Christal J. Thomas November 7, 2010 BCMB 409/ Section 001 Professor: Tom Dockendorff Bacteria: Communication Equals Modification Bacteria are organisms that are extremely copious upon this planet. They are tiny and most are single celled organisms that can survive in just about any environment. Anywhere from plants to the human body is where these organisms can be discovered. Some of the strangest places that support bacterial life include places that have extremes of temperature. These bacteria are also very strange, much different from bacteria found living in and around humans.
For example bacteria that live in extreme cold, like the North Pole, use methane as their substrates; and the ones that live in the deep sea use hydrogen sulphide. While most bacteria can live without oxygen (anaerobic bacteria) or whether they are aerobic bacteria that require oxygen. These particular bacteria use carbon-based sugars as main energy source. Even with so much diversity among bacteria, the most interesting part is how they communicate. Cell communication is a central mechanism in bacteria cells because it provides examples of parasitism relationship among different organisms such as S. aureus.
It gives a means to control infections through a complex mechanism which is found in Pseudomonas aeruginosa. This touches on some variety of processes, including genetic transfer, antibiotic production growth and pathogenesis. Many bacteria cells possess a unique communication system that allows them to exchange information within their own cell or among other cells. There have been recent studies about the communication among bacteria cells by using small molecules in order to communication with each other. These small molecules could consist of amino acid, peptide chains, or fatty acid derivatives.
Moreover, these molecules aid the cells into exhibiting multi-cellular behaviors. The bacterium has the ability to sense diffusible signal molecules that are generated by other cells to modulate specific adaptive responses that enhance their survival rate. The ability of a signal bacterium to communicate among its neighbors, by using different mechanisms, creates a unified response that is essential for its survival and development. One unique characteristic of cell-cell communication is the liberation of light by the organism.
This process is known as bioluminescence, but the most studding aspect of the biochemical pathway is its regulation. Recent studies have explained how the actions of the ATP-dependent Lon protease of E. coli were involved with the downregluation of the Vibrio fischeri Lux operon. In addition, scientists have discovered that LuxR was a target Lon protease. Therefore, to show that this discovery was valid, they used a plasmid model to show the effects of Lon protease on V. fischeri LuxICDABEG expression. The use of Lux operon genes or DNA fragments encoding either full-length LuxR or its carboxyl-terminal end helped in this process.
The more potent activator is the full-length LuxR protein than its carboxyl terminus. Up-regluation was seen at intracellular concentrations approximately two orders of magnitude lower than its carboxyl terminus. The regulatory mechanism has two aspects that work together in a synergistic manner. The first aspect focus on how cells communicate to cells nearby that they exist, and each cell must decide if there are several cells present. This determination is carried out by a signal molecule that is formed by each cell and then ejected into the surrounding media.
This signal molecule is dictated as an autoinducer. Secondly, the cellular machinery has to be able to receive the message. Once it is able to do this action, it will translate and act upon the message. Since the response from the cell gears towards the activation of the Lux operon, then this machinery must be a transcription factor interacting with the Lux operon. The LuxR is an operon encoded by the LuxR gene. Two regulatory genes’ construed products were identified as homologous to the LuxR regulatory protein. These regulatory genes are known as gdmRl and gdmRll.
Besides these genes, the LuxR protein has two important domains known as the amino terminus domain and the carboxyl terminus domain. These domains interact with the autoinducer and the promoter region on the Lux operon. Genetic transfer is any process in which an organism incorporates genetic material from another organism without being the progeny of that organism. For instance, Pseudomonas aeruginosa is a flexible pathogen that takes advantage of any opportunity it can. This will infect pretty much anything from plants, rodent and so much more. They are freely transmissible .
The genetic transfer of the P. aeruginosa to a plant is just one example. “The antibiotic resistance spectrum of R+ Erwina recipients was similar to those of other bacteria harboring these R factors, but maximum resistance levels differed with each recipient. The spontaneous elimination of these factors from the Erwinia strains and the ability to transfer multiple antibiotic resistances suggest that these exist as plasmids in these hosts. Several, but not all, RP1-carrying Erwinia strains were sensitive to the RP1 specific phage PRR1. The R factor R18-1 was also transferred from P. eruginosa to Erwinia spp. R18-1 was unstable in all Erwinia strains. Stable strains were isolated in which R18-1 could not be eliminated by sodium dodecyl sulfate and could not be transferred to other strains,” (Cho, 1975). For horizontal (lateral) gene transfer (Picture 1) occurs when an organism exchange genetic information from another organism without being the offspring of that organism. Bacterial cells in a biofilm may cooperate metabolically and evolve as a community by horizontal gene transfer (Kukavica-Ibrulj, 2008). Just like in the chronic lung infection in the rat model.
By looking at the potential contributions of the Picture 1. Current tree of life demonstrating horizontal (lateral) gene transfer. associated bacteria to the pathogenicity of P. aeruginosa, the virulence of P. aeruginosa in the presence of an oropharyngeal flora (OF) bacterium was tested in vivo using the agar bead rat lung infection model (Duan, 2003). A group of rats with only P. aeruginosa PAO1 was compared with two other groups with only CF004 alone and PAO1 plus CF004 (a Streptococcus strain isolated from cystic fibrosis sample) Fig. 1. A. The rat lungs co-infected with P. eruginosa and OF strain CF004 (group 2) showed significantly more consolidation than that infected with P. aeruginosa alone (group 1) or OF alone (group 3) (P < 0. 0001, unpaired t-test). B. P. aeruginosa (solid bars) and CF004 (grey bars) bacterial loads (cfu) in the lungs of the three groups. to analyze the results of the infection. After seven days, lung pathology was performed on the rats. The results indicated consolidation accumulation of pulmonary edema fluid and/or infiltration of inflammatory cells. More consolidation denotes to more severe lung injury (Fig. 1A). There was no specific change in P. eruginosa loads in the co-infected group compared to the group infected with just the P. aeruginosa (P = 0. 4) (Fig. 1B). Likewise, oropharyngeal flora loads remained unchanged in the co-infection group and when it is presence alone (P = 0. 9). Among the genes affected by OF are a relatively large number of well-characterized P. aeruginosa virulence factor genes or genes relevant to P. aeruginosa pathogenicity (Duan, 2003). For instance, lasB plays a part in the inflammatory damage of the respiratory epithelia and impedes the host immunological defenses by up-regulating of it to a maximum of sevenfold throughout a 24 hour time course.
XcpP was the most activated gene in this group. The up-regulation PA1282 and PA1882 (two probable multidrug efflux genes) also took place. Yet, the importance of bacterial antibiotic resistance is not always associated with efflux pumps but can contribute to a pathogen’s invasiveness by assisting export of virulence determinants. Resistant is becoming more wide spread among bacterial strains; which include a current drug of choice for methicillin-resistant Staphylococcus aureus (MRSA) called vancomuycin. Resistant towards many antibiotics is a difficult problem with the P. eruginosa bacterium. Therefore, drugs with new mechanisms of action are urgently needed to combat the growth of antibiotic-resistant bacteria (Jiang, 2008). However, some bacteria can form communities enmeshed in a self-produced polymeric matrix by attaching themselves to surfaces and producing a biofilm. Biofilm is a difficult problem that is brought on by the bacteria P. aeruginosa. It is a common environmental microorganism that has acquired the ability to take advantage of weaknesses in the host immune system to become an opportunistic pathogen in humans (Jiang, 2008).
The pulmonary tract, urinary tract, burns, wounds, and other blood infections are usually infected by P. aeruginosa. Its motility by swimming, swarming, and twitching and its capacity of forming a biofilm are recognized as playing vital roles in the ability of the bacterium to adapt to and colonize various ecological niches, including the human lung (Kukavica-Ibrulj, 2008). In another rat study, the use of PAO1, PA14 (a human isolate), and LESB58 (hyper-virulent human cystic fibrosis isolate) was used to identify pathogenicity of P. aeruginosa.
The findings of two pathogenicity islands (PAPI-1 and PAPI-2) and a broad degree of conservation of virulence genes were identified. These pathogenicity islands may contribute to the augmented extra-Fig. 2. In vivo growth curves for P. aeruginosa strains PAO1 (A), PA14 (B), and LESB58 (C) in the rat model of chronic lung infection for 14 days. Rats were infected with agarose-embedded bacteria at 1 x 106 CFU for each strain. At different time points (1, 3, 7, and 14 days post-infection), five animals were used from each group and CFU were determined from infected lungs. air copulation of the highly virulent PA14 strain. In contrast, the PAO1 strain is highly transmissible and aggressive virulent epidemic strain. It has a more extensive spectrum of antibiotic resistance, and a better adaptation to the cystic fibrosis lung. Bacterial growth was monitored by determining colony-forming unit (CFU) from lung tissues at a certain point of time from day 1 to day 14 post-infection. This was experimented to compare the capacities of the strains to initiate and establish a chronic lung infection in vivo. As depicted in Fig. , the overall growth curves were similar for the three strains tested, with a peak of CFU at day 1 and a reduction in CFU from day 3 to day 7 (kukavica-Ibrulj, 2008). At day seven, a plateau was established and there were fewer variations in CFU from that day to day 14. A lower count of bacterial was found in PA14, expect on day 3. The CFU was higher at day 3 for LESB58 but much lower at day 7 and 14 post-infection. This revealed that different P. aeruginosa strains were able to initiate and maintain an infection in the rat lung. Fig. 3. Localization and persistence of PAO1, PA14, and LESB58 in the rat lung at 7 days post-infection.
Rats were infected with P. aeruginosa strains embedded in agarose beads, the lungs were fixed and investigated histologically, and bacteria were localized by indirect immunofluorescence. (A, E, and I) HE-stained rat lung histology at 7 days after infection with agarose-embedded PAO1 (A), PA14 (E), and LESB58 (I). Inflammatory cell infiltrations are evident in the thickened alveolar septa of rat lung for the PAO1 and PA14 strains, while for the lungs infected with LESB58, the recruitment of neutrophils is predominantly in the bronchial lumen, where the beads are still localized. C, G, and K) At day 7, P. aeruginosa bacterial macro-colonies were detected by indirect immunofluorescence (IF) (red) in the thickened alveolar septa of rat lungs infected with strains PAO1 (C) and PA14 (G), while for LESB58, (K) bacterial colonies were still present in the agar beads. (B, F, and J) DAPI (blue) staining of the same tissue sections. (D, H, and L) Merge of the DAPI-stained slides (blue) and bacteria localized by IF (red). Bars, 50 ? m. In order to locate these bacteria, histological and immunofluorescence methods were used to assist in this process.
These methods were performed at days 1 and 3 to show that the bronchial lumen is where PAO1, PA14, and LESB58 bacteria induced an intense inflammatory response. At day seven, data showed PAO1 and PA14 cells in the alveolar region, where they can form biofilm/macro-colonies with extensive inflammation in sub-mucosa and alveoli (Fig. 3D). In comparison, LESB58 bacterial cells were still available in the bronchial lumen (Fig. 3L). There bacteria cells were not found in the same location when the chronic infection was established, even though equal CFU was utilized to form the chronic lung infection.
Measuring the virulence of the three P. aeruginosa strains was done by mixing equal ratios of each strain in agar beads. Next, the mixture obtained was inoculated into the rat’s lung and bacteria was enumerated form the lungs at the seventh day post-infection. The competitive analysis among PAO1 and PA14 showed a large variation in lung CFU between each animal and a mean confidence interval (CI) value of 0. 002. This outcome implied a 1,000-fold reduction of PA14 in vivo when in competition with PAO1. The Cl among PAO1 and LESB58 was 0. 002. This result indicated a 10,000-fold diminution of LESB58 in competition with PAO1. There was a 10-fold attenuation of PA14 by LESB58, giving it a CI value of 0. 14. Chemical attenuation can also be considered as a means to address antibiotic-mediated killing of the bacteria. Both Staphylococci and Pseudomonas employ a mechanism known as quorum sensing to regulate the major parts of their arsenal of virulence factors. Quorum sensing inhibitors are connected with the developments of anti-microbial measurements.
Theses inhibitors are not anticipated to produce the austere selection pressure seen with antibiotics. This should, in theory, keep development of resistance to quorum sensing inhibitor drugs at a low level, although the magnitude of quorum sensing inhibitor drug resistance must be investigated in animal models (Bjarnsholt, 2007). Nevertheless, the quorum sensing system helps bacteria to evade the immune system by delaying production of damaging virulence factors until the number of bacteria amassed is sufficient to cause a successful infection (Bjarnsholt, 2007).
Quorum sensing is found in all types of bacteria cells. It is a mechanism used to accurately communicate among different cells once the proper concentration of signaling molecules or chemicals has been achieved. In the late 1900s, researches show that bacteria did not emit light just towards any appreciable amount until the population of cells reached a concentrated culture. The luminous bacteria has about 100 cells or less per milliliter, and do not make appreciable amount of light. A successful concentration of 1010 to 1011 cells per milliliter will emit light.
In the gram-negative bacteria they make certain inducers called N-acyl homoserine lactose (AHL) that is detected by certain receptors. Once AHL binds to these receptors, they activate transcription and those for inducer synthesis. Fatty acid derivatives are utilized as signaling molecules. In contrast, the gram-positive bacterium uses amino acids and short peptides (oligopeptides) as signaling molecules. These molecules are secreted through a transporter called the ATP-binding cassette (ABC) transporter complex. The detectors for these signals are two-component signaling transduction system.
One is known as the sensor kinase, which leads to autophorylation at conserved histidine residue when the autoinducer binds. The other is recognized as the response regulator that works with the sensor kinase to the binding of a regulator to a specific target promoter. The sensor kinase in this case phosphorylates a conserved aspartate residue so the target promoter can interact with the regulator. Each bacterium contains specific mechanisms dealing with quorum sensing. In gram-negative bacteria, if the number of bacteria is low in the cell, then the concentration of AHL is reduced.
However, if the number of bacteria is high in the cell, then the concentration of AHL is increased. Then a critical threshold volume is reached in AHL, if different kinds of bacteria are present. The autoinducer is now able to enter the cell, bind to the LuxR, and cause a positive feedback loop complex. This will make the receptor active. In addition, the positive feedback loop then activates the Lux operon by binding to the RNA polymerase to the promoter region. The LuxR-autoinducer complex and the RNA polymerase synergistically bind to the Lux box and the -10 region of the right promoter.
Once this threshold is exceeded, the transcription of pLux is activated. In the gram-positive bacteria, quorum-sensing mechanism could enable staphylococcal cells to stop producing factors that facilitate binding to a particular surface when the population becomes large, and instead produce secreted factors that might permit the cells to penetrate new sites in which there are more nutrients and less competition (Dunny, 1997). Staphylococcus strains and Streptococcus strains have also been used in experiments for screening of P. aeruginosa genes modulated by OF bacteria.
A low-copy-number plasmid pMS402 was revealed when using a P. aeruginosa random promoter library constructed with the luxCDABE reporter in vitro. The amount of light produced by the clone identifies the activity of any individual promoter. Temporary screening of the P. aeruginosa library can identify differentially regulated promoters by measuring luminescence in a multilabel plate counter. Screening for 3456 P. aeruginosa (ATCC27853) clones for differentially expressed promoters in the presence of two Gram-positive OF bacteria, Streptococcus strain CF004 and Staphylococcus Figure 4.
Scatter plot of the initial screen data at the 7. 5 h time point. P. aeruginosa promoter clones were screened for differential expression in response to the presence of CF004 (A) or CF018 (B). Each point in the plots represents the activity of one individual promoter in the library. The promoters unaffected by the co-culture conditions (blue) fail along the diagonal. Points in red distributed above and below the diagonal indicate promoters with a minimum 2. 5-fold downregluation and upregulation, respectively, in the presence of CF004 or CF018. C.
Red to green colour gradient indicates the levels of expression from high to low. Three classes of regulated promoters are marked: those regulated by both strains (class I), CF004 only (class II) or CF018 only (class III). strain CF018 (Duan, 2003). The results revealed that 280 promoters regulated by the CF004 strain and 252 by the CF018 strain (Fig. 4A & 4B). However, retesting of these positive bacteria confirmed 214 affected promoters by CF004 and 171 by CF018 by resolving gene expression profile combined with growth evaluation. This signifies 6% and 5% of the P. eruginosa clones respectively. Only 152 were common to both bacterium strains. Three classes were drawn from the regulated P. aeruginosa. The first one focus on those regulated by both strains (class I), the second one is only about CF004 (class II), and the third focus on CF018 only (class III) (Fig. 4C). The different regulation of P. aeruginosa promoters by these two strains suggests that there are common as well as unique signals or pathways in the interactions between P. aeruginosa and these Gram-positive bacteria (Duan, 2003). Comparison of these affected promoters and the annotated P. eruginosa PAO1 genome was done alone with GenBank data (http://www. ncbi. nim. nih. gov) to identify the genes regulated. Expression of 48 operons with known or putative gene functions was found in the regulated promoters. In addition, there were 33 characterized promoters that were associated with genes encoding proteins with unknown function. Seven promoters were found at the 5’ end of the annotated genes. They also adjust to the opposite direction; and designated orphan promoters were identified at this location. This could lead to regulating gene expression on the other strand.
No sequence homology in another 14 promoters was identified with the PAO1 genome, for they do not share homology with any other bacterial sequences in the GenBank. In conclusion, bacteria cell to cell communication is a very unique system that exchange information among themselves or towards other cells. The cells are able to transfer genetic material, be pathogenic, and grow due to the mechanism employed by the quorum sensing and/or biofilm. This was shown in the study when growth rate in the rat lung infection model for three widely used P. aeruginosa strains, PAO1, PA14, and LESB58 were evaluated.
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