Cytotoxicity of Shigella dysenteriae 1 for cultured mammalian cells.

The cytotoxicity of an invasive toxigenic wild-type strain of Shigella dysenteriae 1 (3818T) was compared with that of noninvasive, toxigenic strain 38180 and hypotoxigenic strain 725. Cytolysis of HeLa or Henle 407 cells exposed to these strains was measured by release of (3H) uridine from prelabeled monolayers. HeLa cells exposed to noninvasive, toxigenic strain 38180, or to partially purified Shiga toxin were lysed only after a latent period of more than 8 hr. During this period, protein synthesis was inhibited. In contrast, Henle 407 cells that were exposed to strain 38180 or to exogenous Shiga toxin were unaffected. When either Henle 407 or HeLa cells were infected with invasive toxigenic strains, rapid lysis ensued. Quantitative microassay of cytosol toxicity showed that Shiga toxin was produced intracellularly by strain 3818T. The data suggest that cytolysis of infected mammalian cells is caused, at least in part, by intracellular Shiga toxin.

Ultrasound measured renal length versus low dose CT volume in predicting single kidney glomerular filtration rate.

Ultrasound measured renal length and CT measured renal volume are potential surrogate markers for single kidney glomerular filtration rate (SKGFR). The aims of this study are to determine: (1) the repeatability of ultrasound measured length and low radiation dose spiral CT measured volume; (2) the relationship between renal length and volume; and (3) whether length and/or volume is a predictor of SKGFR. 69 patients with suspected renal artery stenosis underwent ultrasound renal length measurement, CT evaluation of renal volume and assessment of SKGFR. 40 patients had ultrasound measurement of length and CT evaluation of volume performed twice on two separate visits. 25 patients also had ultrasound measured renal parenchymal thickness and area. The region of interest was drawn around the kidneys and a threshold set to subtract renal peripelvic fat and renal pelvis. The volume from each slice was summed to obtain the total volume for each kidney. The limits of agreement for ultrasound measured renal length were -1.6 cm to 1.52 cm and that for CT renal volume were -33 ml to 32 ml. There was significant correlation between ultrasound measured length and CT volume (r=0.74, p<0.01). Volume was a better predictor of SKGFR (r(2)=0.57) than length (r(2)=0.48). The combined parameters of ultrasound measured length, area and parenchymal thickness were a better predictor of volume (r(2)=0.81) and SKGFR (r(2)=0.58) than ultrasound measured length on its own. The low dose CT technique was reasonably reproducible and renal volume measurements correlate better with SKGFR than length. Ultrasound predictions of renal volume and SKGFR can be improved by incorporating cross-sectional area and parenchymal thickness. Further investigation is required to refine our low dose CT technique.

Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection.

Shigella causes diarrhea and dysentery through contaminated food and water. Shigella sonnei live vaccine candidates WRSs2 and WRSs3 are attenuated principally by the loss of VirG(IcsA) that prevents bacterial spread within the colonic epithelium. In this respect they are similar to the clinically tested vaccine candidate WRSS1. However, WRSs2 and WRSs3 are further attenuated by loss of senA, senB and WRSs3 also lacks msbB2. As previously shown in cell culture assays and in small animal models, these additional gene deletions reduced the levels of enterotoxicity and endotoxicity of WRSs2 and WRSs3, potentially making them safer than WRSS1. However the behavior of these second-generation VirG(IcsA)-based vaccine candidates in eliciting an immune response in a gastrointestinal model of infection has not been evaluated. In this study, WRSs2 and WRSs3 were nasogastrically administered to rhesus monkeys that were evaluated for colonization, as well as for systemic and mucosal immune responses. Both vaccine candidates were safe in rhesus monkeys and behaved comparably to WRSS1 in bacterial excretion rates that demonstrated robust intestinal colonization. Furthermore, humoral and mucosal immune responses elicited against bacterial antigens appeared similar in all categories across all three strains indicating that the additional gene deletions did not compromise the immunogenicity of these vaccine candidates. Based on data from previous clinical trials with WRSS1, it is likely that, WRSs2 and WRSs3 will not only be safer in human volunteers but will generate comparable levels of systemic and mucosal immune responses that were achieved with WRSS1.

Genetic basis of virulence in Shigella species.

Shigella species and enteroinvasive strains of Escherichia coli cause disease by invasion of the colonic epithelium, and this invasive phenotype is mediated by genes carried on 180- to 240-kb plasmids. In addition, at least eight loci on the Shigella chromosome are necessary for full expression of virulence. The products of these genes can be classified as (i) virulence determinants that directly affect the ability of shigellae to survive in the intestinal tissues, e.g., the aerobactin siderophore (iucABCD and iutA), superoxide dismutase (sodB), and somatic antigen expression (rfa and rfb); (ii) cytotoxins that contribute to the severity of disease, e.g., the Shiga toxin (stx) and a putative analog of this toxin (flu); and (iii) regulatory loci that affect the expression of plasmid genes, e.g., ompR-envZ, which mediates response to changes in osmolarity, virR (osmZ), which mediates response to changes in temperature, and kcpA, which affects the translation of the plasmid virG (icsA) gene which is associated with intracellular bacterial mobility and intracellular bacterial spread. A single plasmid regulatory gene (virF) controls a virulence-associated plasmid regulon including virG (icsA) and two invasion-related loci, i.e., (i) ipaABCD, encoding invasion plasmid antigens that may be structural components of the Shigella invasion determinant; and (ii) invAKJH (mxi), which is necessary for insertion of invasion plasmid antigens into the outer membrane.

Plasmid-associated adherence of Shigella flexneri in a HeLa cell model.

The initial interaction of Shigella flexneri with HeLa cells was studied at 4 degrees C, a temperature that inhibits parasite-directed endocytosis. It was found that invasive strains were 10-fold more adherent to HeLa cells than were isogenic, noninvasive strains which had lost a 140-megadalton plasmid. Adherent strains were also more hydrophobic than were nonadherent strains.

Shigella infection of Henle intestinal epithelial cells: role of the bacterium.

Epithelial cell infection by Shigella flexneri 2a was studied in an in vitro model system. Using the Henle 407 human intestinal epithelial cell line as host cells, a standardized experimental protocol which allowed quantitative measurement of infection was developed. Intravellular residence of infecting organisms was confirmed by indirect fluorescent-antibody staining of unfixed and methanol-fixed (Henle 407) cells and by quantitative bacteriological culture of disrupted host cells after infection. The process of shigella entry into cells was evaluated by chemical or physical modulation of the bacterium under controlled experimental conditions. Shigella were subjected to mild heat, ultraviolet radiation aminoglycoside antibiotics, and immunoglobulins raised against S. flexneri 2a. The data show that heat-stable antigens on the bacterial surface are not solely responsible for infectivity of S. flexneri 2a. Furthermore, it was shown that physiological and synthetic functions of shigellae are required for entry into host cells.

Effect of infection and endotoxicosis on plasma lactate dehydrogenase isozymes in white rats.

Vertical slab electrophoresis in polyacrylamide gels was used to monitor changes in lactate dehydrogenase (LDH) isozymes in plasma of white rats during bacterial infection and endotoxin poisoning. Peritoneal infection with Francisella tularensis and Salmonella typhimurium and administration of S. typhimurium endotoxin stimulated significant increases in plasma LDH-5. Rates of change in enzyme activity after infection were directly related to size of infecting dose and type of agent employed. Infection with 1 median lethal dose of F. tularensis stimulated both an early, temporary and a prolonged, secondary elevation in LDH-5 activity, whereas salmonellosis, endotoxicosis, killed cells, and latex particles elicited only an initial response of short duration. Changes observed in plasma LDH-5 after exposure to these agents suggest that, as a result of phagocytosis or cell damage, peritoneal leukocytes contribute to early increases in plasma enzyme activity, whereas extensive liver involvement is responsible for high secondary LDH-5 levels during progressive tularemic infection.

Protein synthesis in HeLa or Henle 407 cells infected with Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118.

The incorporation of [14C]leucine into protein was studied in two mammalian cell lines which had been infected with strains of Shigella dysenteriae 1, Shigella flexneri 2a, or Salmonella typhimurium W118. These cell lines differed in susceptibility to the effects of exogenously applied Shiga cytotoxin. All invasive shigella strains (which synthesize this toxin to a greater or lesser degree) were found to inhibit protein synthesis in both cell lines with equal efficiency. Leucine accumulation continued in these cells, but the labeled amino acid was preferentially incorporated into bacterial protein. S. typhimurium W118, which has not been shown to elaborate a Shiga-like toxin, had little effect on protein synthesis in infected host cells.

Shigella vaccines.

Shigellosis remains a major public health problem in developing countries. In these nations, the disease affects young children for the most part. The infecting organism causes illness by invading the colonic mucosa. It is closely related to nonpathogenic Escherichia coli, and genetic material can be transferred from one organism to the other, a process increasing the pathogenic potential of the E. coli or reducing the virulence of the strain of Shigella. Knowledge of the genetics of virulence of shigellae enables the construction of living, attenuated oral vaccines that may offer a practical means of controlling the disease.

Invasive enteric pathogens.

Invasive enteric pathogens of the Salmonella or Shigella genera initiate infections by invading the intestinal epithelium. Depending on the species, salmonellae either translocate across the mucosa of the small intestine and cause a systemic febrile disease or they evoke a localized inflammatory response in discreet areas of the infected mucosa. The latter type of infection is characterized by gastroenteritis, and a choleragen-like enterotoxin may contribute to the symptomology. Shigellae can also evoke diarrheal episodes; however, classic shigellosis is characterized by localized invasion of the colonic epithelium, with inflammation and ulceration of the mucosa. Derangement of the colonic mucosa is manifested in the bloody, mucoid stool characteristic of bacillary dysentery. Genetic analysis of invasive enteric pathogens has shown that extrachromosomal elements (plasmids) are required for full expression of virulence in Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, and Shigella flexneri. In the latter species, at least three chromosomal regions are also necessary for virulence.

Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli.

Seven plasmid-coded polypeptides, designated a through g, were identified by two-dimensional nonequilibrium pH gradient electrophoresis of radiolabeled extracts from minicells of virulent Shigella flexneri serotypes 2a and 5 and enteroinvasive Escherichia coli O143. These polypeptides were deemed to be products of 140-megadalton (MDa) virulence-associated plasmids because they were not synthesized in minicells which were not harboring a 140-MDa plasmid or in minicells which were carrying an F lac plasmid of the same incompatibility group. Synthesis of these polypeptides was repressed in minicells incubated at 30 degrees C and in minicells isolated from a noninvasive opaque colonial variant, even though these strains harbored a 140-MDa plasmid. Enriched fractions of polypeptides b, c, and d were obtained from S. flexneri serotype 5 by preparative isoelectric focusing, and polyclonal rabbit antisera recognizing each polypeptide were raised. These antisera were able to detect cross-reacting plasmid-coded polypeptide antigens in S. flexneri serotype 3, Shigella sonnei, and enteroinvasive E. coli O143. In addition, Western blots of minicell extracts from S. flexneri serotype 5 or E. coli O143 indicated that plasmid-coded polypeptides a through d were recognized by convalescent antiserum from a monkey infected with S. flexneri serotype 2a.

Genotypic and phenotypic characterization of an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen.

The construction and characterization of EcSf2a-2, an aroD-deleted Escherichia coli-Shigella hybrid vaccine carrying chromosomal and plasmid genes from Shigella flexneri and expressing S. flexneri 2a somatic antigen in association with E. coli K12 core are described. Expression of hybrid lipopolysaccharide and deletion of aroD resulted in the attenuation of phenotypic characteristics associated with pathogenicity. The addition of an aroD deletion results in a requirement for an aromatic precursor of para-aminobenzoic acid (PABA), an essential bacterial metabolite not present in mammalian tissues. The biosynthesis of hybrid somatic antigen prevents expression of a Sereny-positive reaction by invasive bacteria capable of expressing a plaque-positive phenotype. A functional kcpA gene is required for expression of the plaque-positive phenotype. The presence of an aroD deletion does not interfere with expression of an invasive phenotype; however, in bacteria containing a functional kcpA gene, replication and spread by invading bacteria are limited, preventing development of the plaque-positive phenotype.

Protection against local Shigella sonnei infection in mice by parenteral immunization with a nucleoprotein subcellular vaccine.

Nucleoprotein subcellular (NPS) vaccine, consisting of ribosome-bound O polysaccharide, was prepared from avirulent Shigella sonnei. NPS vaccine was tested for safety and protective activity in the mouse intranasal challenge model of Shigella infection. The vaccine was nontoxic when injected in doses up to 10,000 micrograms, and a single subcutaneous injection of as little as 0.1 micrograms gave significant protection against a lethal intranasal challenge with S. sonnei. These data demonstrate the induction of local protective immunity by parenteral immunization, support the concept of the ribosome as a potent vaccine vector, and give additional evidence for the protective activity of the NPS vaccine against Shigella infection.

Shigella infection of henle intestinal epithelial cells: role of the host cell.

The process of Henle 407 embryonic intestinal epithelial cell infection by Shigella flexneri 2a M42-43 was studied in an in vitro model system. The role of the Henle cell was assessed. It was established that entry of S. flexneri into cells was suppressed by reagents which inhibit uptake of particles by phagocytic cells. The compounds tested included cytochalasin B, dibutyryl-cyclic adenosine monophosphate, choleragen (Vibrio cholera enterotoxin), iodoacetate, and dinitrophenol. Cytochalasin B inhibited infection at concentrations of 1.0 mug/ml or greater. Dibutyryl-cyclic adenosine monophosphate at concentrations of 1 mM and choleragen at 0.1 mug/ml caused significant suppression of infection. Iodoacetate or dinitrophenol, at 0.1 mM concentrations, inhibited internalization of virulent shigellae, and a combination of these compounds inhibited infection at 0.01 mM concentrations. Preincubation of Henle cell monolayers with the combination of iodoacetate and dinitrophenol (0.05 mM) also inhibited infection markedly. The data suggest that infection of epithelial cells by S. flexneri in vitro is accomplished by an endocytic process induced by virulent bacteria. The process appears to be similar to uptake of particles by phagocytic cells. Ultrastructural analysis by transmission electron microscopy provided corroborative evidence of phagocytosis of shigellae by Henle cells in that intracellular bacteria were often observed within membrane-limiting vacuoles resembling phagosomes.

Characterization of virulence marker antigen of Shigella spp. and enteroinvasive Escherichia coli.

Antisera produced in rabbits immunized with an enteroinvasive O143 strain of Escherichia coli were absorbed with an avirulent derivative of the same strain. The resulting sera have been previously shown to recognize enteroinvasive pathogens when used in an enzyme-linked immunosorbent assay. In the current study, Western blots (immunoblots) showed that such an absorbed rabbit antiserum recognized two proteins (IpaB and IpaC) which are encoded by a large, virulence-associated plasmid. These proteins are the apparent constituents of the virulence marker antigen which is expressed by shigellae and enteroinvasive E. coli.