Mutants of Vibrio cholerae was isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulence-deficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.

Human lung fibroblasts develop a cytopathic effect (CPE) when infected with Mycoplasmapneumoniae. This study was designed to determine the relationship between host cell metabolism and the formation of the CPE. Human lung fibroblasts were grown in different serum concentrations, plated at different densities, and grown for different periods of time to alter the metabolic activity of the cells. Deoxyribonucleic acid, ribonucleic acid, and protein syntheses were measured by the ability of the cells to incorporate thymidine, uridine, and leucine, respectively. With each treatment, leucine incorporation remained constant. Thymidine and uridine incorporation was higher when the cells were in high serum, at low cell densities, or grown for 2 or 3 days. The appearance of an observable CPE, which was corroborated with a protein synthesis assay, correlated closely with thymidine and uridine incorporation. A more pronounced CPE was seen when thymidine and uridine incorporation was high. In addition, it was found that the first 2 h after infection by M. pneumoniae were the critical hours in determining whether a CPE would develop. This was accomplished by altering the serum concentration of the culture medium at different times postinfection and thereby altering the metabolism of the fibroblasts. These results demonstrate the importance of the metabolic state of the host cells in studying mycoplasma infections and establish a correlation between the nucleic acid metabolism of the cells and the production of a CPE. 72 h), until the cells become confluent. Once 174

The nucleotide content of normal MRC-5 human lung fibroblasts and fibroblasts infected with Mycoplasma pneumoniae PI 1428 was determined. Nucleotides from control and infected fibroblasts were extracted with 5% trichloracetic acid. After neutralization of the extracts, the nucleotides in the extracts were separated by anion-exchange chromatography. Significant differences were found between the nucleotide content of the control and infected cells. Nucleotide triphosphate levels were twofold higher in the control fibroblasts than in the infected fibroblasts 4 h after the initiation of infection. At the same time, nucleotide diphosphate and monophosphate levels were higher in the infected fibroblasts than in the control fibroblasts. Determination of the energy charge ratio for each set of nucleotides (adenosine, guanosine, cytidine, and uridine) demonstrated a shift of nucleotide content in the infected fibroblasts. Immediately after infection, the energy charge for each set of nucleotides was higher for the control fibroblasts than it was for the infected fibroblasts. This pattern continued throughout the infection period with only minor exceptions. The work presented here indicates a loss of energy charge in fibroblasts infected with M. pneumoniae and may help to explain some of the metabolic changes and cell damage which accompany infection.

The effects of Mycoplasma pneumoniae on host cell metabolism were studied by using two types of host cells, MRC-5 human lung fibroblasts, a normal cell line, and Lesch-Nyhan fibroblasts, a cell line deficient in hypoxanthine-guanine phosphoribosyl transferase (EC 2.4.2.8). The susceptibilities of the two cell types were determined by infecting the cells with M. pneumoniae at different multiplicities of infection (MOI). Our data indicate that the Lesch-Nyhan cells were four times more susceptible to damage by M. pneumoniae than the MRC-5 cells. The effects of different MOIs (10 and 50) on de novo purine synthesis, DNA synthesis, and the development of a cytopathic effect were determined. In both cell types, the higher MOI inhibited de novo purine synthesis to a greater extent than the lower MOI. This correlated closely with the cytopathic effect which developed in the monolayers (i.e., the more the inhibition of de novo purine synthesis, the greater the cytopathic effect which developed). In the Lesch-Nyhan cells, DNA synthesis was completely inhibited by the high MOI, whereas in the MRC-5 cells, DNA synthesis was stimulated by the high MOI. In the MRC-5 cells infected with M. pneumoniae, purine salvage activity increased, as indicated by an increase in adenosine deaminase (EC 3.5.4.4) activity. These data indicate that M. pneumoniae alters host cell metabolism, particularly the nucleic acid metabolic pathways. This may explain in part the mechanism of pathogenesis of M. pneumoniae infection.

MRC-5 human lung fibroblasts maintained in Eagle's basal medium (BME) with either 10% fetal bovine serum (FBS) or 10% newborn bovine serum (NBS) did not respond identically to infection by Mycoplasma pneumoniae. Fibroblasts grown in NBS did not develop any cytopathic effect (CPE) when infected with M. pneumoniae, whereas those maintained in FBS developed a pronounced CPE. There was also a difference in sensitivity to infection for fibroblasts maintained in the two sera before the infection. Fibroblasts maintained in NBS, then transferred to FBS 48 h before infection, were still less sensitive to M. pneumoniae infection than cells maintained constantly in FBS. Mycoplasma pneumoniae attached comparably to the fibroblasts grown in the two sera, so the differences in CPE development could not be attributed to differences in mycoplasma attachment. Measurements of DNA, RNA, and protein syntheses of the fibroblasts grown in NBS and FBS indicate that the cells in NBS were growing more rapidly than those in FBS. A determination of the doubling times shows that the doubling time of cells in NBS was 44 h, whereas that of cells in FBS was 51 h. Polyacrylamide gel electrophoresis of samples of NBS and FBS showed significant differences in serum protein composition. The NBS had several protein bands that were lacking in the FBS. This study demonstrates the importance of serum effects in the study of M. pneumoniae infection.