Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster.

The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigí, N. Piqué, N. Climent, S. Merino, S. Albertí, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581-7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5alpha by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster.

Surface antigen exposure by bismuth dimercaprol suppression of Klebsiella pneumoniae capsular polysaccharide.

The bacterial capsule is an important virulence determinant in animal and plant disease. Bacterial capsule and slime can be inhibited by bismuth compounds, especially when complexed with lipophilic thiol chelators. Bismuth dimercaprol (BisBAL) at 1 ppm of Bi3+ repressed Klebsiella pneumoniae capsule expression in defined medium by nearly 90%, which exposed subsurface structures. The phagocytic index for BisBAL-treated bacteria increased from <10 to 360 bacteria per 100 neutrophils in the presence of complement and anticapsular or anti-O antigen antiserum. BisBAL treatment also enhanced the reactivity of monoclonal antibodies (MAbs) specific for the O1-antigen lipopolysaccharide (LPS) or the LPS core in a dose-dependent manner as indicated by the results of enzyme-linked immunosorbent assays. When anti-O1 MAb was used, the reactivity increased significantly for fully encapsulated O1:K1 or O1:K2 cells but not for O1:K- cells. Deposition of C3b also increased significantly for BisBAL-treated O1:K1 or O1:K2 cells but not for O1:K- cells. Survival of a serum-sensitive strain was <0.1% when nonimmune human serum absorbed with O1:K1 cells was used and 107% when BisBAL-treated cells were used for absorption. Outer membrane proteins were also more accessible on the surface of K. pneumoniae after BisBAL treatment. Thus, at subinhibitory levels, BisBAL inhibited capsule expression, which promoted phagocytosis, enhanced the reactivity of specific antibodies for LPS O antigen, LPS core epitopes, or outer-membrane proteins, and enhanced complement interaction with encapsulated K. pneumoniae. By unmasking bacterial surface structures and enhancing the immune system reactivity to bacteria, bismuth thiols may prove useful as adjuncts for vaccination.

Mesophilic Aeromonas strains from different serogroups: the influence of growth temperature and osmolarity on lipopolysaccharide and virulence.

Growth of mesophilic Aeromonas sp. strains from serogroups O:13, O:33 and O:44 at different temperatures and osmolarity resulted in changes in the lipopolysaccharide (LPS) and virulence of the strains tested, as we had previously reported for strains from serogroup O:34. The effect of osmolarity could be observed when the cells grew at 37 degrees C but not at 20 degrees C. Purified LPS from cells cultivated at 20 degrees C (high or low osmolarity) or at 37 degrees C at high osmolarity was smooth, whereas the LPS extracted from the cells cultivated on low osmolarity was rough. The smooth strains were resistant to the bactericidal activity of non-immune serum, while the rough strains were sensitive and showed better adhesion to Hep-2 cells than the rough strains. Furthermore, the smooth strains were more virulent for fish and mice than the rough strains. For mesophilic Aeromonas sp. strains from serogroups O:1 to O:44, these changes were not observed, except for serogroups O:13, O:33, O:34 and O:44.

Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein.

The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355-3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups.

Isolation of three different bacteriophage from mesophilic Aeromonas sp. that use different types of monopolar flagella as their primary receptor.

Bacteriophage PM4, PM5 and PM6 were isolated on different mesophilic Aeromonas strains. These bacteriophage use the flagellum as their primary bacterial receptor since purified flagella from these strains are able to inactivate these bacteriophages, independently, and the phage-resistant mutants are aflagellate and nonmotile. Furthermore, we showed that these bacteriophage may be useful to initiate the serotyping of mesophilic Aeromonas for the H-antigen (flagellum).

A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5alpha, and clones were screened for serum resistance. One clone was found resistant to serum, to bacteriocin 28b, and to bacteriophages TuIa and TuIb. This clone also showed O antigen in its lipopolysaccharide. Subcloning and sequencing experiments showed that a 2,124-bp DNA fragment containing the rmlD and wbbL genes was responsible for the observed phenotypes. On the basis of amino acid similarity, we suggest that the 288-residue RmlD protein is a dTDP-L-rhamnose synthase. Plasmid pJT102, containing only the wbbL gene, was able to induce O16-antigen production and serum resistance in E. coli DH5alpha. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rJb-50 mutation in E. coli K-12 derivatives, despite the low level of amino acid identity between WbbL and the E. coli rhamnosyltransferase (24.80%). S. marcescens N28b rmlD and wbbL mutants were constructed by mobilization of suicide plasmids containing a portion of rmlD or wbbL. These insertion mutants were unable to produce O antigen; since strain N28b produces O4 antigen, these results suggest that both genes are involved in O4-antigen biosynthesis.

The role of O1-antigen in the adhesion to uroepithelial cells of Klebsiella pneumoniae grown in urine.

We obtained mutants devoid of the O1-antigen, the capsular polysaccharide (K antigen) or both from Klebsiella pneumoniae clinical isolates (urinary infection). These mutants were grown in urine, and their ability to fimbriate and to adhere were studied. Mutants lacking the O1-antigen, independently of the other surface molecules (capsule and fimbriae), showed a great decrease in adhesion to these cells.

The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains.

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.

Influence of osmolarity on lipopolysaccharides and virulence of Aeromonas hydrophila serotype O:34 strains grown at 37 degrees C.

Growth of Aeromonas hydrophila serotype O:34 strains at 37 degrees C at low and high osmolarity resulted in changes in the lipopolysaccharide (LPS) and virulence of the strains tested. We previously described the effect of growth temperature on LPS and virulence of these strains (S. Merino et al., Infect. Immun. 60:4343-4349, 1992). The effect of osmolarity can be observed when the cells grow at 37 degrees C but not when they grow at 20 degrees C. Purified LPS from cells cultivated at 37 degrees C and high osmolarity was smooth, while the LPS extracted from the cells cultivated at low osmolarity was rough. Furthermore, the strains were more virulent for fish and mice when they were grown at high osmolarity than when they were grown at low osmolarity and also showed increased extracellular activities when they were grown at high osmolarity. Finally, cells grown at high osmolarity showed better adhesion to HEp-2 cells than the same cells grown at low osmolarity, and furthermore the cells grown at high osmolarity were resistant to the bactericidal activity of nonimmune serum, while the same cells grown at low osmolarity were sensitive.

Molecular characterization of a 17-kDa outer-membrane protein from Klebsiella pneumoniae.

A cosmid-based genomic library of Klebsiella pneumoniae 52145 (O1:K2) was introduced into Escherichia coli, and clones were screened for the bacteriocin 28b resistance phenotype. One clone was found which conferred partial resistance to bacteriocin 28b. By using Tn5tac1 insertions, it was shown that this phenotype was due to the expression, in E. coli, of an outer-membrane protein (OMP) with an apparent molecular mass of 17 kDa (OmpK17). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 510 bp. The deduced amino acid sequence has 170 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues. When compared with other enterobacterial OMPs, OmpK17 most closely resembles members of a family of small OMPs of Enterobacteriaceae the known functions of which appear to be related to virulence. Immunoblotting experiments showed that OmpK17 is also present in various K. pneumoniae strains belonging to different O and K serotypes.

The role of the capsular polysaccharide of Aeromonas hydrophila serogroup O:34 in the adherence to and invasion of fish cell lines.

The ability of Aeromonas hydrophila serogroup O:34 strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared. The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation. However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade the fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation. Isogenic unencapsulated mutants grown under conditions promoting capsule formation showed a lower ability to invade the fish cell lines than the parental capsulated strains. From these results, we concluded that the capsular polysaccharide is an important factor in intracellular invasion.

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed.

Interaction between complement subcomponent C1q and the Klebsiella pneumoniae porin OmpK36.

The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks.

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.

The role of the capsular polysaccharide of Aeromonas salmonicida in the adherence and invasion of fish cell lines.

The ability of several Aeromonas salmonicida strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared. The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation. However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation. From these results we conclude that the capsular polysaccharide, in these strains, is an important factor for intracellular invasion.

The O:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila.

We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to adhere to Hep-2 cells. We found a high level of adhesion when the strains were grown at 20 degrees C but not when they were grown at 37 degrees C. We previously described that these strains were able to form the O-antigen lipopolysaccharide when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen lipopolysaccharide (rfb mutants), and they showed significantly lower levels of adhesion to Hep-2 cells than the smooth strains. All these results prompted us to conclude that the O-antigen LPS, in these strains, is an important adhesin.

The role of the O-antigen lipopolysaccharide on the colonization in vivo of the germfree chicken gut by Aeromonas hydrophila serogroup O:34.

We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to colonize in vivo the germfree chicken gut. We found a good colonization when the strains were grown at 20 degrees C but not when they were grown at 37 degrees C. We previously described that these strains were able to form the O-antigen lipopolysaccharide (LPS) when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen LPS (rfb mutants), and showed that they were unable to colonize the germfree chicken gut. All these results prompted us to conclude that the O-antigen LPS, in these strains, is a main factor for colonization in this animal model system.

Emerging pathogens: Aeromonas spp.

Aeromonas spp. are Gram-negative rods of the family Vibrionaceae. They are normal water inhabitants and are part of the regular flora of poiquilotherm and homeotherm animals. They can be isolated from many foodstuffs (green vegetables, raw milk, ice cream, meat and seafood). Mesophilic Aeromonas spp. have been classified following the AeroKey II system (Altwegg et al., 1990; Carnahan et al., 1991). The major human diseases caused by Aeromonas spp. can be classified in two major groups: septicemia (mainly by strains of A. veronii subsp. sobria and A. hydrophila), and gastroenteritis (any mesophilic Aeromonas spp. but principally A. hydrophila and A. veronii). Most epidemiological studies have shown Aeromonas spp. in stools to be more often associated with diarrhea than with the carrier state; an association with the consumption of untreated water was also conspicuous. Acute self-limited diarrhea is more frequent in young children, in older patients chronic enterocolitis may also be observed. Fever, vomiting, and fecal leukocytes or erythrocytes (colitis) may be present (Janda, 1991). The main putative virulence factors are: exotoxins, endotoxin (LPS), presence of S-layers, fimbriae or adhesins and the capacity to form capsules.

The presence of capsular polysaccharide in mesophilic Aeromonas hydrophila serotypes O:11 and O:34.

Mesophilic Aeromonas hydrophila from serotypes O:11 and O:34 grown in a glucose-rich medium produce a capsule that can be seen under light and electron microscopy. The purified capsular polysaccharide has a composition qualitatively similar for strains O:11 and O:34, but quantitatively different. The capsular polysaccharides were immunogenic in rabbits, and did not cross-react with specific antibodies against either purified lipopolysaccharide from strains O:34 or O:11 or against the S-layer characteristic of strains from serotype O:11.