Identification of regions of Ail required for the invasion and serum resistance phenotypes.

Yersinia enterocolitica is an enteric pathogen that has served as a model system for the study of microbial pathogenesis. Numerous virulence gene have been identified both on the virulence plasmid and on the chromosome. One of the chromosomal genes that is highly correlated with virulence is ail, a gene identified along with inv in a screen for Y. enterocolitica genes that could confer an invasive phenotype to Escherichia coli. Ail also promotes serum resistance in both E. coli and Y. enterocolitica. Several virulence factors homologous to Ail have been identified in other pathogens, yet very little is known about what constitutes the functional domain(s) of these proteins. Proteins in this family are predicted to consist of eight transmembrane beta-sheets and four cell surface-exposed loops. We constructed and characterized a number of insertion, deletion and point mutations in the regions of ail predicted to encode the cell surface loops. The results from the analysis of these mutants indicate that cell surface loops one and four do not directly promote invasion or serum resistance, whereas mutations in loop three appear to modulate both phenotypes. Analysis of mutations in loop 2 suggests that this surface-exposed loop contains sequences required for serum resistance and invasion. In addition, a peptide derived from the sequence of loop 2 was able specifically to inhibit Ail-mediated invasion in a dose-dependent manner. These results suggest that Ail directly promotes invasion and that loop 2 contains an active site, perhaps a receptor-binding domain. Analyses of the mutations also suggest that the serum resistance and invasion phenotypes may be separable, because there are numerous mutations that affect one phenotype but not the other.

An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae.

Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.

Amino acid substitutions in naturally occurring variants of ail result in altered invasion activity.

Yersinia enterocolitica is the causative agent of a variety of gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis. In addition, systemic infections resulting in high mortality rates can occur in elderly and immunocompromised patients. More than 50 serotypes of Y. enterocolitica have been identified, but only a few of them commonly cause disease in otherwise healthy hosts. Those serotypes that cause disease have been divided into two groups, American and non-American, based on their geographical distributions, biotypes, and pathogenicity. We have been studying two genes, inv and ail, from Y. enterocolitica that confer in tissue culture assays an invasive phenotype that strongly correlates with virulence. Some differences between the American and non-American serotypes at the ail locus were noted previously and have been investigated further in this report. The ail locus was cloned from seven Y. enterocolitica strains (seven different serotypes). Although the different clones produced similar amounts of Ail, the product of the ail gene from non-American serotypes (AilNA) was less able to promote invasion by Escherichia coli than was the product of the ail gene from American serotypes (AilA). This difference is probably due to one or more of the eight amino acid changes found in the derived amino acid sequence for the mature form of AilNA compared with that of AilA. Seven of these changes are predicted to be in cell surface domains of the protein (a model for the proposed folding of Ail within the outer membrane is presented). These results are discussed in relation to the growing family of outer membrane proteins, which includes Lom from bacteriophage lambda, PagC from salmonella typhimurium, and OmpX from Enterobacter cloacae.

Influence of growth temperature on virulence of Legionella pneumophila.

The effect of growth temperature on the virulence of a strain of broth-grown serogroup 1 Legionella pneumophila (Wadsworth F889) was examined by growing the bacterium at different temperatures and then infecting guinea pigs (by intratracheal injection) and guinea pig alveolar macrophages. The 50% lethal dose for guinea pigs infected with 25 degrees C-grown F889 was log10 5.0 CFU and that for 41 degrees C-grown F889 was log10 5.7 CFU, or a fivefold difference. Guinea pig alveolar macrophages were infected in quadruplicate with log10 3.8 CFU of F889 cells grown at either 25 or 41 degrees C. Counts of F889 in the alveolar macrophages infected with 25 degrees C-grown bacteria were 40% greater after 1 day of incubation (P = 2 X 10(-4)) than were counts in the alveolar macrophage suspensions inoculated with 41 degrees C-grown bacteria. However, the counts were not significantly different after 3 days of incubation. Examination of cover slip cultures of guinea pig alveolar macrophages infected with 25 degrees C-grown or 41 degrees C-grown bacteria showed that the bacteria grown at the lower temperature were twice as likely to be macrophage-associated after 1 h of incubation than were the bacteria grown at the higher temperature. Growth at the lower temperature was also associated with a change in reactivity with monoclonal antibodies, but not with a change in plasmid content. Thus, environmental temperature may play an important role in modulating the virulence of L. pneumophila, possibly by affecting bacterial adherence to host cells.

Paleoepidemiologic investigation of Legionnaires disease at Wadsworth Veterans Administration Hospital by using three typing methods for comparison of legionellae from clinical and environmental sources.

Multilocus enzyme electrophoresis, monoclonal antibody typing for Legionella pneumophila serogroup 1, and plasmid analysis were used to type 89 L. pneumophila strains isolated from nosocomial cases of Legionnaires disease at the Veterans Administration Wadsworth Medical Center (VAWMC) and from the hospital environment. Twelve L. pneumophila clinical isolates, obtained from patients at non-VAWMC hospitals, were also typed by the same methods to determine typing specificity. Seventy-nine percent of 33 VAWMC L. pneumophila serogroup 1 clinical isolates and 70% of 23 environmental isolates were found in only one of the five monoclonal subgroups. Similar clustering was found for the other two typing methods, with excellent correlation between all methods. Enzyme electrophoretic typing divided the isolates into the greatest number of distinct groups, resulting in the identification of 10 different L. pneumophila types and 5 types not belonging to L. pneumophila, which probably constitute an undescribed Legionella species; 7 clinical and 34 environmental VAWMC isolates and 2 non-VAWMC clinical isolates were found to be members of the new species. Twelve different plasmid patterns were found; 95% of VAWMC clinical isolates contained plasmids. Major VAWMC epidemic-bacterial types were common in the hospital potable-water distribution system and cooling towers. Strains of L. pneumophila which persisted after disinfection of contaminated environmental sites were of a different type from the prechlorination strains. All three typing methods were useful in the epidemiologic analysis of the VAWMC outbreak.

Clinical utility of a monoclonal direct fluorescent reagent specific for Legionella pneumophila: comparative study with other reagents.

Twenty-four lower respiratory tract samples taken from patients with culture-confirmed Legionella pneumophila infection were examined with three different direct immunofluorescent antisera to L. pneumophila, as were 29 samples from similar sources taken from patients without Legionnaires disease. The reagents studied were Genetic Systems Corp. (GS) monoclonal L. pneumophila conjugate, which reacts with all known serogroups of L. pneumophila, BioDx polyvalent L. pneumophila serogroups 1 through 6 conjugate, and Centers for Disease Control polyvalent pool A L. pneumophila serogroups 1 through 4 conjugate. The specimens had been frozen at -70 degrees C for 0.5 to 5 years. Randomization was used in coding the samples, which were stained and read by an independent observer. All three conjugates correctly identified all positive and negative samples. No difference was noted among the conjugates in the absolute numbers of fluorescent L. pneumophila bacteria per sample. The GS conjugate had a much cleaner background than did the other two reagents. Mean staining intensity scores were 3.4, 3.9, and 3.7 for the GS, BioDx, and Centers for Disease Control conjugates, respectively. This study demonstrates that the diagnostic efficiency of all three conjugates is equivalent. Since the GS conjugate is easier to read, does not cross-react with non-L. pneumophila bacteria, and reacts with serogroups 1 through 10 of L. pneumophila, it appears to be preferable for use in diagnostic testing on nonhistopathologically processed specimens.