A galU mutant of Francisella tularensis is attenuated for virulence in a murine pulmonary model of tularemia.

BACKGROUND: A number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen. RESULTS: Here we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1ß following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT. CONCLUSIONS: Disruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.

UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of Clostridium perfringens phospholipase C.

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.

Reduced levels of uridine diphosphate glucose pyrophosphorylase activity in fetal and neonatal human and guinea pig liver.

Uridine diphosphate glucose pyrophosphorylase (UDPGPP) is the first enzyme in the bilirubin conjugation pathway. A study aiming to screen for red blood cell UDPGPP deficiency in newborns with hyperbilirubinemia was carried out. No individuals with severe UDPGPP deficiency were found, however, levels of UDPGPP in premature and at term newborns were lower than in adults. These findings led to the study of UDPGPP in human fetal, neonatal and adult liver, using guinea pig tissues as a parallel control. UDPGPP activities in fetal and neonatal samples were also significantly lower than in adult ones in both species. Therefore, it is postulated that the reduced levels of UDPGPP in fetal and neonatal liver could be a factor which contributes to the pathogenesis of the physiologic jaundice in human newborns.

Effects of a mutation that eliminates UDP glucose-pyrophosphorylase on the pathogenicity of Erwinia carotovora subsp. carotovora.

A nonpathogenic mutant of Erwinia carotovora obtained by Mu d1 mutagenesis was defective in the ability to utilize several carbon sources. The basis of the mutation was analyzed biochemically and shown to be a defect in the ability to form UDP glucose-pyrophosphorylase. The nonpathogenic phenotype of the mutant was caused by its sensitivity to galactose.