Identification of a two-component VirR/VirS regulon in Clostridium perfringens

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens. To elucidate the precise regulatory network governed by VirR/VirS and VR-RNA, a series of microarray screening using VirR/VirS and VR-RNA-deficient mutants was performed. Finally, by qRT-PCR analysis, 147 genes (30 single genes and 21 putative operons) were confirmed to be under the control of the VirR/VirS-VR-RNA regulatory cascade. Several virulence-related genes for alpha-toxin, kappa-toxin, hyaluronidases, sialidase, and capsular polysaccharide synthesis were found. Furthermore, some genes for catalytic enzymes, various genes for transporters, and many genes for energy metabolism were also found to be controlled by the cascade. Our data indicate that the VirR/VirS-VR-RNA system is a global gene regulator that might control multiple cellular functions to survive and multiply in the host, which would turn out to be a lethal flesh-eating infection.     

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb Hindlll fragment, or the 4.3 kb Pstl fragment resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved In virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.     

Global regulation of gene expression in response to cysteine availability in <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

Background  

Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail.     

Comparative genomics of VirR regulons in Clostridium perfringens strains

Background  

Clostridium perfringens is a Gram-positive anaerobic bacterium causing severe diseases such as gas gangrene and pseudomembranosus colitis, that are generally due to the secretion of powerful extracellular toxins. The expression of toxin genes is mainly regulated by VirR, the response regulator of a two-component system. Up to now few targets only are known for this regulator and mainly in one strain (Strain 13). Due to the high genomic and phenotypic variability in toxin production by different strains, the development of effective strategies to counteract C. perfringens infections requires methodologies to reconstruct the VirR regulon from genome sequences.     

Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney

In our recent study using Wnk4^D561A/+ knockin mice, we determined that the WNK-OSR1/SPAK-NaCl cotransporter (NCC) phosphorylation cascade is important for regulating NCC function in vivo. Phosphorylation of NCC was necessary for its plasma membrane localization. Previously, angiotensin II infusion was shown to increase apical membrane expression of NCC in rats. Therefore, we investigated whether angiotensin II was an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in cultured cells and in vivo kidney. In mpkDCT cells, the phosphorylation of OSR1 and NCC was increased 30 min after the addition of angiotensin II (10^-9-10⁻⁷ M) but returned to baseline after 18 h. In mice, a 5-min infusion of angiotensin II (5 ng/g/min) increased NCC phosphorylation in the kidney at 30 min and 2 h after the injection but returned to baseline 24 h later. This increase was inhibited by angiotensin II receptor blocker (valsartan) but not by aldosterone receptor blocker (eplerenone). Ten-day infusions of angiotensin II (720 ng/day) also increased phosphorylation of OSR1 and NCC in the mouse kidney, and both valsartan and eplerenone inhibited the increased phosphorylation. Although angiotensin II is identified as an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in vivo, aldosterone appears to be the major regulator of this signal cascade in the long-term regulation by angiotensin II.     

The SKHR motif is required for biological function of the VirR response regulator from Clostridium perfringens

The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.     

Further Characterization of Clostridium perfringens Small Acid Soluble Protein-4 (Ssp4) Properties and Expression

Background

Clostridium perfringens type A food poisoning (FP) is usually caused by C. perfringens type A strains that carry a chromosomal enterotoxin gene (cpe) and produce spores with exceptional resistance against heat and nitrites. Previous studies showed that the extreme resistance of spores made by most FP strains is mediated, in large part, by a variant of small acid soluble protein 4 (Ssp4) that has Asp at residue 36; in contrast, the sensitive spores made by other C. perfringens type A isolates contain an Ssp4 variant with Gly at residue 36.

Methodology/Principal Findings

The current study has further characterized Ssp4 properties and expression. Spores made by cpe-positive type C and D strains were found to contain the Ssp4 variant with Gly at residue 36 and were shown to be heat- and nitrite-sensitive; this finding may help to explain why cpe-positive type C and D isolates rarely cause food poisoning. Saturation mutagenesis indicated that both amino acid size and charge at Ssp4 residue 36 are important for DNA binding and for spore resistance. C. perfringens Ssp2 was shown to bind preferentially to GC-rich DNA on gel-shift assays, while Ssp4 preferred binding to AT-rich DNA sequences. Maximal spore heat and nitrite resistance required production of all four C. perfringens Ssps, indicating that these Ssps act cooperatively to protect the spore''s DNA, perhaps by binding to different chromosomal sequences. The Ssp4 variant with Asp at residue 36 was also shown to facilitate exceptional spore survival at freezer and refrigerator temperatures. Finally, Ssp4 expression was shown to be dependent upon Spo0A, a master regulator.

Conclusions/Significance

Collectively, these results provide additional support for the importance of Ssps, particularly the Ssp4 variant with Asp at residue 36, for the extreme spore resistance phenotype that likely contributes to C. perfringens type A food poisoning transmission.     

Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens

The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.     

Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities

The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (∼ 7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.     

Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer,Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions

The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp⁵⁰ residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called “phosphorylation pocket” is not conserved, with its space being occupied by an Arg⁵² from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp⁵⁰ with the highly conserved Arg⁵² and Thr⁹ in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.     

Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1

Background

Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.

Methodology/Principal Findings

Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.

Conclusions/Significance

hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.