FliT selectively enhances proteolysis of FlhC subunit in FlhD4C2 complex by an ATP-dependent protease, ClpXP.

We previously reported that the ClpXP ATP-dependent protease specifically recognizes and degrades the flagellar master transcriptional activator complex, FlhD4C2, to negatively control flagellar biogenesis. The flagellum-related protein, FliT, is also a negative regulator of flagellar regulon by inhibiting the binding of FlhD4C2 to the promoter DNA. We have found a novel pathway of FliT inhibition of FlhD4C2 activity connected to ClpXP proteolysis. An in vitro degradation assay using purified proteins shows that FliT selectively increases ClpXP proteolysis of the FlhC subunit in the FlhD4C2 complex. FliT behaves specifically to ClpXP-dependent proteolysis of FlhC. An in vitro interaction assay detects the ternary complex of FliT-FlhD4C2-ClpX. FliT promotes the affinity of ClpX against FlhD4C2 complex, whereas FliT does not directly interact with ClpX. Thus, FliT interacts with the FlhC in FlhD4C2 complex and increases the presentation of the FlhC recognition region to ClpX. The DNA-bound form of FlhD4C2 complex is resistant to ClpXP proteolysis. We suggest that the role of FliT in negatively controlling the flagellar gene expression involves increasing free molecules of FlhD4C2 sensitive to ClpXP proteolysis by inhibiting the binding to the promoter DNA as well as enhancing the selective proteolysis of FlhC subunit by ClpXP.

The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression.

The flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD4C2 transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1(flhDC) and P5(flhDC), were functional in a wild-type background, while P6(flhDC) was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1(flhDC) initiates flagellar assembly and a negative autoregulatory loop through FlhD4C2-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1(flhDC) also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD4C2 transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.

A role for the calmodulin kinase II-related anchoring protein (αkap) in maintaining the stability of nicotinic acetylcholine receptors.

αkap, a muscle specific anchoring protein encoded within the Camk2a gene, is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChRs). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated, and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells and thus the maintenance of postsynaptic receptor density and synaptic function.

Nicotinic acetylcholine receptor stability at the NMJ deficient in α-syntrophin in vivo.

α-Syntrophin (α-syn), a scaffold protein, links signaling molecules to the dystrophin-glycoprotein complex. Absence of α-syn from the DGC is known to lead to structurally aberrant neuromuscular junctions (NMJs) with few acetylcholine receptors (AChRs) clustered at synaptic sites. Using α-syn knock-out mice, we show that during the first postnatal week, α-syn is not required for synapse formation. However, at postnatal day 6 (P6)-P7, the structural integrity of the postsynaptic apparatus is altered, the turnover rate of AChRs increases significantly, and the number/density of AChRs is impaired. At the adult α-syn(-/-) NMJ, the turnover rate of AChRs is ∼ 4 times faster than wild-type synapses, and most removed receptors are targeted to degradation as few AChRs recycled to synaptic sites. Biochemical analyses show that in muscle cells of adult knock-out α-syn mice, total AChRs and scaffold protein rapsyn are significantly reduced, the 89 kDa and 75 kDa isoforms of tyrosine phosphorylated α-dystrobrevin (α-dbn) 1 (which are required for the maintenance and stability of AChR in α-dbn(-/-) synapses) are barely detectable. Electroporation of GFP-α-dbn1 in α-syn(-/-) muscle cells partially restored receptor density, turnover rate, and the structural integrity of the postsynaptic apparatus, whereas expression of rapsyn-GFP failed to rescue the α-syn(-/-) synaptic phenotype. These results demonstrate that α-syn is required for the maturation and stability of the postsynaptic apparatus and suggest that α-syn may act via α-dbn1.

Calcium/calmodulin kinase II-dependent acetylcholine receptor cycling at the mammalian neuromuscular junction in vivo.

At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Using in vivo time-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIßm isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca(2+) along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJ in vivo.

Sensing by bacterial regulatory systems in host and non-host environments.

Free-living organisms have the ability to gauge their surroundings and modify their gene expression patterns in ways that help them cope with new environments. Here we discuss the physiological significance of recent reports describing the ability of the Salmonella typhimurium PhoP/PhoQ two-component system to recognize and respond to host-derived antimicrobial peptides.

The PmrA/PmrB and RcsC/YojN/RcsB systems control expression of the Salmonella O-antigen chain length determinant.

The lipopolysaccharide (LPS) is the outermost component of the cell envelope in Gram-negative bacteria. It consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a distal polysaccharide termed O-antigen. We report here that the PmrA/PmrB and RcsC/YojN/RcsB two-component systems of Salmonella enterica serovar Typhimurium independently promote transcription of the wzzst gene, which encodes a protein that determines the chain length of the O-antigen. We show that the regulatory proteins PmrA and RcsB footprint partially overlapping regions of the wzzst promoter stimulating transcription from the same start site. Induction of the PmrA/PmrB or RcsC/YojN/RcsB systems increased the fraction of LPS molecules containing 16-35 O-antigen subunits, leading to heightened resistance to serum. The LPS of a rcsB null mutant exhibited an altered mobility in the O-antigen subunits attached to the lipid A-core region when separated on a SDS/PAGE gel, suggesting that RcsB may regulate additional LPS genes. Inactivation of the wzzst gene eliminated the enhanced swarming behaviour exhibited by the rcsB mutant. That multiple regulatory systems control wzzst expression suggests that the Wzzst protein is required under different environmental conditions.

Activation of the RcsC/YojN/RcsB phosphorelay system attenuates Salmonella virulence.

Bacterial pathogens have the ability to sense their presence in host tissues and to promote expression of their virulence factors in a time- and location-dependent manner. However, little is known about those genes whose expression is detrimental and thus suppressed during infection. Here we report that constitutive activation of the RcsC/YojN/RcsB system resulting from a mutation in the rcsC sensor gene dramatically attenuates Salmonella virulence. Mutation of the cognate response regulator gene rcsB restored full virulence to the rcsC constitutive mutant, indicating that virulence attenuation results from aberrant expression of RcsB-regulated genes. The virulence attenuation phenotype was partially dependent on the regulatory gene rcsA, which is necessary for transcription of certain RcsB-regulated genes, and on the RcsB- and RcsA-dependent colanic acid capsule synthesis cps operon. The rcsC constitutive mutant was phagocytized less efficiently by macrophages and it was defective for invasion of non-phagocytic cells and survival within macrophages; but it could protect mice upon challenge with wild-type Salmonella. Our results suggest that a successful infection demands that pathogens turn off expression of products that might interfere with virulence functions.

Signal-dependent requirement for the co-activator protein RcsA in transcription of the RcsB-regulated ugd gene.

The RcsC/YojN/RcsB phosphorelay system controls gene expression in response to a variety of signals, including changes in temperature, osmolarity, and overproduction of membrane proteins. Transcription of certain RcsB-activated genes, such as the capsule synthesis cps operon, requires the co-activator protein RcsA, whereas expression of other RcsB-activated genes is RcsA-independent. We have established previously that a tolB mutation induces transcription of the Salmonella UDP-glucose dehydrogenase ugd gene in an RcsA- and RcsB-dependent manner. This induction is independent of the two-component systems PhoP/PhoQ and PmrA/PmrB, which are required for ugd expression in response to low Mg2+. We now report that the RcsC/YojN/RcsB system is activated in a pmrA mutant experiencing Fe3+ and low Mg2+, resulting in expression of both cps and ugd genes. However, whereas cps transcription remained RcsA-dependent, ugd transcription became RcsA-independent but dependent on the PhoP protein. S1 mapping experiments demonstrated that RcsA-dependent and -independent transcription of the ugd gene use the same promoter. DNase footprinting analysis identified a PhoP-binding site in the ugd promoter. Yet, PhoP-mediated ugd transcription required either the RcsC/YojN/RcsB or the PmrA/PmrB systems.

Control of the Salmonella ugd gene by three two-component regulatory systems.

The Salmonella ugd gene is required for the incorporation of 4-aminoarabinose in the lipopolysaccharide and resistance to the antibiotic polymyxin B. Transcription of the ugd gene is induced by Fe3+ via the PmrA-PmrB two-component system and by low Mg2+ in a process that requires the PhoP-PhoQ two-component system, the PhoP-activated PmrD protein and the PmrA-PmrB system. Here, we establish that mutation of the tolB gene promotes ugd transcription independently of both the PhoP-PhoQ and PmrA-PmrB systems. This activation is mediated by the RcsC-YojN-RcsB phosphorelay and the RcsA protein, suggesting a role for ugd in capsule synthesis. Binding sites for the RcsB, PmrA and PhoP proteins were identified in the ugd promoter. Although the PmrA-PmrB and RcsC-YojN-RcsB systems promoted ugd transcription independently of the PhoP-PhoQ system under different environmental conditions, ugd expression inside macrophages was strictly dependent on PhoP-PhoQ, suggesting that low Mg2+ is a cue for the intracellular environment.

Conflicting needs for a Salmonella hypervirulence gene in host and non-host environments.

The Gram-negative pathogen Salmonella enterica harbours a periplasmic D-Ala-D-Ala dipeptidase (termed PcgL), which confers the ability to grow on D-Ala-D-Ala as sole carbon source. We now demonstrate that inactivation of the pcgL gene renders Salmonella hypervirulent. This phenotype results from the accumulation of peptidoglycan-derived D-Ala-D-Ala in the pcgL mutant and not from an intrinsically faster growth rate. Synthetic D-Ala-D-Ala (but not L-Ala-L-Ala or D-Ala) increased the number of wild-type Salmonella in the liver and spleen of mice within 24 h of injection, suggesting that D-Ala-D-Ala interferes with some aspect of innate immunity. However, the pcgL mutant was unable to grow on D-Ala-D-Ala as sole carbon source and was defective for survival in nutrient-poor conditions. We identified clinical isolates lacking D-Ala-D-Ala dipeptidase activity and unable to grow on D-Ala-D-Ala because of inactivation of the pcgL gene. Our data suggest that genes (such as pcgL) that, when mutated make pathogens more virulent, may be retained because their contribution to pathogen fitness in non-host environments outweighs potential advantages of the hypervirulent vari-ant in the infected host.

Molecular mechanisms of Salmonella pathogenesis.

The success of a pathogen depends on its capacity to enter a host, circumvent host defense barriers and establish infection. The Gram-negative bacterium Salmonella enterica has evolved different strategies to subvert normal host cellular functions, which allow it to enter into and proliferate within host cells.