The highest fold change result was 2. Microarray analysis of B. Glycosyl linkage analysis was also performed [ 55 ]. The predominant glycosyl residue detected was a terminally-linked heptopyranosyl t-Hep at a percentage of Other residues detected were a terminally-linked and a 4-linked glucopyranosyl t-Glcp 4-Glcp at percentages of Although significant advances have been made in the field, melioidosis continues to be a public health concern in many regions of the world [ 59 ].
Completion of the sequencing of the B. Further studies are ongoing to define the pathogenesis of B. To obtain virulence determinants unique to B. Analysis of the subtractive hybridization library revealed that B.
One of the subtraction clones, pDD, demonstrated weak homology to a glycosyltransferase, WbpX, from P. The insert from pDD was cloned into a mobilizable suicide vector for insertional inactivation of the glycosyltransferase gene in wild-type B. The resulting strain, SR, was markedly less virulent than the parent strain in an animal model. We determined that SR harbored a mutation in a glycosyltransferase gene involved in the production of a capsular polysaccharide which we subsequently designated as CPS I.
We then identified the operon involved in the biosynthesis and transport of this capsular polysaccharide CPS I [ 42 ]. The genes identified encode for proteins that are similar to proteins involved in the biosynthesis and export of capsular polysaccharides, particularly those involved in the production of group 3 capsular polysaccharides.
Group 3 capsules are always coexpressed with O serogroups, are not thermoregulated, are transported by an ABC-2 exporter system, and do not contain the kpsU and kpsF genes, and usually the gene clusters map near the serA locus [ 8 ]. Thus far, no serA locus that is associated with the type I O-PS cluster was identified, but this polysaccharide is coexpressed with O antigen and lacks the kpsU and kpsF genes, and genes encoding for a putative ABC-2 transporter have been identified.
The genes involved in the production of group 3 capsules are organized into regions and are divergently transcribed. Regions 1 and 3 are generally conserved and contain genes involved in export of the polysaccharide. These regions flank region 2, which contains the biosynthetic genes and is not conserved between serotypes [ 4 ]. The genetic organization of the CPS I is also similar to that of other capsule gene clusters in that the genes are organized into more than one transcriptional unit and appear to be divergently transcribed [ 42 ].
However, our results suggested that this polysaccharide is a capsule rather than an O-PS moiety. Western blot analysis of proteinase K cell extracts and silver staining showed that this polysaccharide has a high molecular mass kDa and lacks the banding pattern seen with O-PS moieties. This conclusion was further supported by another group of researchers that demonstrated this polysaccharide is a capsule rather than an O-PS component of LPS because it lacks a lipid A moiety and was not capable of macrophage activation [ 49 ].
Studies by our laboratory have indicated that mutants in the production of the core oligosaccharide of the LPS are still capable of producing this polysaccharide [ 48 ]. Based on the above criteria and the genetic similarity to group 3 capsules, we proposed that this polysaccharide is a capsule.
Virulence genes of a number of pathogenic bacteria are located on pathogenicity islands PAIs , regions on the bacterial chromosome that are present in the genome of pathogenic strains but rarely present in those of nonpathogenic strains. These genetic units are often flanked by direct repeats and may be associated with tRNA genes or insertion sequence IS elements at their boundaries.
They may also be associated with the presence of mobility genes, such as IS elements, integrases, transposases, and origins of plasmid replication. These DNA regions are considered to be unstable in that they may be subject to deletion with high frequency or undergo duplications and amplifications [ 7 ]. A number of PAIs have been described for both gram-positive and gram-negative bacteria, and the application of subtraction hybridization has been used to successfully identify such genetic elements [ 7 ].
The subtractive hybridization that was carried out between B. This, combined with the fact that insertional mutagenesis of the glycosyltransferase gene identified by this method resulted in an avirulent strain, suggests that we may have identified DNA sequences from a putative PAI and that the capsular polysaccharide gene cluster may be located on this island. It is possible that B. Capsule production has been correlated with virulence in many bacteria, particularly those causing serious invasive infections of humans [ 61 ].
A number of functions have been suggested for polysaccharide capsules: prevention of desiccation for transmission and survival, adherence for colonization, resistance to complement-mediated phagocytosis and complement-mediated killing, and resistance to specific host immunity due to a poor antibody response to the capsule [ 4 ]. To establish a correlation between capsule production and clinical infection a number of B.
All 55 strains tested were found to produce CPS I by western blot analysis [ 51 ]. In addition 10 strains of B. CPS I production by B. All CPS I mutants tested in the animal model could not be isolated from the blood following infection. Differences in tissue distribution between wild type B. CPS I production was shown to be responsible for persistence in the blood by evasion of the complement cascade and the mechanism for this was determined to be through the reduction of C3b deposition and opsonophagocytosis.
The addition of purified CPS I to serum bactericidal assays showed that the capsule contributes to increased resistance of serum sensitive strains lacking the O-polysaccharide moiety O-PS of LPS to the bactericidal effects of normal human serum. However, CPS I mutants themselves were not found to be serum sensitive because they still produced O-PS, which was previously shown to be responsible for serum resistance, because it prevents lysis by the MAC complex [ 36 ].
This led us to postulate that CPS I was affecting the complement cascade through some other mechanism and it was found that this mechanism was through the reduction of C3b deposition and opsonization [ 50 ].
Both Western blot analysis and immunofluorescence microscopy experiments using a mouse monoclonal antibody to human C3b demonstrated the inhibition of C3b deposition by CPS I. In both experiments C3b deposition was more pronounced on the surface of the CPS I mutant compared to wild type. Also evident was that some C3b deposition occurred in the wild type, but this was expected since bacterial capsules are known to allow the diffusion of some C3b to the bacterial surface and B.
The accumulation of C3b affects the amplification step of the complement cascade and therefore, the less C3b deposited the less C5a is generated for phagocyte recruitment [ 63 ]. This explains the increased clearance of CPS I mutants from the blood.
This conclusion was supported by the fact that B. Effective opsonization of invading bacteria results in enhanced phagocytosis and clearance of organisms form the blood of an infected host [ 52 ]. Quantitative radiolabelled phagocytic assays were also performed to establish a correlation between opsonization of the bacteria and phagocytosis by polymorphonuclear leukocytes. In the presence of serum, the CPS I mutant was more readily phagocytosed than wild type [ 50 ].
The expression of CPS I in the presence of normal human serum was found to be significantly elevated, also confirming that this capsule contributes to survival in the host. The presence of this capsule facilitates survival as well as spreading to other organs, which can explain the overwhelming septicemia that is common in culture-positive melioidosis patients [ 64 ]. Sequence analysis of the genome of B. One of these operons, CPS I , corresponded to the previously identified and characterized mannoheptose capsule that was shown to be responsible for virulence and comprises one of the currently proposed melioidosis and glanders subunit conjugate vaccine [ 28 , 66 , 42 , 50 , 67 ].
This correlates with previous studies that have shown that this capsule is produced by B. Three other putative capsule operons were identified by sequence analysis and all of these operons were found to be present in B. Since these capsules are found in B. CPS III, located on chromosome 2, was found to contain 11 genes involved in the biosynthesis of a polysaccharide and was shown to be present in the genomes of B.
A mutation in the CPS III cluster did not affect production of CPS I and so it can be concluded that this operon encodes for gene products responsible for the biosynthesis of a separate capsule. This capsule was not shown to be highly expressed in vivo by microarray analysis and was not required for virulence in the animal model.
The expression of this capsule was shown to be elevated when incubated in water, but suppressed in the presence of normal human serum [ 55 ]. Previous studies have demonstrated that B. The other two are: a branched 1,4-linked glucan polymer CP-1a and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galatose, glucose, and glucoronic acid CP-2 [ 49 ]. Therefore it is evident that the capsule identified in this study is not one of the previously described capsule structures.
Some of the previously characterized capsules produced by B. Strain variation, differences in expression of the capsules, and discrepancies between purification strategies may also explain why a number of capsules have been shown to be produced by this organism.
Nevertheless, the genes BPSS appear to be involved in the biosynthesis of a capsule with this composition. Studies by another laboratory have also focused on the presence of these capsule clusters in B.
Sarkar-Tyson et al. However, a mutant in this polysaccharide did not demonstrate any difference in hydrophobicity compared to wild-type, indicating that this polysaccharide does not contribute to making the cell surface more hydrophobic, which is an advantageous characteristic for some pathogenic bacteria.
The differences in virulence compared to the current work can be attributed to the use of different animal models; however, all other data seem to indicate that this capsule is not required for virulence.
A study was recently published which outlines the identification of another capsule produced by B. This capsular polysaccharide was also found to be produced by B. The genes involved in the synthesis of this polysaccharide have not yet been identified and work is also underway to determine the role for this novel capsule in the pathogenesis of melioidosis and glanders.
This organism harbors a large genome which explains its ability to survive for long periods of time in the environment as well exist as a significant pathogen in both humans and animals.
The presence of multiple polysaccharide clusters in the genome and the production of multiple capsule structures under differing conditions may contribute to the ability of this organism to adapt to a variety of conditions. As demonstrated in this study, capsule expression is dependent on the particular environment, which indicates that B. Further studies aimed at characterizing the capsules of B. The author would like to thank Dr. Donald Woods and all colleagues in the Burkholderia pseudomallei research community for their support and discussions over the years.
Microarray analysis was performed at the Southern Alberta Microarray Facility. Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers. Login to your personal dashboard for more detailed statistics on your publications. Edited by Desiree Nedra Karunaratne.
We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. Downloaded: Introduction Polysaccharide capsules are structures found on the cell surface of a broad range of bacterial species. Cloning and sequencing of the genetic loci required for CPS I production and export Two methods were used to clone the genes involved in the production and export of type I O-PS.
CPS I promotes survival of B. The B. Distribution of capsule loci between three Burkholderia species Comparative analysis of the genomes of three Burkholderia species, B. Table 1. More Print chapter. The HI test involves three main components: antibodies, influenza virus, and red blood cells that are mixed together in the wells i. See Image 1. A microtiter plate is used to perform the HI test. The plate contains wells i. These wells are arranged according to rows and columns which are identified on the microtiter plate by letters and numbers, respectively.
The rows of the plate can be used to test different influenza viruses against the same set of antibodies. The columns can be used to differentiate between greater dilutions of antibodies, like a scale from low to high going from left to right see Figures 3 and 4 for an example. To study these antibodies, a sample of blood is drawn from the animal, from which serum is obtained.
The HI test measures how well these antibodies recognize and bind to other influenza viruses such as, influenza viruses isolated from flu patients. If the ferret antibodies that resulted from exposure to the vaccine virus recognize and bind well to the influenza virus from a sick patient, this indicates that the vaccine virus is antigenically similar to the influenza virus obtained from the sick patient. This finding has implications for how well the vaccine might work in people.
As previously mentioned, the influenza viruses used in the HI test are obtained from sick people. CDC and other WHO collaborating centers collect specimens from people all over the world to track which influenza viruses are infecting humans and to monitor how these viruses are changing.
They are used in the HI test because influenza viruses bind to them. Normally, RBCs in a solution will sink to the bottom of the assay well and form a red dot at the bottom Figure 2A. This keeps the RBCs suspended in solution instead of sinking to the bottom and forming the red dot. Row A shows that in the absence of virus, RBCs in a solution will sink to the bottom of a microtiter plate well and look like a red dot.
Row B shows that influenza viruses will bind to RBCs when placed in the same solution. When antibodies are pre-mixed with influenza virus followed by RBCs, the antibodies will bind to influenza virus antigens that they recognize, covering the virus so that its HA surface proteins can no longer bind to RBCs Figure 2C. The reaction between the antibody and the virus inhibits i. When the antibodies do recognize and bind to the influenza virus in the solution, this shows that the vaccine virus like the one the ferret was infected with is similar to the influenza virus obtained from the sick patient.
When a circulating influenza virus is antigenically different from a vaccine, the antibodies developed in response to the vaccine virus may not recognize and bind this virus. In the HI test, this will cause hemagglutination to occur see Figure 2B. Circulating influenza viruses tested via the HI test are typically obtained from respiratory samples collected from sick patients.
The HI test assesses the degree of antigenic similarity between two viruses using a scale based on antibody dilution. As previously mentioned, the HI test is performed using a microtiter plate. The microtiter plate contains rows and columns of wells i. Dilutions are marked across the top of the microtiter plate. These dilutions function as a scale for assessing antigenic similarity and immune response.
By testing the ability of greater dilutions of antibody to prevent hemagglutination, scientists measure how well those antibodies recognize and bind to an influenza virus. The higher the dilution, the fewer antibodies are needed to block hemagglutination and the more antigenically similar the two viruses being compared are to each other. The highest dilution of antibody that results in hemagglutination inhibition of the test virus is considered an HI titer Figure 3.
Capsule is located immediately exterior to the murein peptidoglycan layer of gram-positive bacteria and the outer membrane Lipopolysaccharide layer of gram-negative bacteria. In electron microscopy, capsule appears like a mesh or network of fine strands. Most bacterial capsules are composed of polysaccharides i. These polymers are composed of repeating oligosaccharide units of two to four monosaccharides.
Capsules composed of single kinds of sugars are termed homopolysaccharides. For example, the capsule of Streptococcus mutans is made up of glucose polymers. If several kinds of sugars are present in a capsule, then it is called heteropolysaccharides , eg. The capsule of Bacillus anthracis is an exception. This polypeptide capsule is composed of polymerized D-glutamic acid.
The sugar components of polysaccharides vary within the species of bacteria, which determines their serologic types. Example: Streptococcus pneumoniae has 84 different serotypes discovered so far. Slime is a loose network of polymers extending outward from a cell whereas capsule is a dense and well-defined polymer layer surrounding the cell.
Both capsules and slime layers are important for the adherence of microorganisms and subsequent colonization but they differ in some of the properties.
Capsules are anti-phagocytic. They limit the ability of phagocytes to engulf the bacteria. The smooth nature and negative charge of the capsule prevent the phagocyte from adhering to and engulfing the bacterial cell. Polysaccharide capsule is the major virulence factor for Streptococcus pneumonia. Encapsulated strains of S. Nonencapsulated strains are less pathogenic; they are quickly and efficiently ingested and destroyed by phagocytes via the process called phagocytosis.
Bacterial capsule prevents the direct access of lysosome contents with the bacterial cell, preventing their killing. Capsule of gram-positive bacteria such as S.
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