MEKK3-MEK5-ERK5 signaling promotes mitochondrial degradation.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.

Analysis of the role of HP0208, a phase-variable open reading frame, and its homologues HP1416 and HP0159 in the biosynthesis of Helicobacter pylori lipopolysaccharide.

The roles of the three ORFs HP0208, HP0159 and HP1416 in the biosynthesis of Helicobacter pylori 26695 LPS were investigated in this study. These ORFs represent a paralogous family of genes with homology to the Salmonella enterica serovar Typhimurium (hereafter referred to as S. typhimurium) waaJ gene, which encodes an alpha-1,2-glycosyltransferase required for core LPS biosynthesis. HP0208 contains multiple tandem repeats of the dimer 5'GA at its 5' end and its expression is predicted to be subject to phase variation. The number of 5'GA repeats present in this ORF was found to be non-permissive for the expression of HP0208 in the majority of H. pylori strains examined. To determine a role for this ORF in LPS biosynthesis a non-phase-variable, constitutively expressed variant of HP0208 was constructed and introduced into the genome of H. pylori 26695. Analysis of the LPS profile of this strain by Tricine-SDS-PAGE and immunoblotting with anti-Lewis Y antigen (Le(y)) mAbs confirmed a role for HP0208 in the biosynthesis of core LPS. A role for HP0159 and HP1416 in the biosynthesis of core LPS was also established. Although homologous to waaJ, H. pylori HP0208, HP0159 and HP1416 failed to complement an S. typhimurium waaJ mutant, suggesting that these ORFs encode functionally different enzymes.

Phase variation of lic1A, lic2A and lic3A in colonization of the nasopharynx, bloodstream and cerebrospinal fluid by Haemophilus influenzae type b.

The role of phase variation of lic1A, lic2A and lic3A in the ability of Haemophilus influenzae type b to colonize the nasopharynx, bloodstream and cerebrospinal fluid (CSF) of infants was investigated. This was achieved by using PCR to determine the number of 5'-CAAT-3' repeats present in each gene, which is indicative of whether each ORF can be expressed. Multiple PCR products of different intensities were amplified from all three genes at each site sampled. This indicated that the nasopharynx, bloodstream and CSF were colonized by a heterogeneous population of organisms, expressing different combinations of lic genes. At each site however, a predominant PCR product was amplified from each gene, indicating that organisms with this genotype were the most abundant. The number of 5'-CAAT-3' repeats in this predominant product varied depending upon whether organisms were isolated from the nasopharynx, bloodstream or CSF. These observations suggest that the expression of different combinations of lic genes may influence the efficiency with which H. influenzae colonizes the nasopharynx, bloodstream and CSF of infant rats.

The extracytoplasmic sigma factor, final sigma(E), is required for intracellular survival of nontypeable Haemophilus influenzae in J774 macrophages.

Nontypeable Haemophilus influenzae (NTHi) causes a wide variety of respiratory tract infections in humans. It is capable of invading and surviving in epithelial cells and has also been shown to persist in macrophage-like cell line J774A.1. To determine the molecular mechanisms which enable NTHi to survive in an intracellular environment, differential display reverse transcriptase PCR was used to identify genes which were either induced or upregulated by NTHi residing in macrophages. Using this approach, we identified one transcript which was consistently amplified from intracellular NTHi cDNA. Nucleotide sequence analysis of this product revealed that it spanned the 3' and 5' ends of rpoE and rseB, respectively, which form part of the extracytoplasmic stress operon that encodes and regulates expression of alternate sigma factor sigma E (final sigma(E)). To confirm that expression of rpoE was upregulated following uptake of NTHi by macrophages, an rpoE-lacZ transcriptional fusion was constructed, and expression of beta-galactosidase activity in broth-grown NTHi was compared with expression of beta-galactosidase activity in intracellular NTHi. The level of beta-galactosidase activity in NTHi 4 h after phagocytosis by macrophages was found to be 100-fold higher than that of broth-grown organisms, suggesting that genes of the final sigma(E) regulon may be important for persistence of NTHi in mammalian cells. The hypothesis that final sigma(E) plays a role in the intracellular survival of NTHi was subsequently confirmed by the decreased ability of an rpoE insertion mutant to survive in macrophages.

Expression of the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold temperature.

Previous studies have shown that several bacterial species exhibit a multigenic response following temperature downshift (cold shock). Evidence for such a response in Salmonella typhimurium is reported, based on the isolation of a range of low-induction-temperature gene fusions containing Mudlux insertions. The fusions exhibited different levels of basal light at 30 degrees C, and were induced at different rates and to different degrees over several hours following a reduction in temperature to 10 degrees C. Of the Mudlux gene fusions isolated, one was found which produced essentially no light when grown at 30 degrees C but exhibited rapid and high-level induction when the temperature was reduced to 10 degrees C. The target of this gene fusion (which was named cspB) was shown to lie adjacent to the umuDC operon and to encode a homologue of the major cold-shock protein of Escherichia coli, CspA. Luminescence studies revealed that substantial light production occurred from the cspB::Mudlux fusion at or below 22 degrees C but not at higher temperatures, even following a temperature drop from 30 degrees C. Moreover, cspB mRNA levels were found to mimic this pattern of luminescence, suggesting that cspB expression occurs below a defined temperature threshold. The cspB mRNA was also found to be very stable at 10 degrees C but to become highly unstable when the temperature was raised towards the threshold temperature, even in the presence of rifampicin. Existing cellular RNases therefore appear to mediate the decay of cspB mRNA at high temperatures, but are incapable of this at low temperatures.