The trans-activator vir is required for expression of all virulence-associated genes in Bordetella pertussis. The nature of the global regulation of these factors by vir and environmental signals was examined by Northern blot analysis and with j-galactosidase transcriptional fusions in five vir-regulated genes. Northern blots suggested that vir regulates at the level of transcription since Vir-organisms did not exhibit detectable mRNA from vir-regulated loci. Environmental signals such as high levels of salts, nicotinic acid, and 6-chloronicotinic acid or growth at low temperatures were examined. Of all of the cations and anions examined, only SO4 ions eliminated transcription of vir-regulated genes and reduced transcription of vir itself, suggesting that global regulation is obtained by modifying expression of the essential component, vir. Organisms grown on 6-chloronicotinic acid or quinaldic acid did not have detectable transcription from vir-regulated loci. Modulation by nicotinic acid, on the other hand, was strain dependent, acting at the level of transcription in strain 18-323 but not in Tohama I derivatives. Growth at lower temperatures reduced, but did not eliminate, transcription from vir-regulated loci. At 28°C the ratio of pertussis toxin mRNA to recA mRNA (a non-vir-regulated factor) was equivalent to that at 37°C, suggesting that transcription at low temperatures is reduced in a proportional manner and need not involve vir.Bordetella pertussis is the etiologic agent of whooping cough. The bacterium expresses a number of toxins and factors such as pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin (FHA), and dermonecrotic toxin (for a review, see reference 29) which are known to be positively induced by a trans-acting factor, Vir (28). If vir is inactivated by transposon mutagenesis (31), B. pertussis is unable to express any of these virulence factors and is unable to cause disease (30). At a low frequency, B. pertussis undergoes a phase change, such that organisms expressing the virulence factors spontaneously lose this ability. This has been suggested to occur when a frameshift mutation arises in the vir gene (26). Analysis of the sequence of the vir region suggests that there are three open reading frames, two of which show homology to the family of two-component regulator-sensor systems present in pathogenic and nonpathogenic bacteria (17,23). This information suggests that the observed global control by vir in response to environmental input may occur in a similar fashion to regulation by other members of the two-component regulator-sensor family.It has been known for several decades that certain environmental conditions result in the lack of expression of some B. pertussis antigens. In the late 1950s Lacey (13) serendipitously discovered that substituting MgSO4 for NaCl in the growth medium results in a reversible loss of the ability to agglutinate the organism with antisera to Bordetella parapertussis. In a very thorough study, Lacey (13) found that a number of environmental...

Phase-dependent invasive behavior of Bordetella pertussis was demonstrated by recovery of viable organisms from gentamicin-treated HeLa cell monolayers and by transmission electron microscopy. Several mutants of B. pertussis with TnS or TnS lac inserted into various vir-regulated genes were evaluated for differences in their invasive abilities. Mutants lacking filamentous hemagglutinin, pertussis toxin, and two as yet uncharacterized vir-regulated products had levels of invasion significantly lower than that of the parent strain BP338. In contrast, invasion by mutants lacking adenylate cyclase toxin was significantly increased compared with that of wild-type B. pertussis. This increase in invasion was eliminated when concentrations of intracellular cyclic 3'-5' AMP were stimulated by treating HeLa cells with cholera toxin or forskolin. Entry of B. pertussis occurred through a microfilament-dependent phagocytic process, as evidenced by the marked reduction in uptake following treatment of HeLa cells with cytochalasin D. Invasion was inhibited with polyclonal anti-B. pertussis and anti-filamentous hemagglutinin antisera. In addition, a monoclonal antibody against lipooligosaccharide A reduced uptake by 65.5%. The preservation of HeLa cell integrity and the limited replication of intracellular bacteria suggest that invasion may represent a means by which B. pertussis evades an active host immune response.

Eschterichia coli K-12 strains producing high levels of Shiga-like toxin type II (SLT-II) but not SLT-I were previously shown to be virulent in an orally infected, streptomycin-treated mouse model. In this investigation, we tested the virulence of several SLT-II-producing enterohemorrhagic E. coli (EHEC) isolates from patients with hemorrhagic colitis or hemolytic uremic syndrome. All of the strains tested were able to colonize the mouse intestine. However, only two strains were consistently virulent for mice: 091:1121 strain B2F1(Str), which was previously shown to carry two copies of si-II-related toxins, and 091:H21 strain H414-36/89(Strr), which was found in this study to contain three genes from the si-Hi group. The oral 50%o lethal doses of strains B2F1(Strr) and H414-36/89(Strr) when fed to streptomycin-treated mice were less than 10 bacteria. Histological sections from moribund mice fed the 091:H21 strains demonstrated extensive renal tubular necrosis; however, hematological results were not consistent with a diagnosis of hemolytic uremic syndrome. The central role of SLT in the virulence of the 091:1121 EHEC strains was supported by the finding that streptomycin-treated mice preinoculated with monoclonal antibody specific for SLT-II survived oral challenge with either B2Fl(Strr) or H414-36/89(Strr). The basis for the variation in virulence among the SLT-II-producing EHEC strains tested was not determined. However, a correlation between the capacity of an EHEC strain to grow in small intestinal mucus and lethality in the streptomycin-treated mice was observed.

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon TnS lac. TnS lac is a derivative of TnS which generates promoter fusions for ,B-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed I-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from TnS lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes. 2674on August 5, 2020 by guest http://iai.asm.org/ Downloaded from

We analyzed Escherichia coli 0157:H7 isolates from stool samples of five patients who had bloody diarrhea and were infected during a large food-borne outbreak of hemorrhagic colitis in Washington state. The isolates were assessed for Shiga-like toxin profile, adherence and plasmid traits, mouse virulence, capsule, and