Blockade of CD14 aggravates experimental shigellosis.

Shigella infections lead to severe inflammation associated with destruction of colonic mucosa. We assessed the effect of in vivo blockade of CD14 on the outcome of experimental Shigella infection in rabbits. A total of 17 rabbits were divided into two groups: 8 received a single i.v. dose of anti-rabbit CD14 monoclonal antibody prior to infection with an invasive Shigella flexneri strain; the remainder served as controls. The anti-CD14-treated rabbits exhibited more severe tissue destruction and a 50-fold increase in bacterial invasion of the intestinal mucosa when compared to controls. Similar numbers of polymorphonuclear leukocytes were recruited to the intestinal mucosa in both groups despite the massive bacterial invasion seen in the CD14-blocked group. No statistically significant differences were seen in levels of IL-1beta nor in the ratio of IL-1RA/IL-1beta for either group. In contrast, higher quantities of TNF-alpha were observed in the CD14-blocked group. To conclude, anti-CD14 treatment had a detrimental effect on the capacity of Shigella-infected animals to clear the infection.

Parameters underlying successful protection with live attenuated mutants in experimental shigellosis.

Because the use of live attenuated mutants of Shigella spp. represents a promising approach to protection against bacillary dysentery (M. E. Etherridge, A. T. M. Shamsul Hoque, and D. A. Sack, Lab. Anim. Sci. 46:61-66, 1996), it becomes essential to rationalize this approach in animal models in order to optimize attenuation of virulence in the vaccine candidates, as well as their route and mode of administration, and to define the correlates of protection. In this study, we have compared three strains of Shigella flexneri 5--the wild-type M90T, an aroC mutant, and a double purE aroC mutant--for their pathogenicity, immunogenicity, and protective capacity. Protection against keratoconjunctivitis, induced by wild-type M90T, was used as the protection read out in guinea pigs that were inoculated either intranasally or intragastrically. Following intranasal immunization, the aroC mutant elicited weak nasal tissue destruction compared to M90T and achieved protection correlated with high levels of local anti-lipopolysaccharide immunoglobulin A (IgA), whereas the purE aroC double mutant, which also elicited weak tissue destruction, was not protective and elicited a low IgA response. Conversely, following intragastric immunization, only the M90T purE aroC double mutant elicited protection compared to both the aroC mutant and the wild-type strain. This mutant caused mild inflammatory destruction, particularly at the level of Peyer's patches, but it persisted much longer within the tissues. This could represent an essential parameter of the protective response that, in this case, did not clearly correlate with high anti-lipopolysaccharide IgA titers.

Caspase-1 activation of IL-1beta and IL-18 are essential for Shigella flexneri-induced inflammation.

Caspases are intracellular proteases that mediate mammalian cell apoptosis. Caspase-1 (Casp-1) is a unique caspase because it activates the proinflammatory cytokines interleukin (IL)-1beta and IL-18. Shigella flexneri, the etiological agent of bacillary dysentery, induces macrophage apoptosis, which requires Casp-1 and results in the release of mature IL-1beta and IL-18. Here we show that casp-1(-/-) mice infected with S. flexneri do not develop the acute inflammation characteristic of shigellosis and are unable to resolve the bacterial infection. Using casp-1(-/-) mice supplemented with recombinant cytokines and experiments with IL-1beta(-/-) and IL-18(-/-) mice, we show that IL-1beta and IL-18 are both required to mediate inflammation in S. flexneri infections. Together, these data demonstrate the importance of Casp-1 in acute inflammation and show the different roles of its substrates, IL-1beta and IL-18, in this response.

Blockade of CD14 increases Shigella-mediated invasion and tissue destruction.

Shigella is a diarrheal pathogen that causes disease through invasion of the large intestinal mucosa. The endotoxin of the invading bacterium may play a key role in the disease process by causing inflammation and tissue injury during infection. Earlier studies have shown that various animal species lacking functional CD14 were protected against endotoxin-mediated shock. Rabbits experimentally infected with Shigella were used to test the hypothesis that blockade of endotoxin-induced cell activation with anti-CD14 mAb would diminish inflammation and thus disease severity. Unexpectedly, we observed that the intestinal mucosa of anti-CD14-treated animals exhibited a 50-fold increase in bacterial invasion and more severe tissue injury compared with controls. Despite higher bacterial loads in treated animals, the numbers of polymorphonuclear leukocytes that were recruited to the infection site were similar to those in controls. Furthermore, the phagocytic cells of CD14-blocked animals produced IL-1 and TNF-alpha. Moreover, in vitro blockade of CD14 did not impede bactericidal activity. Thus, anti-CD14 treatment interfered with host defense mechanisms involved with removal/eradication of Shigella.

Increased interleukin-1 (IL-1) and imbalance between IL-1 and IL-1 receptor antagonist during acute inflammation in experimental Shigellosis.

Infection by the enteric bacterial pathogen Shigella results in intense mucosal inflammation and destruction of the colonic and rectal epithelium in infected humans. Initial bacterial translocation occurs through the follicle-associated epithelium. Previous experiments suggest that interleukin-1 (IL-1) is crucial to trigger inflammation, particularly in the follicular zones. During the first 4 hours of infection in a rabbit ligated-loop model of intestinal invasion, there are two salient characteristics: (i) a high concentration of IL-1alpha and IL-1beta, both in infected Peyer's patch tissue and in the corresponding efferent mesenteric blood, and (ii) a very low level of expression of IL-1 receptor antagonist (IL-1ra). These may reflect a combination of regulation of expression and secretion of IL-1alpha, IL-1beta, and IL-1ra by both resident and recruited phagocytes and the induction of mononuclear phagocyte apoptosis by Shigella. This low IL-1ra/IL-1 ratio likely accounts for the rapid, uncontrolled inflammation characteristic of shigellosis.

Interleukin-8 controls bacterial transepithelial translocation at the cost of epithelial destruction in experimental shigellosis.

In shigellosis, the network of cellular interactions mediated by a balance of pro- and anti-inflammatory cytokines or chemokines is clearly tipped toward acute destructive inflammation of intestinal tissues by the bacterial invader. This work has addressed the role played by interleukin-8 (IL-8) in a rabbit model of intestinal invasion by Shigella flexneri. IL-8, which is largely produced by the epithelial cells themselves, appears to be a major mediator of the recruitment of polymorphonuclear leukocytes (PMNs) to the subepithelial area and transmigration of these cells through the epithelial lining. Neutralization of IL-8 function by monoclonal antibody WS-4 caused a decrease in the amount of PMNs streaming through the lamina propria and the epithelium, thus significantly attenuating the severity of epithelial lesions in areas of bacterial invasion. These findings are in agreement with our previous work (31). In contrast to the PMNs, the bacteria displayed increased transepithelial translocation, as well as overgrowth in the lamina propria and increased passage into the mesenteric blood. By mediating eradication of bacteria at their epithelial entry site, although at the cost of severe epithelial destruction, IL-8 therefore appears to be a key chemokine in the control of bacterial translocation.

Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB.

We report here that the Shigella invasion plasmid antigen (Ipa)B, which is sufficient to induce apoptosis in macrophages, binds to caspase (Casp)-1, but not to Casp-2 or Casp-3. Casp-1 is activated and its specific substrate interleukin-1beta is cleaved shortly after Shigella infection. Macrophages isolated from Casp-1 knock-out mice are not susceptible to Shigella-induced apoptosis, although they respond normally to other apoptotic stimuli. Shigella kills macrophages from casp-3, casp-11, and p53 knock-out mice as well as macrophages overexpressing Bcl-2. We propose that Shigella induces apoptosis by directly activating Casp-1 through IpaB, bypassing signal transduction events and caspases upstream of Casp-1. Taken together these data indicate that Shigella-induced apoptosis is distinct from other forms of apoptosis and seems uniquely dependent on Casp-1.

Immunogenicity of IpaC-hybrid proteins expressed in the Shigella flexneri 2a vaccine candidate SC602.

We have investigated the capacity of live attenuated Shigella flexneri strains to act as vectors for the induction of local and systemic antibody responses against heterologous epitopes. The S. flexneri IpaC antigen was selected as a carrier protein into which the C3 neutralizing epitope of the poliovirus VP1 protein was inserted in eight sites distributed along IpaC. The resulting IpaC-C3 hybrid proteins were expressed from recombinant plasmids in the S. flexneri 2a vaccine candidate, SC602. Their production was similar to that of wild-type IpaC. All of the hybrid proteins but one were secreted as efficiently as wild-type IpaC. Immunization of mice with each of the recombinant SC602 derivatives reveals that one construct is able to induce serum and local anti-C3 antibodies, showing that at least one permissive site of insertion within IpaC can be defined. Furthermore, mouse-to-mouse variability in the anti-C3 response indicates that the amount of hybrid proteins produced in the host by SC602 should be improved for optimal use of S. flexneri live attenuated strains as mucosal vectors for foreign epitopes.

Induction of anti-carbohydrate antibodies by phage library-selected peptide mimics.

One of the prerequisites for the development of polysaccharide subunit vaccines is the induction of an efficient immune response to carbohydrate antigens like lipopolysaccharide (LPS) or capsular polysaccharide antigens of pathogens. In an attempt to overcome the problems that arise from the T-independent immune response induced by such antigens, selecting peptide sequences that mimic protective carbohydrate epitopes has been proposed. In this study, we investigate a new selection strategy for immunogenic peptide mimics using the phage-displayed peptide library technology. Two monoclonal antibodies (mAb) of the A isotype (mIgA), mIgA C5 and mIgA I3, specific for the O-antigen (O-Ag) part of the human pathogen Shigella flexneri serotype 5a LPS and protective against homologous infection were used to screen two phage-displayed nonapeptide libraries in pVIII. Using mIgA C5, 13 different specific clones were selected, and 6 using mIgA I3; 5 of the latter also interacted in enzyme-linked immunosorbent assay with the first mAb. All of the 19 clones selected were separately used to immunize mice, but only 2 of them, p100c (mIgA I3-specific) and p115 (interacting with both mIgA) were able to induce anti-O-Ag antibodies. The immune response was specific for the O-Ag of the S. flexneri serotype 5a, and also selectively recognized the corresponding bacterial strain. The amino acid sequences of p100c and p115 immunogenic peptide mimics were YKPLGALTH (flanked by two Cys residues) and KVPPWARTA, respectively. These results are the first example of immunogenic mimicry of carbohydrates by phage-displayed peptides, and indicate a new strategy of selection of immunogens for the development of anti-polysaccharide vaccines.

In vivo apoptosis in Shigella flexneri infections.

Shigella flexneri, an etiological agent of bacillary dysentery, causes apoptosis in vitro. Here we show that it also induces apoptosis in vivo. We were able to quantify the number of apoptotic cells in rabbit Peyer's patches infected with S. flexneri by detecting cells with fragmented DNA. Infection with virulent S. flexneri results in massive numbers of apoptotic cells within the lymphoid follicles. In contrast, neither an avirulent strain nor an avirulent strain capable of colonizing Peyer's patches increases the background level of apoptotic cells. Macrophages, T cells, and B cells are shown to undergo apoptosis in vivo. These results indicate that apoptosis may play a crucial role in the pathogenesis of shigellosis.

Infection of rabbit Peyer's patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium.

In order to invade the colonic mucosa, the bacterial pathogen Shigella flexneri must find a site of entry. Experiments with the rabbit ligated intestinal loop model described here confirm that M cells of the follicle-associated epithelium (FAE) that covers lymphoid structures of the Peyer's patches represent a major site of entry for invasive microorganisms. In addition, in an isogenic Shigella background, expression of an adhesive phenotype, or of an invasive phenotype, is required for bacteria to efficiently colonize the FAE. A nonadhesive, noninvasive mutant barely interacted with FAE. Adhesive and invasive strains induced dramatic but different alterations on FAE. Invasive strain M90T caused major inflammation-mediated tissue destruction after 8 h of infection. Adhesive strain BS15 caused limited inflammation, but major architectural changes, characterized by an increase in the size of M cells that became stretched over large pockets containing an increased number of mononuclear cells, were observed. M cells progressively occupied large surface areas of the FAE at the expense of enterocytes. This contributed to enterocytes losing contact with the lumen. These experiments demonstrate that various remodeling patterns may occur in Peyer's patches in response to bacterial pathogens, depending on the virulence phenotype expressed by the pathogenic strain.

Role of interleukin-1 in the pathogenesis of experimental shigellosis.

The effect of human recombinant interleukin-1 receptor antagonist on intestinal inflammation, tissue destruction, and bacterial invasion during experimental shigellosis caused by Shigella flexneri was studied in the rabbit-ligated loop infection model. Intravenous infusion of the inhibitor at a dose of 2 mg/kg per h, was initiated 30 min before intestinal loops were ligated and infected, and continued during the 8-h period of infection. The animals treated with IL-1 receptor antagonist showed a striking decrease in inflammation, destruction, and bacterial invasion of their tissues, both at the level of the villous intestine and Peyer's patches. This is conclusive evidence that interleukin-1 plays a critical role in the pathogenesis of shigellosis. This proinflammatory cytokine is here proposed as a major trigger of the inflammatory reaction which is characteristic of this invasive disease of the intestine, due to the particular interaction existing between S. flexneri and macrophages.

Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri.

Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection.

OmpB (osmo-regulation) and icsA (cell-to-cell spread) mutants of Shigella flexneri: vaccine candidates and probes to study the pathogenesis of shigellosis.

Genetic and molecular data now available on the pathogenic properties of Shigella flexneri allow rational design of live attenuated vaccine strains. The genes required at given steps of the infection process can be selectively mutated to impair the bacterium's capacity to interact with intestinal epithelial cells and/or survive within intestinal tissues in general. We have tested two mutations in S. flexneri serotype 5a (M90T) which, alone or in combination, have yielded promising results when evaluated as vaccine prototypes in orally infected macaque monkeys. The first mutation, icsA, blocks intracellular and cell-to-cell spread of the micro-organism. This mutant (SC560) appeared reasonably well tolerated and elicited protection against homologous challenge. The second mutation, ompB, disconnects the bacterium from one of its major environmental regulatory factors, osmolarity. This mutant (SC433) still caused slight dysenteric symptoms in vaccinees. It was also perfectly protective. When these two mutations were combined, the double mutant (SC445), was perfectly tolerated but failed to protect one out of five animals. These studies bring interesting prospects of the possibility of immunizing against shigellosis. In addition to providing new possibilities for vaccine design, construction and evaluation of these mutants allowed substantial progress in understanding the pathogenesis of shigellosis.

Construction and evaluation of live attenuated vaccine strains of Shigella flexneri and Shigella dysenteriae 1.

Shigellosis is an invasive disease of the human colon which is particularly prevalent among children of the developing world. No proper vaccine is available to protect against this enteric disease. It is currently accepted that only live strains with attenuated virulence administered orally may elicit protective immunity at the level of the colonic mucosa, which is the exclusive site of multiplication of causative microorganisms such as Shigella flexneri and Shigella dysenteriae 1. We have constructed such vaccine candidates based on the destruction of virulence genes responsible for selected steps of the infection process. In S. flexneri, a combination of two mutations impairing cell-to-cell spread (icsA) and aerobactin production and transport (iuc, iut) which support growth within tissues provide a well tolerated and protective vaccine prototype against shigellosis in macaque monkeys. In S. dysenteriae 1, similar mutations are currently being introduced, in addition to one which eliminates the catalytic activity of Shiga toxin. These mutants and others will be tested soon in human phase I trials.

Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis.

A Shigella flexneri serotype 5 strain deficient in the production of the iron-containing superoxide dismutase FeSOD (sodB) and a catalase-negative (katFG) S. flexneri serotype 5 strain were isolated. Both strains were examined for increased sensitivity to oxygen stress by using assays involving killing by mouse peritoneal macrophages and human polymorphonuclear leukocytes as well as infection of rabbit ileal loops. The sodB mutant was extremely sensitive to killing by phagocytes when compared with the wild-type parent, M90T. The catalase mutant also showed an increased sensitivity to killing, but to a much lesser extent. Upon infection of rabbit ileal loops and subsequent histopathological examination, the sodB mutant caused very little detectable damage to intestinal villi. The pattern of infection was roughly similar to that of BS176, an avirulent plasmidless derivative of M90T. The katFG mutant, on the other hand, showed a high degree of destruction, similar to that caused by M90T. This evidence suggests that the superoxide dismutase encoded by sodB may play an important role in the pathogenesis of S. flexneri. In contrast, catalases appear to make a limited contribution to virulence.

Construction and evaluation of a double mutant of Shigella flexneri as a candidate for oral vaccination against shigellosis.

Based on studies on the genetic and molecular basis of Shigella flexneri invasive properties, we have constructed and evaluated a double mutant of S. flexneri serotype 5 for utilization as a live attenuated oral vaccine against shigellosis. The first mutation, icsA, blocks intracellular spread of bacteria as well as cell-to-cell infection. It affects the capacity of the invasive pathogen to form large abscesses in epithelia. The second mutation, iuc, eliminates production of the siderophore aerobactin thus impairing growth of the bacterium within tissues. This double mutant, SC5700 appeared safe when administered intragastrically to macaque monkeys as three doses (5 x 10(10) c.f.u. each) at weekly intervals. Protection against a challenge by the wild type isolate (M90T) was observed 4 weeks after the last vaccine inoculation. Duration of carriage was considerably reduced as compared to the control group in which all animals had developed severe dysentery. Seroconversion against serotype 5 LPS as well as S. flexneri virulence associated polypeptides was also observed.

Mucoid phenotype of Klebsiella pneumoniae is a plasmid-encoded virulence factor.

We have previously reported that the presence of a 180-kilobase plasmid encoding production of aerobactin was correlated with the virulence of Klebsiella pneumoniae K1 and K2 isolates. This work demonstrates that a variant of a K2 strain which has lost this plasmid, pKP100, becomes avirulent. Labeling of this plasmid with the mobilizable, replication-defective element pME28, used here as a mobilizable transposon, allowed the transfer of this plasmid into a plasmidless derivative. Virulence was restored upon reacquisition of this tagged plasmid, pKP101. In addition to aerobactin production, another phenotype could be correlated with the presence of this virulence plasmid: the mucoid phenotype of the bacterial colonies. Both wild-type and plasmidless strains are encapsulated, but only the former presented mucoid colonies. Participation of this phenotype in the virulence of K. pneumoniae was demonstrated by constructing a mutant altered in the plasmid gene encoding this phenotype. The resulting strain demonstrated a 1,000-fold decrease in virulence. Introduction of the recombinant plasmid pKP200 carrying the gene encoding this mucoid phenotype into Escherichia coli HB101 also led to the production of a mucoid phenotype. Rocket immunoelectrophoresis demonstrated that in E. coli this phenotype was due to the production of colanic acid. On the other hand, neither the overproduction of K2 capsular polysaccharide nor the presence of colanic acid was detected in mucoid strains of K. pneumoniae. We conclude that this mucoid phenotype is definitely an important virulence factor of K. pneumoniae. It is due to the plasmid-encoded production of a substance which is different from colanic acid and the capsular polysaccharide of K. pneumoniae.

Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox- mutant of Shigella dysenteriae 1.

A Tox- mutant of Shigella dysenteriae 1, SC501, was genetically engineered by cloning the Shiga toxin operon, inserting a cassette into the A subunit gene, and exchanging this in vitro-mutagenized sequence with the wild-type gene. SC501 produced a low amount of residual cytotoxicity which was not neutralized by a rabbit immune serum directed against Shiga toxin. Invasion of cultured cells demonstrated that Shiga toxin had no effect on the rate of intracellular growth of bacteria or on the rapid killing of invaded host cells. On the other hand, several significant differences were observed in macaque monkeys infected intragastrically with either the wild-type strain or its mutant. The production of Shiga toxin by the invading strain was correlated with the presence of blood within stools, a sharp drop in blood polymorphonuclear cells, and histopathological alterations, such as the destruction of capillary vessels within the connective tissue of the colonic mucosa, severe inflammatory vasculitis of the peritoneal mesothelium, and major efflux of inflammatory cells to the intestinal lumen. It is proposed that Shiga toxin influences the severity of bacillary dysentery by inducing colonic vascular damage, which accounts for bloody stools, intestinal ischemia, and inflation of a polymorphonuclear intestinal compartment during the infectious process.

Possible role of a defect in hepatic bilirubin glucuronidation in the initiation of cholesterol gallstones.

Abnormally low activity of hepatic bilirubin UDP-glucuronosyltransferase was found in 25% of 81 unselected patients with gallstones, as compared with only 3% in 35 controls. At the time of cholecystectomy, the stones were taken for analysis in 48 of 81 patients, and a bile sample was obtained in 42 of them. Among the stones, 75% were cholesterol stones, 15% pigment stones, and 10% 'intermediate' stones. Low hepatic conjugating activity was not preferentially associated with a given type of stone. No relation was found between the enzymic deficiency and the biliary cholesterol saturation index. A high proportion of biliary bilirubin monoglucuronide (over 40%) was found in four of seven patients with low transferase activity, as was earlier demonstrated in patients with overt Gilbert's syndrome. Raised biliary bilirubin monoglucuronide was also found in three patients, out of 46, who had normal transferase but raised biliary beta-glucuronidase activity. There was no evidence that deficient bilirubin conjugation could be a consequence of gallstones: the activity of another hepatic microsomal enzyme, glucose 6-phosphatase, taken as a reference, was measured in 12 patients and was always normal. Taking into account the very high frequency of a bilirubin centre in the cholesterol stones (87% of the cases in the present series), it is suggested that the increased proportion in poorly soluble biliary bilirubin monoglucuronide, which was associated with defective conjugation, could act as a trigger for gallstone initiation, regardless of the final composition of the stone.