Identification and characterization of Yersinia enterocolitica genes induced during systemic infection.

Yersinia enterocolitica is one of three pathogenic Yersinia species that share a tropism for lymphoid tissues. However, infection of an immunocompromised host is likely to result in a systemic infection, which is often fatal. Little is known about the bacterial proteins needed to establish such an infection. The genes that encode these virulence factors are likely to be active only during systemic infection. A library of random cat fusions was used to inoculate BALB/c mice. Fusions expressed during a systemic infection were enriched by the administration of chloramphenicol-succinate. Y. enterocolitica isolates recovered from the mice were tested for chloramphenicol resistance in vitro. Fusions that were inactive in vitro were analyzed further and found to represent 31 allelic groups. Each was given a sif (for systemic infection factor) designation. Based on homology to known proteins, the sif genes are likely to encode proteins important for general physiology, transcription regulation, and other functions. During systemic infections, 13 of the sif-cat fusions were able to outcompete the wild type in the presence of chloramphenicol-succinate, confirming that the fusions were active. The in vitro expression of several sif genes was determined, showing modest changes in response to various growth conditions. A mutation in sif15, which encodes a putative outer membrane protein, caused attenuation during systemic infection but not during colonization of the Peyer's patches. Comparisons between the Y. enterocolitica sif genes and the previously identified hre genes imply that very different groups of genes are active during a systemic infection and during colonization of the Peyer's patches.

The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli.

The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram-negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. However, its presence in some non-pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions. We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS. Surprisingly, the entry of wild-type E. coli into stationary phase is accompanied by a several-hour-long period of acute sensitivity to hydrogen peroxide. Induction of the RpoS regulon helps to diminish that sensitivity. While mutants of E. coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase. This sensitivity required prior growth in air. Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide. The molecular details await elucidation.

Balance between endogenous superoxide stress and antioxidant defenses.

Cells devoid of cytosolic superoxide dismutase (SOD) suffer enzyme inactivation, growth deficiencies, and DNA damage. It has been proposed that the scant superoxide (O2-) generated by aerobic metabolism harms even cells that contain abundant SOD. However, this idea has been difficult to test. To determine the amount of O2- that is needed to cause these defects, we modulated the O2- concentration inside Escherichia coli by controlling the expression of SOD. An increase in O2- of more than twofold above wild-type levels substantially diminished the activity of labile dehydratases, an increase in O2- of any more than fourfold measurably impaired growth, and a fivefold increase in O2- sensitized cells to DNA damage. These results indicate that E. coli constitutively synthesizes just enough SOD to defend biomolecules against endogenous O2- so that modest increases in O2- concentration diminish cell fitness. This conclusion is in excellent agreement with quantitative predictions based upon previously determined rates of intracellular O2- production, O2- dismutation, dehydratase inactivation, and enzyme repair. The vulnerability of bacteria to increased intracellular O2- explains the widespread use of superoxide-producing drugs as bactericidal weapons in nature. E. coli responds to such drugs by inducing the SoxRS regulon, which positively regulates synthesis of SOD and other defensive proteins. However, even toxic amounts of endogenous O2- did not activate SoxR, and SoxR activation by paraquat was not at all inhibited by excess SOD. Therefore, in responding to redox-cycling drugs, SoxR senses some signal other than O2-.

[Rifampicin toxicity in HIV-infected patients: A study of its incidence and the risk factors]. / Toxicidad de la rifampicina en pacientes con infección VIH. Estudio de la incidencia y factores de riesgo.

OBJECTIVE: Evaluate the effect of HIV infection in the appearance of toxicity in patients treated with rifampin, analysing the involved elements in its genesis. METHODS: We realized a comparative study of the epidemiologic and clinical characteristics, and the incidence of adverse reactions to rifampin (between 1986-1993), comparing the seropositive patients treated with rifampin, during more than 3 months, with one control group, of equal number of patients, without evidence of HIV infection, taken at random, with epidemiologic characteristics (age and sex) similar to the first group and also treated with rifampin during a similar period. In the group with HIV infection, we analysed the related epidemiologic, clinical and analytic characteristics, in a way statistically significative, with the appearance of toxicity to rifampin. RESULTS: The risk of toxicity to rifampin was associated significantly to HIV infection (p < 0.01), without finding any other distinguishing characteristics among the analysed groups. Indicative parameters of advanced HIV infection: advanced clinical stage, minor level of lymphocytes CD4+, total leukocytes, total lymphocytes and quotient CD4+/CD8+, also high levels of beta 2-microglobulinemia and [correction of 2-microglobulina e] IgA, and a negative protein purified derivative test (PPD) were found statistically related with the appearance to toxicity to rifampin. Patients with number of lymphocytes CD4+ between 20-50/mm3, showed a major predisposition of suffering toxicity to rifampin. CONCLUSION: HIV infection involved a notably increase of toxicity risk to rifampin. Clinical or analytic parameters associated with advanced illness conditioned an increase of this risk, essentially among patients with number of CD4+ between 20-50/mm.

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.