The two-component signal transduction system and its regulation in Candida albicans.

Candida albicans, which can cause superficial and life-threatening systemic infections, is the most common opportunistic fungal pathogen in the human microbiome. The two-component system is one of the most important C. albicans signal transduction pathways, regulating the response to oxidative and osmotic stresses, adhesion, morphogenesis, cell wall synthesis, virulence, drug resistance, and the host-pathogen interactions. Notably, some components of this signaling pathway have not been found in the human genome, indicating that the two-component system of C. albicans can be a potential target for new antifungal agents. Here, we summarize the composition, signal transduction, and regulation of the two-component system of C. albicans to emphasize its essential roles in the pathogenesis of C. albicans and the new therapeutic target for antifungal drugs.

Dissecting the Role of VicK Phosphatase in Aggregation and Biofilm Formation of Streptococcus mutans.

VicRK (WalRK or YycFG) is a conserved 2-component regulatory system (TCS) that regulates cell division, cell wall biosynthesis, and homeostasis in low-GC Gram-positive bacteria. VicRK is also associated with biofilm formation of Streptococcus mutans on the tooth surface as it directly regulates the extracellular polysaccharide (EPS) synthesis. Of the 2 components, VicK possesses both autokinase and phosphatase activities, which regulate the phosphorylation and dephosphorylation of the regulator VicR in response to environmental cues. However, the dual mechanism of VicK as the autokinase/phosphatase in regulating S. mutans' responses is not well elucidated. Previously, it has been shown that the phosphatase activity depends on the PAS domain and residues in the DHp domain of VicK in S. mutans. Specifically, mutating proline at 222 in the PAS domain inhibits VicK phosphatase activity. We generated a VicKP222A mutant to determine the level of VicR-P in the cytoplasm by Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis. We show that in VicKP222A phosphatase, attenuation increased phosphorylated VicR (VicR-P) that downregulated glucosyltransferases, gtfBC, thereby reducing the synthesis of water-insoluble polysaccharides (WIS-EPS) in the biofilm. In addition, VicKP222A presented as long-rod cells, reduced growth, and displayed asymmetrical division. A major adhesin of S. mutans, SpaP was downregulated in VicKP222A, making it unable to agglutinate in saliva. In summary, we have confirmed that VicK phosphatase activity is critical to maintain optimal phosphorylation status of VicR in S. mutans, which is important for cell growth, cell division, EPS synthesis, and bacterial agglutination in saliva. Hence, VicK phosphatase activity may represent a promising target to modulate S. mutans' pathogenicity.

Identification of proteins regulated by acid adaptation related two component system HPK1/RR1 in Lactobacillus delbrueckii subsp. bulgaricus.

Lactobacillus delbrueckii subsp. bulgaricus is currently one of the most valuable lactic acid bacteria (LAB) and widely used in global dairy industry. The acid tolerance and adaptation ability of LAB is the key point of their survival and proliferation during fermentation process and in gastrointestinal tract of human body. Two component system (TCS) is one of the most important mechanisms to allow bacteria to sense and respond to changes of environmental conditions. TCS typically consists of a histidine protein kinase (HPK) and a corresponding response regulator (RR). Our previous study indicated a TCS (JN675228/JN675229) was involved in acid adaptation in L. bulgaricus. To reveal the role of JN675228 (HPK1)/JN675229 (RR1) in acid adaptation, the target genes of JN675228 (HPK1)/JN675229 (RR1) were identified by means of a proteomic approach complemented with transcription data in the present study. The results indicated that HPK1/RR1 regulated the acid adaptation ability of bacteria by means of many pathways, including the proton pump related protein, classical stress shock proteins, carbohydrate metabolism, nucleotide biosynthesis, DNA repair, transcription and translation, peptide transport and degradation, and cell wall biosynthesis, etc. To our knowledge, this is the first report with the effect of acid adaptation-related TCS HPK1/RR1 on its target genes. This study will offer experimental basis for clarifying the acid adaptation regulation mechanism of L. bulgaricus, and provide a theoretical basis for this bacterium in industry application.

Haem-Based Sensors of O2: Lessons and Perspectives.

Haem-based sensors have emerged during the last 15 years as being a large family of proteins that occur in all kingdoms of life. These sensors are responsible mainly for detecting binding of O2, CO and NO and reporting the ligation status to an output domain with an enzymatic or macromolecule-binding property. A myriad of biological functions have been associated with these sensors, which are involved in vasodilation, bacterial symbiosis, chemotaxis and biofilm formation, among others. Here, we critically review several bacterial systems for O2 sensing that are extensively studied in many respects, focusing on the lessons that are important to advance the field.

Drug discovery targeting heme-based sensors and their coupled activities.

Heme-based sensors have emerged during the last 20years as being a large family of proteins that occur in all kingdoms of life. A myriad of biological adaptations are associated with these sensors, which include vasodilation, bacterial virulence, dormancy, chemotaxis, biofilm formation, among others. Due to the key activities regulated by these proteins along with many other systems that use similar output domains, there is a growing interest in developing small molecules as their regulators. Here, we review the development of potential activators and inhibitors for many of these systems, including human soluble guanylate cyclase, c-di-GMP-related enzymes, Mycobacterium tuberculosis DevR/DevS/DosT (differentially expressed in virulent strain response regulator/sensor/dormancysurvival sensorT), the Rev-erb-α and ß nuclear receptor, among others. The possible roles of these molecules as biochemical tools, therapeutic agents, and novel antibiotics are critically examined.

Anti-proliferative effect on a colon adenocarcinoma cell line exerted by a membrane disrupting antimicrobial peptide KL15.

The antimicrobial and anticancer activities of an antimicrobial peptide (AMP) KL15 obtained through in silico modification on the sequences of 2 previously identified bacteriocins m2163 and m2386 from Lactobacillus casei ATCC 334 by us have been studied. While significant bactericidal effect on the pathogenic bacteria Listeria, Escherichia, Bacillus, Staphylococcus, Enterococcus is exerted by KL15, the AMP can also kill 2 human adenocarcinoma cells SW480 and Caco-2 with measured IC50 as 50 µg/ml or 26.3 µM. However, the IC50 determined for KL15 on killing the normal human mammary epithelial cell H184B5F5/M10 is 150 µg/ml. The conformation of KL15 dissolved in 50% 2,2,2-trifluroroethanol or in 2 large unilamellar vesicle systems determined by circular dichroism spectroscopy appears to be helical. Further, the cell membrane permeability of treated SW480 cells by KL15 appears to be significantly enhanced as studied by both flow cytometry and confocal microscopy. As observed under a scanning electron microscope, the morphology of treated SW480 cells is also significantly changed as treating time by 80 µg/ml KL15 is increased. KL15 appears to be able to pierce the cell membrane of treated SW480 cells so that numerous porous structures are generated and observable. Therefore, KL15 is likely to kill the treated SW480 cells through the necrotic pathway similar to some recently identified AMPs by others.

Comparative analysis of two component signal transduction systems of the Lactobacillus acidophilus group

The Lactobacillus acidophilus group is a phylogenetically distinct group of closely related lactobacilli. Members of this group are considered to have probiotic properties and occupy different environmental niches. Bacteria generally sense and respond to environmental changes through two component systems (TCSs) which consist of a histidine protein kinase (HPK) and its cognate response regulator (RR). With the use of in silico techniques, the five completely sequenced L. acidophilus group genomes were scanned in order to predict TCSs. Five to nine putative TCSs encoding genes were detected in individual genomes of the L. acidophilus group. The L. acidophilus group HPKs and RRs were classified into subfamilies using the Grebe and Stock classification method. Putative TCSs were analyzed with respect to conserved domains to predict biological functions. Putative biological functions were predicted for the L. acidophilus group HPKs and RRs by comparing them with those of other microorganisms. Some of TCSs were putatively involved in a wide variety of functions which are related with probiotic ability, including tolerance to acid and bile, production of antimicrobial peptides, resistibility to the glycopeptide antibiotic vancomycin, and oxidative condition.

Comparative Analysis of two Component Signal Transduction Systems of the Lactobacillus Acidophilus Group.

The Lactobacillus acidophilus group is a phylogenetically distinct group of closely related lactobacilli. Members of this group are considered to have probiotic properties and occupy different environmental niches. Bacteria generally sense and respond to environmental changes through two component systems (TCSs) which consist of a histidine protein kinase (HPK) and its cognate response regulator (RR). With the use of in silico techniques, the five completely sequenced L. acidophilus group genomes were scanned in order to predict TCSs. Five to nine putative TCSs encoding genes were detected in individual genomes of the L. acidophilus group. The L. acidophilus group HPKs and RRs were classified into subfamilies using the Grebe and Stock classification method. Putative TCSs were analyzed with respect to conserved domains to predict biological functions. Putative biological functions were predicted for the L. acidophilus group HPKs and RRs by comparing them with those of other microorganisms. Some of TCSs were putatively involved in a wide variety of functions which are related with probiotic ability, including tolerance to acid and bile, production of antimicrobial peptides, resistibility to the glycopeptide antibiotic vancomycin, and oxidative condition.

Coronatine Gene Expression In Vitro and In Planta, and Protein Accumulation During Temperature Downshift in Pseudomonas syringae.

The plant pathogenic bacterium Pseudomonas syringae PG4180 synthesizes high levels of the phytotoxin coronatine (COR) at the virulence-promoting temperature of 18 °C, but negligible amounts at 28 °C. Temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of a response regulator, CorR, the histidine protein kinase CorS, and a third component, termed CorP. We analyzed at transcriptional and translational levels the expression of corS and the cma operon involved in COR biosynthesis after a temperature downshift from 28 to 18 °C. Expression of cma was induced within 20 min and increased steadily whereas corS expression was only slightly temperature-dependent. Accumulation of CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis. A transcriptional fusion of the cma promoter to a promoterless egfp gene was used to monitor the cma expression in vitro and in planta. A steady induction of cma::egfp by temperature downshift was observed in both environments. The results indicate that PG4180 responds to a temperature decrease with COR gene expression. However, COR gene expression and protein biosynthesis increased steadily, possibly reflecting adaptation to long-term rather than rapid temperature changes.