The ATP-Dependent Lon Protease of Salmonella enterica Serovar Typhimurium Regulates Invasion and Expression of Genes Carried on Salmonella Pathogenicity Island 1

An early step in the pathogenesis of Salmonella enterica serovar Typhimurium infection is bacterial penetration of the intestinal epithelium. Penetration requires the expression of invasion genes found in Salmonella pathogenicity island 1 (SPI1). These genes are controlled in a complex manner by regulators in SPI1, including HilA and InvF, and those outside SPI1, such as two-component regulatory systems and small DNA-binding proteins. We report here that the expression of invasion genes and the invasive phenotype of S. enterica serovar Typhimurium are negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis in Escherichia coli. A disrupted mutant of lon was able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD. The lon mutant also showed a dramatic enhancement in transcription of the SPI1 genes hilA, invF, sipA, and sipC. The increases ranged from 10-fold to almost 40-fold. It is well known that the expression of SPI1 genes is also regulated in response to several environmental conditions. We found that the disruption of lon does not abolish the repression of hilA and sipC expression by high-oxygen or low-osmolarity conditions, suggesting that Lon represses SPI1 gene expression by a regulatory pathway independent of these environmental signals. Since HilA is thought to function as a central regulator of SPI1 gene expression, it is speculated that Lon may regulate SPI1 gene expression by proteolysis of putative factors required for activation of hilA expression

CS-8958, a Prodrug of the New Neuraminidase Inhibitor R-125489, Shows Long-Acting Anti-Influenza Virus Activity▿

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for the treatment of and prophylaxis against influenza. In this paper, the new potent NA inhibitor R-125489 is reported for the first time. R-125489 inhibited the NA activities of various type A and B influenza viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses. The survival effect of R-125489 was shown to be similar to that of zanamivir when administered intranasally in a mouse influenza virus A/Puerto Rico/8/34 infection model. Moreover, we found that the esterified form of R-125489 showed improved efficacy compared to R-125489 and zanamivir, depending on the acyl chain length, and that 3-(O)-octanoyl R-125489 (CS-8958) was the best compound in terms of its life-prolonging effect (P < 0.0001, compared to zanamivir) in the same infection model. A prolonged survival effect was observed after a single administration of CS-8958, even if it was given 7 days before infection. It is suggested that intranasally administered CS-8958 works as a long-acting NA inhibitor and shows in vivo efficacy as a result of a single intranasal administration

The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis in Salmonella enterica Serovar Typhimurium

The ClpXP protease is a member of the ATP-dependent protease family and plays a dynamic role in the control of availability of regulatory proteins and the breakdown of abnormal and misfolded proteins. The proteolytic activity is rendered by the ClpP component, while the substrate specificity is determined by the ClpX component that has ATPase activity. We describe here a new role of the ClpXP protease in Salmonella enterica serovar Typhimurium in which ClpXP is involved in the regulation of flagellum synthesis. Cells deleted for ClpXP show “hyperflagellate phenotype,” exhibit overproduction of the flagellar protein, and show a fourfold increase in the rate of transcription of the fliC encoding flagellar filament. The assay for promoter activity of the genes responsible for expression of the fliC showed that the depletion of ClpXP results in dramatic enhancement of the expression of the fliA encoding sigma factor ς(28), leaving the expression level of the flhD master operon lying at the top of the transcription hierarchy of flagellar regulon almost normal. These results suggest that the ClpXP may be responsible for repressing the expression of flagellar regulon through the control of the FlhD/FlhC master regulators at the posttranscriptional and/or posttranslational levels. Proteome analysis of proteins secreted from the mutant cells deficient for flhDC and clpXP genes demonstrated that the ΔflhD mutation abolished the enhanced effect by ΔclpXP mutation on the production of flagellar proteins, suggesting that the ClpXP possibly defines a regulatory pathway affecting the expression of flagellar regulon that is dependent on FlhD/FlhC master regulators

In Vivo Pharmacodynamic Activity of Tomopenem (formerly CS-023) against Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus in a Murine Thigh Infection Model▿

Tomopenem (formerly CS-023) is a novel carbapenem with broad-spectrum activities against diverse hospital pathogens, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). We examined the in vivo pharmacodynamic characteristics of tomopenem against P. aeruginosa and MRSA by using a neutropenic murine thigh infection model with P. aeruginosa 12467 (MIC, 1 μg/ml) and MRSA 12372 (MIC, 2 μg/ml). The mice had 106 to 107 CFU/thigh of each strain 2 h after inoculation and were treated for 24 h with a fractionated administration of tomopenem given at intervals of 3, 6, 12, and 24 h. The serum protein binding of tomopenem was 17.4%. The efficacy of tomopenem in both infection models was enhanced by frequent dosing, which indicates that the efficacy is driven by the time above MIC (TMIC). In a sigmoid model, the cumulative percentages of the 24-h period that the concentrations of free, unbound fractions of the drug exceeded the MIC under steady-state pharmacokinetic conditions (f%TMICs) were best correlated with efficacy when R2 was 0.79 and 0.86 against P. aeruginosa and MRSA, respectively. Other pharmacokinetic and pharmacodynamic (PK-PD) indexes for the free, unbound fractions, the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) and the maximum concentration of the drug in serum divided by the MIC (Cmax/MIC), showed poor correlation with efficacy when R2 was ≤0.42. The f%TMIC values required for a static effect, 1-log kill, and 2-log kill against P. aeruginosa were 29, 39, and 51, respectively, which were similar to those for meropenem, for which the values were 24, 33, and 45, respectively. Against MRSA, the values for tomopenem were 27, 35, and 47. In conclusion, the pharmacodynamic characteristics of tomopenem were similar to those of meropenem against P. aeruginosa, and there was no difference between the target values for P. aeruginosa and MRSA required for efficacy in this study