RODNEY A. WELCH
The type I extracellular secretion system in gram-negative bacteria was originally called the RTX family because the members first described were the hemolysins and leukotoxins produced by E. coli, Pasteurella haemolytica, Actinobacillus species, and Bordetella pertussis (76). Following their discovery, other functional classes of exoproteins were found to share the classic nonapeptide Ca2+-binding repeats, C-terminal secretion signal, and dedicated three-gene transport system. The extracellular transport of RTX proteins requires an ABC-type inner membrane protein (B protein), a periplasm-spanning channel protein (D protein), and a TolC homolog (79). Besides the E. coli hemolysin and related toxins, the type I secretion family encompasses exoproteins with activities involving nodulation, proteolysis, lipolysis, heme-binding, or bacteriocin production (1, 19, 21).
Many of the RTX toxins have toxic activities limited to specific types of host cells and animal species (e.g., P. haemolytica leukotoxin). The E. coli hemolysin is broadly active toward many different cell types (erythrocytes, leukocytes, endothelial cells, etc.) (14, 34, 41, 69) from a wide variety of species, including humans and ruminants. Among E. coli or Salmonella strains there are no known examples of strict RTX leukotoxins in which lytic activity is limited to white blood cells. However, the pO157-encoded EhxA RTX toxin produced by E. coli O157:H7 strains has a mixture of cellular and host specificities unlike any of the other RTX toxins (5). Currently there is little direct evidence linking EhxA expression to the pathogenesis of E. coli O157:H7 hemorrhagic colitis and hemolytic-uremic syndrome.
Early during the discovery of the RTX family it appeared that the RTX toxins were limited to extraintestinal pathogens (76). However, with the discovery of the E. coli O157:H7 EhxA and V. cholerae RtxA toxins it became evident that RTX toxins are likely to provide a selective advantage for pathogens infecting practically any niche in the body. This point continues to be relevant with the recent discovery of RTX-like gene sequences in the genomes of different E. coli pathotypes and Salmonella strains. For example, there are RTX B, D, and TolC-like genes linked to very large open reading frames (ORFs) encoding RTX-like proteins in Salmonella enterica serovar Typhi CT18 (ORF STY4458, accession no. AAL23085). These RTX-like proteins have lengthy tandem-arrayed repeats that are different from those seen for either the E. coli hemolysin or V. cholerae RtxA proteins. The functions of these candidate RTX exoproteins are unknown, but the absence of a linked RTX C-like gene suggests that these particular proteins are not likely to undergo a posttranslational fatty acid modification process. This suggests that they lack the cytotoxic activities typically attributed to the acylated RTX toxins.
The extracellular targeting sequence recognized by the dedicated HlyB, HlyD, and TolC export machinery occurs within the C-terminal 60-amino-acid residues. This region has been the object of extensive studies (16, 24, 38, 65, 80). The hemolysin export sequence cannot be recognized as a specific primary sequence, but it is structurally defined only in that it possesses an amphipathic α-helix followed by a charge-rich linker sequence of 8 to 10 residues with the α-helix positioned approximately 45 residues from the C terminus (28).
As discussed above, posttranslational acylation of the RTX protoxin by HlyC-like proteins (51) is a common requirement for cytotoxic activities. For at least the E. coli hemolysin and the B. pertussis hemolysin/adenylate cyclase, the sites of these modifications are known (25, 29, 44, 58). Hackett et al. found that the B. pertussis hemolysin/adenylate cyclase RTX toxin is uniformly palmitoylated at CyaAK983 (25). In the recombinant E. coli background, the position is palmitoylated 87% of the time, and the remaining molecules are myristoylated. In addition, in the E. coli background, a second position, CyaAK860, is palmitoylated 60% of the time. The functional consequences of these acylation variations are that the CyaA prepared from the E. coli background is less erythrolytic but similar to the toxin purified from the Bordetella background in levels of invasive adenyl cyclase-mediated toxicity (26). In contrast, in in vitro reactions using proHlyA and HlyC, active HlyA can be produced that is modified with saturated C14, C16, and C18 fatty acids. C14 in the reaction provides the highest level of erythrolytic activity (29). Quite surprisingly, however, when native, in vivo modified HlyA prepared from either the Goebel or Welch laboratories is examined, the actual fatty acids used at positions 563 and 689 are saturated 14-, 15-, and 17-carbon amide-linked side chains (44) found on 68%, 26%, and 6% of the HlyA molecules, respectively. The modification of HlyA with odd-numbered carbon fatty acids is a unique observation. It also indicates that the in vivo HlyA species are very heterogeneous in structure. The functional significance of the different fatty acid species is unknown. The suspicion is that they may contribute to host and cell type specificity or interactions in host cell lipid-modified signaling pathways.
It is critical to point out in summary that four acylation-independent events occur in E. coli hemolysin biogenesis and cytotoxicity. They are Ca2+ binding to the repeats, extracellular secretion, pore formation in artificial lipid bilayers, and loose association of the hemolysin with target cells (4, 7, 12, 46, 49, 51). Disappointingly, despite efforts by many different laboratories, the evidence is scant as to the secondary, tertiary, and multimeric structures for the E. coli RTX toxins. The absence of a resolved structure for HlyA has thwarted the structure and functional analyses of large sets of hemolysin mutants collected in many laboratories.
Despite numerous studies, the mechanism for hemolysin cytotoxicity remains unresolved. It is unlikely that erythrocytes are a significant target for the hemolysin during E. coli disease; however, many critical observations have been made with this convenient reagent. The significance of these observations for elucidation of important hemolysin structural and functional activities is uncertain. It is clear that the E. coli hemolysin causes rapid ion fluxes in treated cells. Jorgensen and colleagues (33) found that, within 2 min of treating erythrocytes with the E. coli hemolysin, Ca2+ ions begin to accumulate intracellularly and K+ ions are released from the cell into the medium. The ion flow appears to be selective because several different externally applied, small radiolabeled molecules (e.g., 32PO4) do not enter the cell. It is also certain that the final destruction of erythrocytes is by colloidal osmotic lysis (8). For other, more relevant, host cells the most popular hypothesis explaining hemolysin cytotoxicity is that a large sharp and unregulated Ca 2+influx initiates cytoskeletal destruction with multiple necrotic sequelae (reviewed in reference 75). Biochemical and physical definition of these events has not taken place. A recent observation by Uhlen and coworkers adds a new layer of complexity to the Ca2+ ion influx mechanism. They observed that semipurified hemolysin preparations taken from culture supernatants cause a continuous, low-frequency oscillation in intracellular [Ca2+] in primary rat renal tubular cells (73). Based on the use of specific inhibitors, the oscillatory effect is attributed to Ca2+ ion influx through L-type calcium channels and intracellular stores controlled by inositol triphosphate (13) and not a hemolysin-mediated transmembrane pore. The calcium oscillatory behavior induces proinflammatory cytokines, interleukin-6 (IL-6) and IL-8. The Ca2+ ion oscillatory effect occurs over a small, 10-fold concentration range of hemolysin where, below a threshold of input toxin, intracellular [Ca2+] does not change but at too high a hemolysin concentration the cultured cells lyse.
Although the hemolysin causes necrotic death at high concentrations that is characterized as lysis, apoptosis-like events can be observed at a lower, narrow window of "sublytic" concentrations for many different RTX toxins (35, 36, 39, 48, 67). If RTX toxins are apoptotic agents, it is not clear that they act by recognized apoptotic mechanisms. Classic tumor necrosis factor receptor (Fas)- or Bcl-2-mediated apoptotic cell death occurs 2 to 3 days after the induction event with intermediary signs of apoptosis, such as DNA fragmentation occurring nearly a day after induction. On the other hand when cells are treated with sublytic amounts of RTX toxins, apoptotic processes such as DNA fragmentation occur within 3 to 6 h (48, 67, 68). The difficulty lies in assessing the set of toxin concentrations and time conditions under which the RTX toxins are likely to act in vivo. It is possible that hemolysin-mediated cellular cytotoxicity is a mixed event in which, during the presence of an active infection and presumably continuous hemolysin production, there are instances and sites where sufficient amounts of hemolysin cause apoptosis and, in other instances, higher hemolysin concentrations lead to necrosis.
Helena Ostolaza and colleagues have obtained results consistent with the hypothesis that the abundant erythrocyte surface molecule glycophorin acts as a receptor for the E. coli hemolysin (17). In addition, the same group has shown that the small, C-terminal 22-amino-acid hemolysin segment from residues 914 to 936 acts as the toxin ligand for glycophorin (18). Lally and coworkers demonstrated that the host cell surface leukocyte function-associated antigen (LFA-1) participates in RTX toxin activity (40). Anti LFA-1 antibodies were shown to neutralize cellular toxicity of the Actinobacillus actinomycetemcomitans leukotoxin. The role of LFA-1 in this toxin's action was further supported when a normally non-LFA-1-expressing and A. actinomycetemcomitans leukotoxin- or E. coli hemolysin-insensitive human erythroleukemia cell line, K562, was transformed with a recombinant plasmid encoding the two subunits (CD11a and CD18) of LFA-1, leading to toxin sensitivity. These results are consistent with LFA-1 acting as receptor for these two RTX toxins. The attraction of the RTX toxin ligand-LFA-1 receptor hypothesis is that sequence divergence among RTX toxins and the CD11a and CD18 subunits could account for the different host and cell type specificities observed for the RTX leukotoxins mentioned above. Jeyaseelan and coworkers performed a similar set of experiments with the P. haemolytica leukotoxin, which confirmed that bovine-specific forms of LFA-1 help mediate its toxicity (30). Although no new reports have supported LFA-1 in cytotoxicity of the E. coli hemolysin, our laboratory has observed that the SK-β2-7 cell that fails to express LFA-1 because of a mutation in CD18 (74) is 32-fold reduced in sensitivity to the E. coli hemolysin (unpublished results). The reduced, but sustained, sensitivity of this cell line suggests that the E. coli hemolysin may use multiple, different receptor molecules or that possibly LFA-1 acts as a coreceptor where another molecule can bind significant amounts of the toxin. All in all, the results are consistent with the hypothesis that LFA-1 acts as a receptor for RTX toxins. In an alternative hypothesis that has not been tested, LFA-1 serves as a component of a cytoskeletal structure or as a component in a signaling pathway that RTX toxins disrupt.
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