Two Kdo-Heptose Regions Identified in Hafnia alvei 32 Lipopolysaccharide: the Complete Core Structure and Serological Screening of Different Hafnia O Serotypes

Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {l-α-d-Hepp-(1→4)-[α-d-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between →4,7)-α-Kdop and →2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.Lipopolysaccharide (LPS) (endotoxin) is the main surface antigen and an important virulence factor of most of the gram-negative bacteria that are pathogenic for humans and animals (46). LPS contributes greatly to the structural integrity of bacteria and constitutes a “pathogen-associated molecular pattern” for host infection (46). As one of the most potent natural activators of the innate immune system, LPS is recognized by different classes of receptors present on macrophages, monocytes, B and T cells, neutrophils, endothelial cells, and epithelial cells (46). Endotoxins stimulate these cells to produce multiple inflammatory mediators responsible for immunotoxicity (e.g., tumor necrosis factor alpha, interleukin 1 [IL-1], IL-6, IL-8, gamma/alpha interferon, NO, platelet-activating factor, and endorphins). Interaction of LPS with the CD14/Toll-like receptor 4/MD-2 receptor complex constitutes a major mechanism responsible for the innate immune response to infection by gram-negative bacteria (1, 46). A large amount of LPS released into the bloodstream triggers the excessive inflammatory response of the innate immune system, leading to sepsis and septic shock (6). High levels of inflammatory mediators have profound effects on the cardiovascular system, kidneys, lungs, liver, central nervous system, and coagulation system. Following their action, renal failure, myocardial dysfunction, acute respiratory distress syndrome, hepatic failure, and disseminated intravascular coagulation can occur, which may result in death (6). Despite intense research on the etiology and treatment of sepsis, its severe form still carries a high mortality rate (6, 46).Hafnia alvei has been reported to be an opportunistic human pathogen. This gram-negative bacterium and its LPS are among the identified causative agents of bacteremia and septicemia in humans and animals (19). For the years 2001 to 2003, up to 42 cases of H. alvei bacteremia were reported annually in the United Kingdom. Most of them were monomicrobic infections, and in ∼33% of the cases, H. alvei was isolated, not only from the blood, but also from hepatic abscesses, pancreatic pseudocyst fluid, sputum, feces, and central venous catheters (19). Besides bacteremia and sepsis, which seem to be the most common syndromes reported, H. alvei is also associated with respiratory diseases and mixed hospital infections in humans. Since the gastrointestinal and respiratory tracts represent very common habitats for hafniae, most cases of H. alvei bacteremia originate there. H. alvei sepsis is also a serious clinical problem in the animal production industry, as infections of H. alvei can be severe, causing septicemia in commercial laying hens, pullets, and rainbow trout (19).Our knowledge of the pathogenicity of H. alvei is limited. LPS is the major virulence factor in cases of H. alvei septicemia and bacteremia (19). Studies of other virulence factors of H. alvei have reported only on the iron-scavenging mechanism, mannose-sensitive/mannose-resistant hemagglutinins, and plasmids encoding bacteriocins and alveicins (19).Most of the elucidated structures of H. alvei LPS are smooth-type molecules built up of O-specific polysaccharide, core oligosaccharide (OS), and lipid A. The O antigens of H. alvei are subdivided into 40 O serotypes (2, 28, 42). The structures of the O-specific polysaccharides from 30 serologically different H. alvei strains have been elucidated (15, 24, 26, 28, 42).So far, four types of core OS have been identified for H. alvei LPSs (9, 17, 25, 27, 30, 43). The most common core OS, isolated by mild acidic hydrolysis from LPSs of smooth H. alvei strains, is a hexasaccharide composed of two d-Glc, three l,d-Hep, and one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues. Two l,d-Hep residues are substituted by phosphoethanolamine (PEtn) and phosphoryl (P) groups (9, 17, 25). In LPSs isolated from H. alvei PCM 1185 and 1204, core OSs are terminated with d-Galp instead of d-Glcp (27). LPSs of H. alvei containing nontypical core OSs, identical with those found in LPSs of Escherichia coli R4 (strains 23 and 1222) and Salmonella enterica Ra (strain 39), were also identified (43).The chemical structures of H. alvei O-specific chains and core OSs were elucidated using fractions obtained by mild acid hydrolysis of LPS. The procedure was optimized for the delipidation of LPS, exploiting the susceptibility of a ketosidic linkage between the inner core and lipid A to acid. However, other acid-labile linkages within the LPS could also be affected, leading to partial degradation of the isolated molecules.The presence of Kdo-containing OSs among fractions obtained by mild acid hydrolysis of LPSs, other than previously identified core OSs, makes the structural analysis of entire H. alvei LPSs difficult. Two types of trisaccharides were previously identified: (i) l-α-d-Hepp-(1→4)-[α-d-Galp-(1→7)]-α-Kdop (l-α-d-Hep is α-l-glycero-d-manno-heptose) for strains 2, 1211, 32, and 1192 (16, 23) and (ii) α-d-Galp-(1→2)-l-α-d-Hepp-(1→4)-α-Kdop for strains 1188 and 1196 (22). These Kdo-containing motifs were never located in any of the LPS segments. Thus, it is of interest to complete the structure of H. alvei LPSs with the locations of such acid-labile motifs in the structures of LPSs isolated from these bacteria.We now report on structural studies of de-N,O-acylated LPS of H. alvei 32 containing a carbohydrate backbone of lipid A, core OS, an additional trisaccharide in the outer region of the core OS, and all of the acid-labile substituents. Additionally, data obtained previously for a trisaccharide isolated from H. alvei 32 LPS (16) was complemented with detailed ¹H and ¹³C nuclear magnetic resonance (NMR) analyses and the assignment of all proton and carbon signals. Screening for the presence of these acid-labile trisaccharides, identical with those found in the strain 32 LPS, was performed on 37 different O serotypes of H. alvei LPSs with antibodies against the conjugate of the de-N,O-acylated H. alvei 32 endotoxin fragment with bovine serum albumin (BSA), specific for the isolated trisaccharide.(Part of this work was presented at the 21st International Carbohydrate Symposium, Cairns, Australia, 7 to 12 July 2002, and the 3rd German-Polish-Russian Meeting on Bacterial Carbohydrates, Wroclaw, Poland, 6 to 9 October 2004.)