1 Department of Biological Sciences, Faculty of Science, The National University of Singapore, Singapore 117543
2 Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan 162-8640
3 Tropical Marine Science Institute, The National University of Singapore, Singapore 117543
Correspondence
K. Y. Leung
dbslky{at}nus.edu.sg
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ABSTRACT |
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INTRODUCTION |
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All of the known capsule assembly systems seen in Gram-negative bacteria are well represented in Escherichia coli. Capsules in E. coli have been categorized into four groups based on genetic and biosynthetic criteria (Whitfield & Roberts, 1999). The genes for group II type capsules can be divided into three distinct regions (Roberts et al., 1988
; Roberts, 1996
). Genes in regions I and III are for capsule transport, and genes in region II are for capsule biosynthesis. Regions I and III are conserved among various group II capsule clusters of a broad range of bacterial species, while region II is serotype specific (Roberts, 1996
). These unique characteristics can be used to develop a DNA-based detection method for various bacterial species. The presence of capsules in motile aeromonads was first reported by Martinez et al. (1995)
. The first capsule gene cluster was cloned, sequenced and identified as the group II type from Aeromonas hydrophila PPD134/91 in our laboratory (Zhang et al., 2002
). The purified capsular polysaccharides of PPD134/91 were found to be capable of conferring resistance to serum-mediated killing of avirulent strain PPD35/85, but had no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells.
In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was identified by electron microscopy and genetic analyses. Two types of group II capsules (IIA and IIB) were identified based on their genetic organization in region II. Group IIA capsules in our randomly chosen A. hydrophila strains were mainly found in the O : 18 and O : 34 serogroups, while group IIB capsules were found in the O : 21 and O : 27 serogroups. The presence of these group II capsules strongly correlated with the serum and phagocyte survival abilities in A. hydrophila.
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METHODS |
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Survey of the distribution of group II-like capsules.
Oligonucleotide primers used in this study (Fig. 1, Table 2
) were designed based on regions I and III of the group II capsule gene cluster of PPD134/91 (Zhang et al., 2002
). Primers 79DP5S1 and 79DP5 were used to detect ORFC, and primers 79A5 and CPS13 for ORFL. PCR was performed by using Advantage Genomic Polymerase Mix (Clontech), following a three-step cycle protocol: 25 s at 94 °C; 32 cycles of 15 s at 94 °C, 30 s at 62 °C, and 4 min at 72 °C. PPD134/91 was used as the positive control. Only those samples which had one band at the same position as the positive control were taken as positive.
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Determining the organization of region II genes of group II capsules.
Seven pairs of primers were designed based on the sequences of the seven genes in region II (ORFEK) of the PPD134/91 capsule gene cluster (Fig. 1, Table 2
). PCR was performed to detect their presence in nine capsule gene clusters of A. hydrophila. Only those samples which had one band at the same position as that of PPD134/91 were taken as positive. The 3' and 5' end sequences of each PCR product were sequenced to confirm the identities of these genes. DNA sequencing and analysis were carried out as described previously (Srinivasa Rao et al., 2001
).
Cloning the capsular biosynthesis regions from motile A. hydrophila strains.
Region II of group II capsule gene clusters from A. hydrophila was cloned using long-range PCR with primers 79B1 (ORFD) and 79B2 (ORFL) (Fig. 1, Table 2
). PCR was performed by using Advantage Genomic Polymerase Mix (Clontech) and carried out under the following conditions: one hold at 94 °C for 1 min followed by 32 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1020 min. The amplified fragments were purified using QIAquick PCR Purification Kit (Qiagen) and cloned into the pGEM-T Easy Vector (Promega). The recombinant DNA molecules were transformed into E. coli DH5
competent cells and sequenced.
Survival assay in tilapia serum and phagocyte intracellular replication assay.
Naive tilapia [Oreochromis aureus (Steindachner)] serum was used to perform the serum resistance assay. Bacteria were prepared and treated with 50 % tilapia serum as described previously (Leung et al., 1995). The survival of A. hydrophila was calculated by dividing the number of viable bacteria after a 1 h serum treatment by the number of bacteria before treatment. Bacteria with survival values greater than 1 were considered serum resistant, while those with values below 1 were considered serum sensitive. The intracellular replication assay was performed as described by Srinivasa Rao et al. (2001)
. Phagocytes were isolated from the head kidney of naive blue gourami [Trichogaster trichopterus (Pallas)]. Thirty minutes after infection, the phagocytes were washed once with Hanks' balanced salt solution (Sigma) and then incubated for 1·5 h in fetal calf serum-supplemented fresh L-15 medium with 100 µg gentamicin ml-1. The gentamicin treatment killed extracellular bacteria but did not affect the viability of intracellular organisms. The intracellular population of bacteria was assayed at 2 and 6·5 h. The phagocytic index was calculated by dividing the intracellular population of A. hydrophila at 6·5 h by the population at 2 h. The data were obtained from three independent experiments.
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RESULTS AND DISCUSSION |
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Genetic organization of regions I and III of the group II capsule
To further verify the presence of group II capsule gene clusters in these ten A. hydrophila strains, the genetic organization of regions I and III of these strains was further examined. DNA fragments for ORFAB, ORFAC and ORFAD in region I, and ORFLM in region III, were amplified using various sets of primers and the sizes were compared with those in PPD134/91 (Fig. 1, Table 2
). All of the ten strains except JCM3990 had the same PCR profiles as PPD134/91. The results indicate that all the capsule gene clusters in nine of these strains share a common genetic organization in their regions I and III and consequently confirm them as having PPD134/91-like group II capsule gene clusters. JCM3990 and six other strains that produced non-group II capsules did not give any PCR products of expected sizes. A Southern blot analysis using probes prepared from regions I and III of PPD134/91 further confirmed the presence of group II capsule gene clusters in these nine strains, but not in JCM3990 and the six other non-group II capsule-producing strains (Table 1
).
Two types of capsule biosynthesis gene clusters in nine group II capsule-producing strains
Region II DNA from the ten group II capsular positive strains (including PPD134/91) was cloned by long-range PCR using primers 79B1 and 79B2 (Table 1, Fig. 1
). These strains were divided into two types based on the sizes of their PCR products. The first type included PPD134/91, PPD64/90, JCM3980, JCM3996, 307-01 and 309-01. They had similar PCR bands as PPD134/91, which was about 10 kb in size (data not shown). The other type consisted of four strains, namely PPD88/90, PPD11/90, JCM3983 and SL118-79, which had band sizes of about 5 kb in their PCR products (data not shown).
Seven pairs of primers were designed based on the seven capsule biosynthesis genes in region II of PPD134/91 (Fig. 1, Table 2
). PCR was performed on all the nine strains of A. hydrophila that produced group II capsules to survey the distribution of the biosynthesis genes among these strains. The results (Table 3
) showed that, except for ORFG' in PPD64/90, PCR products of similar size were present in six group II capsule-producing strains (first type). The 3' and 5' end sequences of these capsule biosynthesis genes were also sequenced to confirm their identities (data not shown). Our results confirmed that all of the PPD134/91 group II capsule biosynthesis genes were present in JCM3980, JCM3996, 307-01, 309-01 and PPD64/90. Due to the similarity in their capsule gene organization, these six strains were named group IIA capsule-producing strains. None of the capsule biosynthesis (region II) genes in PPD134/91 was present in the other three strains, namely PPD11/90, JCM3983 and SL118-79 (Table 3
), and their region II gene clusters were much shorter than those of group IIA capsule-producing strains. These were named group IIB capsule-producing strains. The genetic organization of the region II gene cluster in PPD88/90 was unusual. Four out of seven genes in PPD134/91 (ORFE, F, G, K) were found in PPD88/90, with three genes (ORFHJ) deleted and an addition of an ORF with unknown function (ORFN, position 16182361, in accession no. AY177619) (Fig. 1
, Table 3
). This could reflect the heterogeneity of capsule biogenesis in A. hydrophila.
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Cloning and sequence analysis of region II genes of PPD11/90 and JCM3983
Genes in region II of PPD11/90 and JCM3983 were sequenced for detailed analysis of the group IIB capsule clusters. Region II of PPD11/90 was 5424 bp long, including five ORFs, namely ORFO, P, Q, R and S (Fig. 1, Table 4
; accession no. AY144595). The first ORF of region II, ORFO, was separated from region I by 79 bp. The second ORF (ORFP) was separated from the first one by 53 bp. ORFQ was separated from ORFP by 91 bp, and from ORFR by 361 bp. The last ORF (ORFS) of region II was separated from ORFR by 69 bp, and from the first ORF of region III (ORFL) by 205 bp. The mean G+C content of PPD11/90 region II cluster was 35 mol%, which is low compared to the mean G+C content for A. hydrophila. The G+C contents of ORFO, P, Q, R and S were 41 %, 35 %, 36 %, 33 % and 34 %, respectively.
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Five pairs of primers were designed based on the five ORFs of region II genes of PPD11/90. PCR was performed on PPD134/91, PPD88/90, JCM3983, SL118-79 and PPD11/90. Four of the region II genes in PPD11/90 (ORFO, P, Q, R) were present in JCM3983 and SL118-79. None of them was present in PPD134/91 and PPD88/90, suggesting that PPD88/90 is closer to the group IIA rather than the group IIB capsule. Only four ORFs were found in the region II gene cluster of strain JCM3983. However, the corresponding ORFQ and R of PPD11/90, which encoded putative glycosyltransferase and putative galactosyl phosphotransferase, were merged to form a putative bifunctional enzyme (ORFQ'R') in JCM3983. This ORF might have evolved from ORFQ and ORFR by mutation or recombination.
Predicted gene function of region II genes in the group IIB capsule
The first ORFs (ORFO) of region II in PPD11/90 and JCM3983 share about 70 % identity in their amino acid sequences with GalU of Yersinia pestis and other bacteria such as E. coli and Salmonella enterica subsp. enterica serovar Typhi (Table 4). These two ORFs share 77 % identity in their DNA sequences and 91 % identity in their amino acid sequences. galU encodes the enzyme UTP-glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, UDPG : PP), which catalyses the formation of UDP-glucose (UDP-Glc) from glucose 1-phosphate and UTP. UDP-Glc is required for the interconversion of glucose and galactose (Gal) by the Leloir pathway (Frey, 1996
) and is also the substrate for the synthesis of UDP-galacturonic acids (UDP-GalA). Glc, Gal and UDP-GalA are the common components for the biosynthesis of capsular polysaccharides (Mollerach et al., 1998
). A defect in UDPG : PP can impair the biosynthesis of capsules (Mollerach et al., 1998
) and decrease the virulence of bacteria (Genevaux et al., 1999
). It is reasonable to predict that UDPG : PP is a key component in the biosynthesis pathways of PPD11/90 and JCM3983 capsules.
ORFP in PPD11/90 and JCM3983 have 76 % and 81 % identities in their nucleotide and amino acid sequences, respectively. Both of them have sequence similarities to LcbA of Neisseria meningitidis, the capsular polysaccharide synthesis gene of Actinobacillus pleuropneumoniae, SacB of N. meningitidis serogroup A, and hexose transferase of Streptococcus thermophilus NCFB2393. Specific functions have not been assigned to these proteins. However, the functions of SacB of N. meningitidis serogroup A and hexose transferase of S. thermophilus have been postulated (Swartley et al., 1998; Almiron-Roig et al., 2000
). Mutational studies have shown that SacB is essential for the expression of group A capsules in N. meningitidis. It is hypothesized that SacB is a polymerase linking individual UDP-ManNAc monomers together. The sequences of orfP from PPD11/90 and JCM3983 are much shorter than SacB (by 186 and 182 aa). Detailed biochemical analysis will be required to confirm the functions of ORFP.
The amino acid sequence of the product of ORFQ of PPD11/90 showed similarity to glycosyltransferase of S. thermophilus (EpsH) (Germond et al., 2001) and Lactobacillus delbrueckii subsp. bulgaricus (EpsJ) (Lamothe et al., 2002
). A conserved domain database search (http://www.ncbi.nlm.nih.gov/BLAST/) showed that ORFQ (of 144 aa) has sequence similarity to putative glycosyltransferase of Rhizobium meliloti ExpA2 (Becker et al., 1997
) and other bacterial glycosyltransferases. Thus, ORFQ may be a putative glycosyltransferase. ORFR of PPD11/90 had sequence similarity to EpsJ of Lactococcus lactis subsp. cremoris (van Kranenburg et al., 1999
), which appeared to be involved in its exopolysaccharide biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier. ORFS of PPD11/90 had sequence similarity to O-acetyltransferase of Vibrio cholerae type 2 strain V208 (Nesper et al., 2002
). This protein may play a role in the modification of the capsular polysaccharides of this bacterium.
A conserved domain database search using the amino acid sequence of ORFQ'R' of JCM3983 showed that there is a glycosyltranferase domain in its N-terminus (173 aa, positions 20712589 in accession no. AY177683). The N-terminal sequence (213 aa, positions 20622700 in accession no. AY177683) had similarity to glycosyltransferase of Strep. thermophilus (EpsH) and Lb. delbrueckii subsp. bulgaricus (EpsJ) (Table 4). The 330 aa sequence (positions 20593048 in accession no. AY177683) from the N-terminus of ORFQ'R'shared 70 % amino acid identity when compared with ORFQ of PPD11/90. The 266 aa sequence (positions 34934290 in accession no. AY177683) from the C-terminus of ORFQ'R' shared 55 % amino acid identity with ORFR of PPD11/90. The C-terminus sequence (343 aa, positions 32114239 in accession no. AY177683) had sequence similarity to galactosyl phosphotransferase of Lc. lactis subsp. cremoris (EpsJ) (Table 4
). Thus, ORFQ'R' may be a bifunctional glycosyltransferase and galactosyl phosphotransferase. The function of ORFT of JCM3983 could not be predicted since no good sequence similarity was obtained from searches in available databases using both its DNA and amino acid sequences.
In summary, the G+C content and database search results indicated that large portions of genes in these region II gene clusters of PPD11/90 and JCM3983 are homologous and may have evolved from the same source.
Serum resistance and phagocyte killing
Capsulation is a crucial virulence determinant for a number of bacterial species, providing protection from serum and phagocyte killing. The capsule increased the degree of virulence of Pasteurella piscicida for fish and conferred resistance to serum killing (Magarinos et al., 1996). Acapsular mutants of P. multocida were removed efficiently from the blood and other host organs, and were readily taken up by murine peritoneal macrophages, but the capsulated wild-type showed significant resistance to phagocytosis (Boyce & Adler, 2000
). Differences in capsule types were also found to have significant effects on pneumococcal (Kadioglu et al., 2002
) and pathogenic E. coli infections (Kim et al., 1992
; Russo et al., 1994
). Furthermore, certain group II capsular serotypes in pathogenic E. coli have been documented to contribute to pathogenesis in systemic models of infection (Kim et al., 1992
; Russo et al., 1994
).
Similarly, the group II capsules of A. hydrophila were found to act as important anti-serum killing and anti-phagocytic factors in our infection model. Table 5 shows that seven out of ten group II capsule-producing A. hydrophila strains (those other than PPD64/90, JCM3983 and SL118-79) were resistant to serum and phagocyte killing. On the other hand, four out of six randomly chosen acapsular strains were sensitive to serum and phagocyte killing. Two acapsular strains, namely L31 and PPD122/91, and a non-group II capsule-producing strain (PPD70/91), also survived in serum and phagocyte killing, suggesting that the group II capsule may not be the only factor contributing to the resistance. It is interesting to note that most of the group IIA capsule-producing strains belong to the O : 18 and O : 34 serogroups in our 33 randomly chosen A. hydrophila isolates, while the group IIB strains belong to the O : 21 and O : 27 serogroups. Among the group II capsule-producing strains, group IIA capsules have a stronger correlation with conferring resistance to serum and phagocyte killing than do group IIB capsules. It is unclear as to the correlation of O-serogroups and types of capsules, and the exact virulence properties of groups IIA and IIB capsules. More strains and further experiments are needed to draw any conclusion.
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Conclusion
We have reported a DNA-based method for the detection of group II capsules in A. hydrophila. These group II capsules can be divided into two types, IIA and IIB, with group IIA-producing strains having a strong correlation with conferring resistance to serum and phagocyte killing. Knowledge of the composition of the various capsular polysaccharides and of the function of the encoded proteins of each type is important. This will provide insight into the dynamics of the capsule biosynthesis pathway, the evolution of serotypes, and their roles in bacterial pathogenesis. However, the functional relevance of these capsule genes and the mechanisms by which the capsular polysaccharides are synthesized remain to be determined.
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Received 22 November 2002;
revised 15 January 2003;
accepted 17 January 2003.
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