Diverse virulence traits underlying different clinical outcomes of Salmonella infection (original) (raw)

LPS. The genes encoding the enzymes that synthesize the sugars and organize them into the polysaccharide sidechains on the LPS are clustered together at the rfb locus on the chromosome. The Kauffmann-White scheme that is used to serotype Salmonella (as well as Escherichia coli and other strains; see Donnenberg and Whittam, this series, ref. 4) is based on antigenic polymorphisms of LPS (O) and flagella (H). Although the core structure of the LPS (lipid A) is largely conserved in all Salmonella, the polysaccharide side chains are highly polymorphic because the rfb region is polymorphic (5). Classically, the antibodies used to group salmonellae are raised in rabbits that have been immunized with heat-killed bacteria. The exact structure of many of the carbohydrate antigens is not known and may be difficult to discern, because epitopes found on helical polysaccharides may not be linear (6).

Almost all human infections (>95%) are caused by Salmonella that are in groups (defined by O antigen structures) A, B, C, D, and E. Interestingly, groups A, B, and D have the same common trisaccharide backbone structure of their LPS: D mannose α1→4 L rhamnose β1→3 D galactose α1→2. The only difference between the three LPS structures is the di-deoxyhexose that is attached to the 3 position of the mannose. In group A the di-deoxyhexose is paratose, in B it is abequose, and in D it is tyvelose. In group E Salmonella, the backbone structure is made up of the same trisaccharide, but there is no di-deoxyhexose. The group C LPS structure is made up of a linear polymer of four mannose residues and one _N_-acetyl glucosamine residue.

Although a few serotypes of Salmonella have an outer capsular layer (notably S. typhi), in most serotypes LPS forms the layer around the bacterium that protects it from the environment. LPS interacts with both antibodies and with complement. Rough Salmonella mutants, which lack this protective layer, activate complement by the alternative pathway, leading to killing of the bacteria by the membrane attack complex of complement. Not surprisingly, rough mutants are avirulent. Naturally occurring infections are caused by Salmonella with a complete LPS, and they are resistant to killing by complement (7). However, activated complement protein C3 covalently binds to the terminal sugars on the LPS and acts as an opsonin. In the case of groups A, B, and D Salmonella, these terminal sugars are di-deoxyhexoses, which therefore modulate the interaction with complement (C3) (8). Changing the structure of the di-deoxyhexose from tyvelose to abequose increases the amount of C3 that binds to the bacteria, which in turn increases phagocytosis by macrophages (9). This change also affects the virulence of these bacteria in mice (10), which is due largely to differences in phagocytosis and killing by polymorphonuclear leukocytes (J. Fierer, unpublished observations). It is clearly not true that group B salmonellae are more virulent in humans, as S. typhi is a group D Salmonella. Human complement is much more active than mouse complement, which may explain why C3 activation and binding is a limiting step in mice but not humans.

Much of the antibody response to Salmonella infection is directed against the LPS, and especially against the di-deoxyhexoses, if they are present. The terminal sugar fits into the antigen binding site of the antibody, where hydrogen bonds form between aromatic amino acids and the OH groups on the di-deoxyhexose (11). Although the relative importance of antibodies and T lymphocytes in suppressing Salmonella infections is controversial (12), there is ample evidence that antibodies protect both humans and mice against Salmonella (13, 14). Furthermore, in most experimental systems, immunity to Salmonella is O antigen–specific (15, 16). Thus, it might benefit the bacteria to change the structure of their LPS, which is indeed observed, although not during the course of a single infection. O-antigen variation between isolates can occur because of lysogenic conversion by bacteriophages or mutations in chromosomal genes. Most of the variants are O-acetylated or glucosylated sugars, which can change the LPS structure enough to create new antigens. For instance, OafA is a chromosomal enzyme that O-acetylates abequose creating a new antigen (O5) (17). There is a phage that adds glucose to some of the galactose residues on the repeating trisaccharide unit in S. typhimurium which creates antigen O1, a common alteration. Phages can also change the linkages between the sugars. None of these substitutions is ever stoichiometric, so both the original and the modified LPS are expressed on the same bacteria. Very little is known about the regulation of these enzymatic modifications.

Because the antibody response is largely directed toward the O antigen, the polymorphism of O antigens could allow Salmonella that modify these antigens to infect hosts that already have antibody to unmodified O antigens. For instance, most of the antibody response to OafA+S. typhimurium is directed to O-acetylated (O5) abequose (18). Mice immunized with an Oaf– mutant are more resistant to that mutant than to an isogenic Oaf+ strain, and vice versa (18). It is also conceivable that antigenic variations in the LPS may be advantageous to the Salmonella that are actively infecting a host and so are under attack by antibodies to LPS, but there is no evidence that bacteria without modifying enzymes are less virulent in mice. The only known example of lysogeny increasing the virulence of Salmonella involves phage 14 in S. choleraesuis (19). This phage increases polymerization of the repeating LPS units, creating much longer LPS side chains, which makes the bacteria resistant to serum bactericidal activity and hence more virulent in mice. Increasing the chain length of LPS in S. enteritidis also increases its virulence, but the genetic mechanism controlling that process is not known. Because LPS also provides the attachment site for the phage, lysogenized strains that express phage-encoded enzymes are sufficiently altered in their LPS structure that they are not susceptible to superinfection.

Although the lipid A that is attached to the LPS core is conserved in Salmonella, it has recently been shown that the enzymes that make the lipid substitutions on the core galactosamines are regulated in Salmonella by the two component regulator PhoP/Q (20). Mutants that are constitutively on (Phoc) and constitutively off (PhoP–) are both avirulent in mice. The Phoc mutant adds an amino-arabinose to the core disaccharide, hydroxylates a myristic acid, and adds a palmitate to one of the amino-galactose core sugars (20). The PhoP– strain cannot make these lipid A modifications. Salmonella with the modified LPS are both more resistant to antimicrobial peptides and their lipid A is less stimulatory for macrophages (20).

Flagellin. Another potent antigen on Salmonella is flagellin. Interestingly, most _Salmonella_-specific CD4+ T lymphocytes that are generated in response to Salmonella infection are directed at flagellin epitopes (21). There is a great deal of variation in the central portion of the flagellin genes, while the NH2 and COOH termini are highly conserved. This variation is used to define specific flagellar antigens, which are used in the Kauffmann-White scheme to serotype Salmonella. It is not known whether the variations in flagellin structure affect virulence.

Most Salmonella have two sets of flagellin genes that are distinct, and they switch expression between the two at a rate of 10–3 to 10–5, so that only one allele is expressed by any given bacterium. The operon that controls the synthesis of phase 1 flagella also encodes a repressor of phase 2 flagellin synthesis (22). The switch to phase 2 flagellin is controlled by a gene that promotes an inversion of a segment of DNA in the phase 1 operon that prevents transcription of the operon, shutting off synthesis of both phase 1 flagellin and the repressor of the phase 2 operon (22). It is possible that this switching mechanism may be a way for the bacteria to temporarily avoid cellular immunity. While antibody to flagellin is not protective, flagellin may be an important T-cell target.

In addition to being antigenic, flagellae from both S. typhi and S. typhimurium also stimulate macrophages to make TNF-α (23). Surprisingly, the phase 2 flagellin from S. typhimurium is not as potent as the phase 1 flagellin. This suggests that flagellar phase variation may be a mechanism to downregulate inflammation in the host. The inflammatory potential of individual flagellin proteins made by different Salmonella serotypes has not been extensively compared.

Fimbriae. Salmonella make several classes of fimbriae, including the so-called long polar fimbriae (Lpf), which mediate attachment of Salmonella to Peyer’s patches in the mouse. Expression of Lpf in Salmonella undergoes phase variation, such that the bacteria alternate between expressing and not expressing Lpf. Norris and Bäumler recently provided the first demonstration of the biological importance of this phase variation. These authors showed that the lpf genes in S. typhimurium and S. enteritidis are highly conserved and Lpf proteins are antigenically crossreactive (24). Furthermore, while immunization with an Lpf-expressing strain of S. typhimurium did not protect against a subsequent challenge with S. enteritidis, it did select for phase variants of S. enteritidis not expressing Lpf. This demonstrates the advantage to the pathogen of being able to switch off synthesis of immunogenic surface proteins, and it also suggests why Salmonella have so many different and apparently redundant fimbriae.