The Human Antimicrobial Peptide LL-37 Binds Directly to CsrS,a Sensor Histidine Kinase of Group A Streptococcus,to Activate Expression of Virulence Factors

Group A Streptococcus (GAS) responds to subinhibitory concentrations of LL-37 by up-regulation of virulence factors through the CsrRS (CovRS) two-component system. The signaling mechanism, however, is unclear. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. We identified a 10-residue fragment (RI-10) of LL-37 as the minimal peptide that retains the ability to signal increased expression of GAS virulence factors, yet it has no detectable antimicrobial activity against GAS. Substitution of individual key amino acids in RI-10 reduced or abrogated signaling. These data do not support the hypothesis that CsrS detects LL-37-induced damage to the bacterial cell membrane but rather suggest that LL-37 signaling is mediated by a direct interaction with CsrS. To test whether LL-37 binds to CsrS, we used the purified CsrS extracellular domain to pull down LL-37 in vitro, a result that provides further evidence that LL-37 binds to CsrS. The dissociation of CsrS-mediated signaling from membrane damage by LL-37 fragments together with in vitro evidence for a direct LL-37-CsrS binding interaction constitute compelling evidence that signal transduction by LL-37 through CsrS reflects a direct ligand/receptor interaction.     

Regulation of virulence by a two-component system in group B streptococcus

Group B Streptococcus (GBS) is frequently carried in the gastrointestinal or genitourinary tract as a commensal organism, yet it has the potential to cause life-threatening infection in newborn infants, pregnant women, and individuals with chronic illness. Regulation of virulence factor expression may affect whether GBS behaves as an asymptomatic colonizer or an invasive pathogen, but little is known about how such factors are controlled in GBS. We now report the characterization of a GBS locus that encodes a two-component regulatory system similar to CsrRS (or CovRS) in Streptococcus pyogenes. Inactivation of csrR, encoding the putative response regulator, in two unrelated wild-type strains of GBS resulted in a marked increase in production of beta-hemolysin/cytolysin and a striking decrease in production of CAMP factor, an unrelated cytolytic toxin. Quantitative RNA hybridization experiments revealed that these two phenotypes were associated with a marked increase and decrease in expression of the corresponding genes, cylE and cfb, respectively. The CsrR mutant strains also displayed increased expression of scpB encoding C5a peptidase. Similar, but less marked, changes in gene expression were observed in CsrS (putative sensor component) mutants, evidence that CsrR and CsrS constitute a functional two-component system. Experimental infection studies in mice demonstrated reduced virulence of both CsrR and CsrS mutant strains relative to the wild type. Together, these results indicate that CsrRS regulates expression of multiple GBS virulence determinants and is likely to play an important role in GBS pathogenesis.     

Variation in the group B Streptococcus CsrRS regulon and effects on pathogenicity

CsrRS (or CovRS) is a two-component regulatory system that controls expression of multiple virulence factors in the important human pathogen group B Streptococcus (GBS). We now report global gene expression studies in GBS strains 2603V/R and 515 and their isogenic csrR and csrS mutants. Together with data reported previously for strain NEM316, the results reveal a conserved 39-gene CsrRS regulon. In vitro phosphorylation-dependent binding of recombinant CsrR to promoter regions of both positively and negatively regulated genes suggests that direct binding of CsrR can mediate activation as well as repression of target gene expression. Distinct patterns of gene regulation in csrR versus csrS mutants in strain 2603V/R compared to 515 were associated with different hierarchies of relative virulence of wild-type, csrR, and csrS mutants in murine models of systemic infection and septic arthritis. We conclude that CsrRS regulates a core group of genes including important virulence factors in diverse strains of GBS but also displays marked variability in the repertoire of regulated genes and in the relative effects of CsrS signaling on CsrR-mediated gene regulation. Such variation is likely to play an important role in strain-specific adaptation of GBS to particular host environments and pathogenic potential in susceptible hosts.     

Regulation and function of pilus island 1 in group B streptococcus

Group B streptococcus (GBS) pili may enhance colonization and infection by mediating bacterial adhesion to host cells, invasion across endothelial and epithelial barriers, and resistance to bacterial ingestion and killing by host phagocytes. However, it remains unclear how pilus expression is regulated and how modulation of pilus production affects GBS interactions with the human host. We investigated the regulation and function of pilus island 1 (PI-1) pili in GBS strain 2603. We found that PI-1 gene expression was controlled by the CsrRS two-component system, by Ape1, an AraC-type regulator encoded by a divergently transcribed gene immediately upstream of PI-1, and by environmental pH. The response regulator CsrR repressed expression of Ape1, which is an activator of PI-1 gene expression. In addition, CsrR repressed PI-1 gene expression directly, independent of its regulation of Ape1. In vitro assays demonstrated specific binding of both CsrR and Ape1 to chromosomal DNA sequences upstream of PI-1. Pilus gene expression was activated by acidic pH, and this effect was independent of CsrRS and Ape1. Unexpectedly, characterization of PI-1 deletion mutants revealed that PI-1 pili do not mediate adhesion of strain 2603 to A549 respiratory epithelial cells, ME180 cervical cells, or VK2 vaginal cells in vitro. PI-1 pili reduced internalization and intracellular killing of GBS by human monocyte-derived macrophages, by approximately 50%, but did not influence complement-mediated opsonophagocytic killing by human neutrophils. These findings shed new light on the complex nature of pilus regulation and function in modulating GBS interactions with the human host.     

The Mg2+ transporter MagT1 partially rescues cell growth and Mg2+ uptake in cells lacking the channel-kinase TRPM7

Magnesium (Mg(2+)) transport across membranes plays an essential role in cellular growth and survival. TRPM7 is the unique fusion of a Mg(2+) permeable pore with an active cytosolic kinase domain, and is considered a master regulator of cellular Mg(2+) homeostasis. We previously found that the genetic deletion of TRPM7 in DT40 B cells results in Mg(2+) deficiency and severe growth impairment, which can be rescued by supplementation with excess extracellular Mg(2+). Here, we show that gene expression of the Mg(2+) selective transporter MagT1 is upregulated in TRPM7(-/-) cells. Furthermore, overexpression of MagT1 in TRPM7(-/-) cells augments their capacity to uptake Mg(2+), and improves their growth behavior in the absence of excess Mg(2+).     

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.     

A Bacterial Pathogen Co-opts Host Plasmin to Resist Killing by Cathelicidin Antimicrobial Peptides

The bacterial pathogen Group A Streptococcus (GAS) colonizes epithelial and mucosal surfaces and can cause a broad spectrum of human disease. Through the secreted plasminogen activator streptokinase (Ska), GAS activates human plasminogen into plasmin and binds it to the bacterial surface. The resulting surface plasmin protease activity has been proposed to play a role in disrupting tissue barriers, promoting invasive spread of the bacterium. We investigated whether this surface protease activity could aid the immune evasion role through degradation of the key innate antimicrobial peptide LL-37, the human cathelicidin. Cleavage products of plasmin-degraded LL-37 were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Ska-deficient GAS strains were generated by targeted allelic exchange mutagenesis and confirmed to lack surface plasmin activity after growth in human plasma or media supplemented with plasminogen and fibrinogen. Loss of surface plasmin activity left GAS unable to efficiently degrade LL-37 and increased bacterial susceptibility to killing by the antimicrobial peptide. When mice infected with GAS were simultaneously treated with the plasmin inhibitor aprotinin, a significant reduction in the size of necrotic skin lesions was observed. Together these data reveal a novel immune evasion strategy of the human pathogen: co-opting the activity of a host protease to evade peptide-based innate host defenses.     

The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor.

The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327-547) showed constitutive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB-D56N formed a stable complex with MalE-CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45-176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 46.3 kJ mol-1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2- is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.     

The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima

The bacterial histidine autokinase CheA contains a histidine phosphotransfer (Hpt) domain that accepts a phosphate from the catalytic domain and donates the phosphate to either target response regulator protein, CheY or CheB. The Hpt domain forms a helix-bundle structure with a conserved four-helix bundle motif and a variable fifth helix. Observation of two nearly equally populated conformations in the crystal structure of a Hpt domain fragment of CheA from Thermotoga maritima containing only the first four helices suggests more mobility in a tightly packed helix bundle structure than previously thought. In order to examine how the structures of Hpt domain homologs may differ from each other particularly in the conformation of the last helix, and whether an alternative conformation exists in the intact Hpt domain in solution, we have solved a high-resolution, solution structure of the CheA Hpt from T. maritima and characterized the backbone dynamics of this protein. The structure contains a four-helix bundle characteristic of histidine phosphotransfer domains. The position and orientation of the fifth helix resembles those in known Hpt domain crystal and solution structures in other histidine kinases. The alternative conformation that was reported in the crystal structure of the CheA Hpt from T. maritima missing the fifth helix is not detected in the solution structure, suggesting a role for the fifth helix in providing stabilizing forces to the overall structure.     

O2- and NO-sensing mechanism through the DevSR two-component system in Mycobacterium smegmatis

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.     

Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis

The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.