Melioidosis may present as an acute pneumonia or an acute septicemia, which is the most severe form of the disease. The disease may also manifest as a chronic infection involving long-lasting suppurative abscesses in numerous sites in the body. Infection with B.
Both the chronic and subclinical forms generally remain undiagnosed until activated by a traumatic event or a decrease in immunocompetence [ 25 ]. At the time our studies were initiated some cell-associated antigens had been identified and characterized in B.
The EPS produced by B. The role of EPS in virulence was not known, but sera from patients with melioidosis had been shown to contain antibodies against EPS [ 30 ]. Two other EPS structures were also identified; a branched 1,4-linked glucan polymer CP-1a and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galactose, glucose, and glucuronic acid CP-2 [ 33 ]. The genes involved in the synthesis of these capsules, and the role of these capsules in virulence had not been identified.
The LPS of B. Based on biochemical, immunological, and genetic data, B. However, these two organisms differ in a number of ways and have been classified into two different species [ 38 ]. The rRNA sequence of B. The biochemical profiles of these two species differ in that B. The most distinct difference between these two species, however, is their relative virulence.
It has also been shown that the two species can be differentiated based on their propensity to cause disease in humans. Environmental strains isolated in Thailand that are able to assimilate L-arabinose are not associated with human infection, whereas clinical isolates are not able to utilize L-arabinose [ 41 ].
To identify the genetic determinants that confer enhanced virulence in B. Subtractive hybridization was carried out between the virulent B. The genomic DNA sample from B. Tester and driver DNAs were digested and subjected to two rounds of hybridization. The remaining unhybridized sequences were considered tester-specific sequences. To enrich for tester-specific sequences, excess driver DNA was added in the hybridizations.
Screening of the subtraction library revealed a number of DNA sequences unique to B. Fifteen distinct plasmid inserts from the library were sequenced.
One of the plasmid inserts, pDD, was found to share limited homology with WbpX, a glycosyltransferase, from Pseudomonas aeruginosa [ 43 ]. The resulting plasmid, pSR, was mobilized into wildtype B. Since the insert from pDD was found to demonstrate homology to a glycosyltransferase from P. Since three carbohydrate structures had been previously purified and characterized, antibodies to each of these polysaccharides were available.
Immunogold electron microscopy studies using rabbit polyclonal sera specific for a type I O-PS—flagellin conjugate was performed on the parent strain, b, and SR Figure 1. Unlike B. Western blot analysis of proteinase K-digested whole cells from B. These results indicated that we had identified and insertionally inactivated a gene involved in the synthesis of the type I O-PS of B.
SR was tested for virulence in the Syrian hamster model of acute septicemic melioidosis. The LD 50 for SR after 48 h was 3. This demonstrated that SR is severely attenuated for virulence in this animal model of melioidosis and that type I O-PS is a major virulence determinant of B. Immunogold electron microscopy of B. Bacteria were reacted with polyclonal rabbit antiserum directed against an O-PS—flagellin protein conjugate absorbed with B.
Original magnification x, Two methods were used to clone the genes involved in the production and export of type I O-PS. We also used transposon mutagenesis to clone the genes involved in production of the polysaccharide; this was done to obtain any unlinked genes that may be involved in polysaccharide production. Six mutants were identified, and the DNA flanking the transposon insertion was cloned and sequenced.
Sequence analysis of the cloned fragments revealed the presence of 26 potential open reading frames involved in the synthesis and export of type I O-PS [ 42 ]. The open reading frames that predicted proteins involved in polysaccharide biosynthesis were found to demonstrate homology to proteins involved in the synthesis of a polysaccharide structure composed primarily of mannose.
The other reading frames in the locus predicted proteins involved in the transport of capsular polysaccharides in a variety of bacteria, particularly those that produce group 2 and group 3 capsular polysaccharides [ 8 ]. The genes responsible for the production of type I O-PS were found to be similar to other loci encoding for capsular polysaccharides in that they are divergently transcribed [ 4 ].
The gene cluster involved in the production of this polysaccharide is also similar to group 3 capsule gene clusters in that there are no genes encoding KpsF and KpsU, which are present in group 2 capsule gene clusters [ 8 ]. However, the organization of the B. The biosynthetic genes identified are not organized into one continuous transcriptional unit; instead, wcbB , manC , and wcbP are separated from the rest of the biosynthetic genes. The genes involved in the production of this polysaccharide were named according to the bacterial polysaccharide gene nomenclature scheme [ 47 ].
The gene products associated with this cluster are shown in Figure 4. Mutations constructed in a number of these genes confirmed their role in the production of this polysaccharide [ 42 ].
However, our results suggested that this polysaccharide was a capsule rather than an O-PS moiety. The genes involved in the production of this capsule demonstrated strong homology to the genes involved in the production of capsular polysaccharides in many organisms, including N.
In addition, the export genes associated with this cluster are not associated with the previously characterized O-PS gene cluster [ 36 ]. Western blot analysis of proteinase K cell extracts and silver staining demonstrated that this polysaccharide has a high molecular mass kDa and lacks the banding pattern seen with O-PS moieties. Studies by our laboratory 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 group 3 capsule and designated this capsule CPS I. This conclusion was further supported by Isshiki et al who separated this polysaccharide from a smooth lipopolysaccharide preparation of B.
These experiments were facilitated by constructing a deletion strain harbouring a mutation in one of the CPS I genes and by complementation of this strain. An in-frame deletion was constructed in wcbB , a gene which encodes a glycosyltransferase, resulting in the capsule-minus strain SZ To confirm the role of wcbB in the biosynthesis of capsule, SZ was complemented by the introduction of a wild-type copy of the wcbB gene cloned into the mobilizable broad-host-range plasmid pBHR1 MoBiTec.
Western blot analysis of proteinase K-digested whole cells was performed using mouse monoclonal antibody directed to B. Similar to the capsule minus strain SR and B. Complementation of SZ by providing the wild type wcbB gene in trans restored capsule production.
Whole-cell extracts from the complemented strain SZ pSZ reacted to the capsule antibody producing the kDa band corresponding to the B. Out of the 55 clinical strains tested for capsule production, 52 were found to produce this capsule. Three strains, a, c, and a were found to be negative for capsule production, similar to B.
However, one of the capsule genes, wzt2 , was successfully amplified from these three strains and following inoculation in the animal model, all three of these strains were found to produce capsule by western blot analysis.
This indicated that CPS I production may be regulated in some strains and its expression may be induced in vivo. Therefore all of the 55 clinical strains of B. Syrian golden hamsters were inoculated intraperitoneally with 10 1 to 10 5 cells of either wild type B. After 48 h, the LD 50 values were calculated, and the blood of the infected animals was diluted and plated for bacterial quantitation.
The addition of purified capsule significantly increased the virulence of the capsule mutant strain SR In contrast, the LD 50 value for SR without the addition of purified capsule was calculated to be 3. In addition, purified capsule enhanced the survival of SR in the blood. Bacteria could not be detected in the blood of hamsters inoculated with SR alone. This number was comparable to the number of wild-type B.
The LD 50 value for the capsule mutant strain SZ containing an in frame deletion of the wcbB gene was calculated to be 9. Complementation of this strain restored virulence in the animal model, resulting in an LD 50 value of 12 CFU, comparable to that of wild type B.
Furthermore, the number of bacteria in the blood of animals infected with the complemented strain, SZ pSZ , was determined to be 4. To further demonstrate the role of the capsule in infection by B.
Animals were inoculated with 10 2 CFU of either wild-type B. At different time points, the animals were sacrificed, and the numbers of bacteria in the blood, liver, lungs, and spleen of each animal were determined. As seen in Figure 2 , the numbers of B. By 24 h, the numbers of b bacteria recovered from the blood, lung, liver, and spleen increased, while SR was detected only in the spleen Figure 2B.
By 48 h, very high numbers of b bacteria were recovered from all of the organs taken, representing a dramatic increase compared to the inoculum Figure 2C. In contrast, all of the organs taken from hamsters infected with SR contained fewer bacteria Figure 2C. Of particular interest was the fact that the number of SR bacteria recovered from the blood at 48 h was lower than in the inoculum, suggesting that the capsule mutant was cleared from the blood more effectively than the wild type.
The number of SR bacteria recovered from the spleen was higher than the number of SR bacteria in the blood, suggesting that SR was being cleared from the blood and sequestered in the spleen.
The difference in virulence between the two strains can be attributed to capsule production, since the CPS I mutant strain was found to have a growth rate similar to that of the wild-type strain b [ 50 ].
Differences in tissue distribution between B. Female Syrian hamsters three per group were inoculated intraperitoneally with 10 2 CFU of either strain, and at 12, 24, and 48 h, two groups of animals were sacrificed and bacterial quantitation of the tissues was determined. The data represent the average number of bacteria found in each tissue and the standard deviation for a given time point. To define the role of the capsule for persistence in the blood, serum bactericidal assays were performed with the addition of purified capsule to determine if capsule had an effect on the survival of serum-sensitive strains of B.
For these experiments, we utilized a double mutant that we constructed in the laboratory, SLR5, which lacks both capsule and O-polysaccharide, since the capsule mutant SR was previously found to be serum resistant [ 42 ]. Since capsule mutants of B. To investigate the effect of capsule on C3b deposition, the amount of C3b deposited on the surfaces of wild-type B. Similar results were observed with the capsule mutant SZ, a strain containing an in-frame deletion of the wcbB gene.
Optical densitometry measurements were performed in order to quantitate the difference in C3b deposition between the strains. In addition, there was a shift in the molecular mass of C3b, which normally runs at kDa, indicating a covalent attachment of the molecule to the bacterial surface. The nature of this attachment was not investigated; however, C3b is thought to covalently attach to the bacterial surface through an ester or amide linkage [ 50 , 52 ].
Immunofluorescence microscopy analysis was also performed to demonstrate the difference in C3b deposition between the capsule mutant and the wild type. During the colonization experiment, cages were changed daily.
At days 1, 3, 6 and 10 after inoculation, faeces was collected, homogenized in 0. The UTI model has been described in detail previously [20].
Briefly, mice were anaesthetized by intraperitoneal administration of an 0. The catheter was carefully pushed horizontally through the urethral orifice until it reached the top of the bladder, and the bacterial suspension slowly injected into the bladder. The catheter was immediately removed after inoculation and the mice subjected to no further manipulations until sacrifice, 24 h after inoculation.
The Mann—Whitney U test was used for statistical evaluation. P values less than 0. Spontaneous NCVs of K. The non-capsulated phenotype of the NCVs was confirmed by light microscopy after negative capsule staining. The parent strains were found to express a prominent capsule structure surrounding the bacterial cell, whereas a well-defined capsule structure could not be visualized in the NCVs Fig.
Furthermore, expression of capsular antigens in the NVCs and respective parent strains was tested by the capsular swelling technique. While the parent strains of C and C reacted clearly with specific K35 and K2 capsule antiserum, respectively, no reaction was observed for the NCVs Fig.
The NCVs were found to be identical to their respective parents with respect to biochemical reactions and in vitro growth rates. To examine the stability of the non-capsulated phenotype of the NCVs, 10 successive passages in LB broth were performed followed by subculture on solid medium.
The phenotype of the NCVs was found to be stable, since reversion to the capsulated phenotype was never observed. Visualization of capsule expression in K. Bars represent 2. A pronounced difference in interaction with the epithelial cell lines was observed between the NCVs and their respective parent strains. The percent adhesion to the intestinal HCT-8 and bladder T cell lines was approximately fold higher for the NCVs compared to the parent strains Table 1.
Likewise, the percent internalization by both cell lines was significantly higher for the NCVs than for the parent strains Table 1. No effect of the addition of mannose to the cell culture medium on the adhesion or invasion ability of the NCVs or the parent strains was observed data not shown. Influence of capsule expression on K. To investigate the influence of capsule in K. Reversion to the capsulated phenotype was not observed for the NCVs during colonization of the intestine, as mucoid K.
GI colonization abilities of K. Means and standard errors of the means are shown. Symbols for day 0 represent size of the inoculum. As no difference was observed in the colonization ability when fed individually to mice, the NCV and parent of each strain were also fed simultaneously to the same mice to investigate the role of capsule during competition. At no time significant differences in the number of wild-type and NCVs in faeces were detected, indicating that expression of capsule is not essential in K.
The role of capsule in K. After 24 h, the mice were sacrificed and the bacterial counts in infected bladders determined Fig. The importance of capsule was furthermore established by competitive experiments. Sets of 10 mice were inoculated with a mixture of equal numbers of the parent and NCV of each strain.
After 24 h, the mice were sacrificed and the numbers of parent and NCV in infected bladders determined by selective plating. In all infected bladders, a higher bacterial count of the parent compared to the NCV was detected Table 1. Bacterial counts recovered from bladders of sets of five mice inoculated separately with C, C or their respective NCVs.
In the present study, the role of capsule on the virulence of two K. The NCVs were isolated from translucent parts of bacterial colonies after prolonged growth on agar plates as described previously [3 , 4 , 7 , 8 , 15]. We confirmed the NCVs as being NCVs of the parent strains by negative capsule staining, as well as by lack of reaction with capsule specific antisera.
Whether NCVs arise as a consequence of mutations in genes involved in either regulation of capsule expression or capsule biosynthesis is not known. However, the non-capsulated phenotype is stable, as neither successive passage in culture medium nor passage in mice induced reversion to the capsulated phenotype.
Future genetic studies may characterize the exact nature of the NCVs. A striking difference in the ability of the NCVs to bind to the intestinal cell line HCT-8, as well as the bladder cell line T, compared to their parent strains was observed in both strains. Thus, in both cell lines, the percentage of adhering bacteria was fold higher for the NCVs compared to their respective parent strains. This is in agreement with recent studies reporting the expression of capsule to reduce the ability of K.
It is obvious to speculate whether the reduced ability of encapsulated bacteria to adhere may be due to masking of adhesins on the bacterial surface by the capsule. A recent study demonstrated an inverse relationship between capsule and type 1 fimbriae expression in K. Capsulated strains exhibited little type 1 fimbriae activity whereas type 1 fimbriae expression in non-capsulated strains was pronounced.
Expression of capsule has also been found to down-regulate expression of the CF29K adhesin in K. The results of these studies suggest that some form of co-regulation between expression of adhesins and capsule exists in K.
In the present study, type 1 fimbriae did not seem to be involved in adhesion of either the NCVs or the parent strains to cultured epithelial cells, as addition of d -mannose to the cell culture medium did not influence the number of adhering bacteria. Although traditionally considered an extracellular pathogen, recent studies have described the ability of K.
The in vivo relevance of this phenomenon is so far uncharacterized, but the ability to invade host cells may play an important role in K. The NCVs of both strains were found to invade both cultured epithelial cell lines in significantly higher numbers than their respective parent strains. That capsule interferes with the internalization of K. The ability of the NCVs to colonize the intestine of streptomycin treated mice was identical to their respective parent strains.
This is in accordance with a previous study where non-capsulated mutants of K. In one study, the colonization ability of a capsule defective mutant of K. Here, the capsule defective mutant was found to form aggregates during growth in the intestine, which could explain the inability of this particular mutant to effectively colonize the intestine.
To our knowledge, the role of capsule as a virulence factor in K. In the present study the role of capsule in UTI was examined by monitoring the infection abilities of NCVs and parent strains individually, and by competitive experiments. Mice were inoculated with a mixture of equal numbers of the NCV and parent of each strain and the ratio of NCV to parent in infected bladders subsequently monitored.
This strategy is advantageous as the differences in bacterial counts in infected bladders of different mice may otherwise complicate interpretation of the results. In all infected bladders the bacterial counts of the NCVs were significantly lower than of the respective parent strain. These results establish that capsule plays a significant role in K.
In conclusion, the results of this study demonstrate that expression of capsule impedes interactions of K. Furthermore, by use of animal models the capsule was established as a significant virulence factor in UTI, but had no influence on the ability of K. The significantly higher ability of the NCVs to adhere to and invade epithelial cells in vitro was not associated with higher infectivity in the animal models.
The results of this study hereby illustrate the caution needed when extrapolating from results of in vitro studies and emphasize the use of in vivo models in studies of microbial virulence.
We thank the undergraduate student Ninell Pollas Mortensen for her participation in the work. Montgomerie J. Google Scholar. Domenico P. Salo R. Cross A. Cunha B. Podschun R. Penner I. Ullmann U. Simoons-Smit A. Verweij-van Vught A. MacLaren D. Williams P. These capsules are typically composed of only one polysaccharide and lie outside the outer membrane of gram-negative cells and the peptidoglycan layer of gram-positive cells.
In general, individual bacteria do not exhibit variation of these antigens as has been described for the variant glycoproteins of trypanosomes C ross These include determining access of molecules and ions to the bacterial cell envelope and the cytoplasmic membrane, the promotion of adherence to the surfaces of inanimate objects or living cells and the formation of biofilms and microcolonies C osterton and I rwin Among certain gram-positive and gram-negative bacteria, capsules have evolved distinctive structural and functional characteristics which are of cardinal importance in the pathogenesis of infections of animals, plants and insects S utherland Unable to display preview.
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This is a preview of subscription content, log in to check access. Alper C, Abramson N, Johnston RB Increased susceptibility to infection associated with abnormalities of complement-mediated functions and of the third component of complement C3. Acta Chem. Structural characterization of the capsular polymer of strain Eagan. Cross GAM Antigenic variation in trypanosomes. Infect Immun in press Google Scholar.
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