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The capsulation (cap) locus of Haemophilus influenzae type e (Hie) was characterized and sequenced. No IS1016 element was found to flank the locus. The 18.2-kb locus included 14 open reading frames (ORFs), which were grouped into three functional regions. Eight new ORFs (named ecs1 to ecs8) were identified in the Hie capsule-specific region II.
In the post-Haemophilus influenzae serotype b (Hib) vaccine era, concern about the potential emergence of non-vaccine-preventable strains has arisen (1, 17, 20, 23, 26). In encapsulated H. influenzae strains, the genes for the production of the polysaccharide capsules are organized in a capsulation (cap) locus, which consists of three different functional regions (11, 13). Regions I and III are common to all capsular types and contain genes necessary for transport and process of the capsular material, while region II contains serotype-specific biosynthesis genes (7, 10, 18, 19, 25).
Invasive disease caused by H. influenzae serotype e (Hie) strains has recently been observed in Italy, suggesting the importance of further molecular investigations on Hie cap locus (4, 5). It is recognized that the Hie capsule is a copolymer of the repeat unit of an N-acetylglucosamine and N-acetylmannosamine uronic acid (22, 24), but the genes involved in the polysaccharide biosynthesis have neither been identified nor characterized.
In the present study, we characterized the Hie cap locus for the first time. Eleven invasive Hie strains isolated in Italy during the period of January 2000 to December 2008 were analyzed. The strains were identified as type e by PCR capsular genotyping (6).
PCR amplification of the 5′ and 3′ end junctions of the Hie cap locus was performed by using primer sets “capfSodC/bexBrev” and “hcsBfrw/HI1637,” respectively (Table (Table1).1). The resulting PCR products were sequenced and analyzed. All 11 Hie strains were found to have the cap locus in the identical chromosomal location as that of H. influenzae serotype f (Hif), associated with the same flanking genes (sodC at the 5′ end and HI1637 at the 3′ end), confirming previous investigations (19). Sequence analysis of the two end junctions also revealed that they contained no sequences reminiscent of the insertion element IS1016. It is well known that this element provides the molecular substrate for amplifications of the cap gene sequences (11). Most Hib strains, in which the cap locus lies between direct repeats of IS1016, possess a duplication of the capsule genes (11, 12). The finding that our Hie strains' lack of IS1016 sequences flanking the cap locus is remarkable, since reasonably the locus cannot be amplified.
The complete cap locus from the strain Hie 274 (isolated from the cerebrospinal fluid of a patient with meningitis) was sequenced. To cover the entire Hie cap locus, overlapping amplicons ranging from 1,500 bp to 9 kb were obtained by PCR analysis using several primer pairs based on the published sequences of the Hib and Hif cap loci (GenBank accession numbers AF549213 and AF549211, respectively) (Table (Table1).1). Each amplicon was then subcloned into pCR4-TOPO (TOPO TA cloning kit or TOPO XL PCR cloning kit; Invitrogen, Milan, Italy). Both strands of the insert from each plasmid were sequenced by the primer walking service at Eurofins MWG Operon (Ebersberg, Germany). The nucleotide sequences were assembled and analyzed using DNAMAN sequence analysis software (version 5.2; Lynnon Corp., Quebec, Canada). Nucleotide and deduced amino acid sequences were compared to other known sequences databases by using the National Center for Biotechnology Information BLAST programs. The entire Hie cap locus was shown to be 18.2 kb in length. It contained 14 open reading frames (ORFs) which, by analogy with other capsule loci, were grouped into three distinct regions (I, II, and III) (Fig. (Fig.1).1). A comparison of the deduced proteins from the Hie cap locus genes with the corresponding gene products from Hib and Hif as well as with proteins from other bacterial species is shown in Table Table22.
Overall, region I exhibited 90% and 96% sequence identity to the previously described region I from Hib and Hif, respectively (18, 19). Region I included four ORFs, which were named bexA, bexB, bexC, and bexD. Although the putative proteins of genes bexABCD were nearly identical (from 91 to 98% identity) to the region I corresponding gene products from both Hib and Hif (Table (Table2),2), some polymorphism at nucleotide sequence level was observed. The bexA gene from the Hie cap locus exhibited 95% identity to bexA from Hif but only 84% identity to bexA from Hib, in agreement with a previous study demonstrating bexA nucleotide sequence diversity among different H. influenzae serotypes (27).
Overall, region III showed 91% and 93% sequence identity to the previously described region III from Hib and Hif, respectively (18, 19). Region III contained two ORFs, which were named hcsA and hcsB. Their deduced amino acid sequences exhibited high identity (from 90 to 96% identity) with the corresponding products from both Hib and Hif region III (Table (Table2).2). Recently, both HcsA and HcsB proteins have been demonstrated to be crucial for transport of capsular polysaccharide from the periplasm to the bacterial surface across the outer membrane (21).
Overall, region II showed no sequence identity to the previously described specific capsular regions from other H. influenzae serotypes (7, 18, 19). On the contrary, high overall sequence identity (67%) was found with the capsule biosynthetic-specific region II from Pasteurella multocida B:2 (accession number AF169324), indicating that the genetic organization of the whole region is similar (2, 3). The G+C content of the DNA in the Hie cap locus region II is 31.3%, significantly different from that of both regions I and III (38% and 39.4%, respectively) and from the overall background for the H. influenzae species (38%), suggesting that region II might be more recently acquired. However, since the G+C content of DNA of P. multocida cap locus region II is 35%, this microorganism was probably not the direct source of the region II for Hie. Although we cannot rule out a common evolutionary origin of the two polysaccharide biosynthetic regions followed by a partial diversification of their DNA content, no data are available to support this hypothesis. Region II contained 8 ORFs, which were named ecs1 to ecs8 (for serotype e capsule-specific genes) (Table (Table2).2). The deduced products of ecs1 and ecs2 had homology with putative UDP-N-acetyl-d-glucosamine 2-epimerase and UDP-N-acetyl-d-mannosaminuronic acid dehydrogenase enzymes, respectively, which catalyze the two-step conversion of UDP-N-acetyl-d-glucosamine to N-acetyl-d-mannosaminuronic acid, as previously demonstrated with Escherichia coli (14). The encoded protein by the ecs3 gene showed similarity to glycosyltransferases (Table (Table2),2), which are involved in polymerization of the sugar monomers in several bacterial species (8, 9). Considering that the structure of the Hie capsular polymer is composed of repeating units of N-acetylglucosamine and N-acetylmannosamine uronic acid (22, 24), it is likely that the products of the ecs1, ecs2, and ecs3 genes play an essential role in the biosynthesis of serotype e polysaccharide. No specific putative functions were assigned to the remaining 5 ORFs (ecs4 to ecs8), although similarity with other deduced products in the database was detected, including the predicted products of the genes bcbDEFGI from cap locus region II from P. multocida (2), (Table (Table2).2). Further studies of functional activities of the Hie cap locus region II genes are required.
Although Hie strains belong to the phylogenetic division I of the encapsulated H. influenzae strains (15), the Hie cap locus shares two remarkable features of the division II cap loci: chromosomal location and lack of association with the IS1016 insertion element, confirming the previously described genetic distance of Hie from all other division I H. influenzae strains (16). The availability of the Hie cap locus sequences may be regarded as a powerful tool to be used in further investigations on molecular detection and characterization of the Hie isolates.
The nucleotide sequence for the Hie cap locus from this study has been deposited in the EMBL nucleotide sequence database under the accession number FM882247.
This work was partially supported by Ministry of Health-CCM project 116 “Surveillance of Invasive Bacterial Diseases.”
We are very grateful to Tonino Sofia for editorial assistance.
Published ahead of print on 27 January 2010.