic acid (required to generate E. coli’s 3D biofilm structure) and type I pili result in increased formation of biofilm (Jackson et al., 2002).

Because initial stages of cell attachment during biofilm formation in E. coli require flagella and motility, researchers studied effects of ΔmotB mutation that renders cells nonmotile (Jackson et al., 2002; Pratt & Kolter, 1999). WT csrA with motility disrupted were unable to form biofilm compared to csrA mutant decreased biofilm formation. Deletion of type I pilus had no additional effects on csrA motB mutant suggesting that csrA mutant can form biofilm without requirements of extracellular and or/surface factors (Jackson et al., 2002).

Glycogen synthesis and catabolism mediated by CsrA: csrA gene is known to be a repressor of glycogen biosynthesis, and studies have shown that CsrA plays regulatory roles in glycogen biosynthesis and catabolism during the early stationary phase of growth (T. Romeo et al., 1993). These processes showed an acceleration in the csrA mutants (Yang, Liu, & Romeo, 1996). Since CsrA represses biosynthetic genes, glg, Jackson et al. sought to evaluate the role of intracellular glycogen during biofilm formation. What researchers found was that csrA mutation did not impact biofilm formation by the glgA (glycogen synthase) knock out mutant. Further studies on glg genes showed that CsrA effects on biofilm formation were through intracellular regulation of glycogen biosynthesis and catabolism (Jackson et al., 2002).

Biofilm dispersal of planktonic cells: Biofilm planktonic cells can spread infections to distal areas of the body. Therefore, the role CsrA on biofilm dispersal is of interest. Researchers investigated three different conditions and results showed that csrA expression signal for biofilm dispersal.

CsrA expression regulates biofilm formation: The experiments above suggest that CsrA effects E. coli biofilm development through various regulations. Jackson et al. followed up with an investigation to observe if there are any modification to csrA expressions during the development of biofilm by using a chromosomal csrA’-‘lacZ translational fusion construct. Researchers monitored β-galactosidase activity in planktonic cells and sessile cells. Results showed β-galactosidase activity decreased in planktonic cells before biofilm appeared and remained consistent after that. In the sessile biofilm, β-galactosidase activity sharply declined during the first few hours but once biofilm growth ceased, activity moderately increased. Data suggests that csrA’-‘lacZ translational fusion is dynamically regulated during biofilm formation (Jackson et al., 2002).

Conclusions & Summary

Research suggests that QS regulation plays a role in biofilm formation for several organisms under specific sets of conditions, but it is not the only type of control exploited by the bacterial community (Kjelleberg & Molin, 2002). Differentiated biofilm is likely the result of multiple interactions. The authors, Kjelleberg and Molin, posed several questions in their paper that still need to be answered such as a need to evaluate whether there are organisms that form biofilm independently from QS or other global regulators, investigating if discrete events during biofilm development target specific genes in response to QS or what proportion of QS-regulated genes are controlled directly (Kjelleberg & Molin, 2002).

Biofilm formation is a complex process, but research continues to uncover regulatory mechanisms that play important roles in its development. The above experiments conducted by Jackson et al. demonstrate the importance of the RNA-binding protein, CsrA, on E. coli biofilm formation. Compared to previous research on E. coli biofilm regulatory factors, Rpos (a sigma factor required for transcription of station phase genes) and OmpR (outer membrane protein regulator), CsrA had a more significant impact on biofilm formation (Adams & McLean, 1999; Jackson et al., 2002; Vidal et al., 1998). CsrA and its gram-negative bacteria homologs repress biofilm formation, in contrast to CsrB activation of biofilm formation by antagonizing CsrA. Evidence for CsrA repression of biofilm formation in uropathogenic strains of E. coli and C. freundii (pathogenic relatives of E. coli K-12) was also noted in ectopic expression studies by Jackson et al. Further investigation may be beneficial for the public health sector.

Disruption to the csrA gene stimulated biofilm formation regardless of the presence of extracellular or surface factors that are known to be involved in biofilm development. In another study, Jackson et al. explored the influence of the CsrA protein on genes that participate in curli and type I pili synthesis. Under certain conditions, CsrA influence gene expression for curli and type I pili. Therefore, CsrA may affect multiple properties during biofilm development (Jackson et al., 2002).

Experimental results from Jackson et al. provide evidence that CsrA impact biofilm formation in E. coli through regulation of glycogen metabolism. Disrupted glgA in mutant csrA had comparable biofilm growth as seen in WT. Glycogen phosphorylase assays were conducted to evaluate the need for glycogen during biofilm formation. It was observed that catabolism of glycogen, through the activity of glycogen phosphorylase, was a requirement for the creation of biofilm (Jackson et al., 2002). Researches concluded that CsrA influences the flux of carbon into glycogen and from glycogen into glucose-1-phosphate. Jackson et al. also believe that glycogen is the energy and carbon source utilized for synthesizing adhesins and other factors during stationary phase growth. Further research is suggested to investigate the mechanisms of glycogen on biofilm formation in other pathogens.

CsrA serves as a repressor of biofilm formation but also as an activator of biofilm dispersal under a variety of conditions. Future research is suggested to investigate compounds that can behave like CsrA or csrA. These compounds could offer therapeutic benefits that inhibit biofilm formation or to take advantage of the dispersal process to target antibiotics.

Lastly, Jackson et al. explored whether biofilm development modifies csrA expression by studying the chromosomally encoded csrA’-‘lacZ translational fusion. The csrA’-‘lacZ translational fusion was observed to be dynamically regulated during the development of biofilm leading researchers to pose that intracellular availability of the CsrA protein may play a role in directing biofilm formation. Researchers concluded that the global regulator protein, CsrA, influence central carbon flux in E. coli biofilm formation and further insights into its role would be beneficial for elucidating regulatory mechanisms of biofilm development and dispersal.

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Quorum Sensing and Bacterial Biofilm Formation. (2021, Aug 24). Retrieved from https://samploon.com/quorum-sensing-and-bacterial-biofilm-formation/

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