Synchronized flowering in pomegranate, following pruning, is associated with expression of the FLOWERING LOCUS T homolog, PgFT1.

Flowering in angiosperms is a crucial event that marks the transition from the vegetative to the reproductive phase. In many perennials, pruning is an important horticultural practice that induces synchronized and profuse flowering. In pomegranate, vegetative growth immediately after pruning is associated with activation of PgCENa, a flowering suppressor of the phosphatidyl ethanolamine binding protein (PEBP) family, while a reduction is associated with synchronous flowering. We show that flowering in pomegranate is activated by expression of another PEBP family member, PgFT1, a homolog of the FLOWERING LOCUS T (FT) gene that promotes flowering. PgFT1 shows a rapid reduction in expression during the extensive vegetative growth immediately after pruning but shows robust expression during synchronous flowering post-pruning, in flower-bearing shoots but not in branches that do not bear flowers. A continuous low-level flowering in the absence of pruning is associated with continuous but reduced expression of PgFT1. Flowering by heterologous expression of PgFT1 in Arabidopsis is affected by a single amino acid change in the C-terminal region of PgFT1, which upon correction, promotes flowering in Arabidopsis. Our study provides insights into the molecular mechanisms by which pruning affects flowering pathways in tropical perennial fruit plants such as pomegranate.

An Antibacterial Nanorobotic Approach for the Specific Targeting and Removal of Multiple Drug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) causes diseases ranging from skin infections to lethal sepsis and has become a serious threat to human health due to multiple-drug resistance (MDR). Therefore, a resistance-free antibacterial therapy is necessary to overcome MDR MRSA infections. In this study, an antibacterial nanorobot (Ab-nanobot) is developed wherein a cell wall-binding domain (CBD)-endolysin, acting as a sensor, is covalently conjugated with an actuator consisting of an iron oxide/silica core-shell. The CBD-endolysin sensor shows an excellent specificity to detect, bind, and accumulate on the S. aureus USA300 cell surface even in a bacterial consortium, and in host cell infections. Ab-nanobot specifically captures and kills MRSA in response to medically approved radiofrequency (RF) electromagnetic stimulation (EMS) signal. When Ab-nanobot receives the RF-EMS signal on the cell surface, actuator induces cell death in MRSA with 99.999% removal within 20 min by cell-wall damage via generation of localized heat and reactive oxygen species. The in vivo efficacy of Ab-nanobot is proven using a mice subcutaneous skin infection model. Collectively, this study offers a nanomedical resistance-free strategy to overcome MDR MRSA infections by providing a highly specific nanorobot for S. aureus.

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains.

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci.

Precise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellular Staphylococcus aureus We found that the difference between the two methods for the recovery of intracellular CFU of S. aureus was about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

Identification of 2',4'-Dihydroxychalcone as an Antivirulence Agent Targeting HlyU, a Master Virulence Regulator in Vibrio vulnificus.

The emergence of antimicrobial resistance and rapid acclimation allows Vibrio vulnificus to rapidly propagate in the host. This problematic pathological scenario can be circumvented by employing an antivirulence strategy, treating Vibrio infections without hindering the bacterial growth. We developed a genome-integrated orthogonal inhibitor screening platform in E. coli to identify antivirulence agents targeting a master virulence regulator of V. vulnificus. We identified 2',4'-dihydroxychalcone (DHC) from the natural compound library and verified that it decreases the expression of the major toxin network which is equivalent to the ∆hlyU deletion mutant. 2',4'-DHC also reduced the hemolytic activity of V. vulnificus which was tested as an example of virulence phenotype. The electrophoretic mobility shift assay confirmed that 2',4'-DHC specifically targeted HlyU and inhibited its binding to PrtxA1 promoter. Under in vivo conditions, a single dose of 2',4'-DHC protected ~50% wax-worm larvae from V. vulnificus infection at a non-toxic concentration to both V. vulnificus and wax-worm larvae. In the current study, we demonstrated that an orthogonal reporter system is suitable for the identification of antivirulence compounds with accuracy, and identified 2',4'-DHC as a potent antivirulence agent that specifically targets the HlyU virulence transcriptional regulator and significantly reduces the virulence and infection potential of V. vulnificus.

Structural and functional study of ChuY from Escherichia coli strain CFT073.

The uropathogenic Escherichia coli strain CFT073 contains multiple iron and heme transport systems, which facilitate infection of the host urinary tract. To elucidate the molecular and cellular function of ChuY, a hypothetical gene in the heme degradation/utilization pathway, we solved the crystal structure of ChuY at 2.4 Å resolution. ChuY has high structural homology with human biliverdin and flavin reductase. We confirmed that ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. A chuY deletion-insertion strain showed reduced survival potential compared to wild-type and complemented strains in mammalian cells. Current results suggest ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of E. coli CFT073.

Molecular characterization of CONSTANS-Like (COL) genes in banana (Musa acuminata L. AAA Group, cv. Grand Nain).

The CONSTANS (CO) family is an important regulator of flowering in photoperiod sensitive plants. But information regarding their role in day neutral plants is limited. We report identification of nine Group I type CONSTANS-like (COL) genes of banana and their characterization for their age dependent, diurnal and tissue-specific expression. Our studies show that the Group I genes are conserved in structure to members in other plants. Expression of these genes shows a distinct circadian regulation with a peak during light period. Developmental stage specific expression reveals high level transcript accumulation of two genes, MaCOL3a and MaCOL3b, well before flowering and until the initiation of flowering. A decrease in their transcript levels after initiation of flowering is followed by an increase in transcription of other members that coincides with the continued development of the inflorescence and fruiting. CO binding cis-elements are observed in at least three FT -like genes in banana suggesting possible CO-FT interactions that might regulate flowering. Distinct tissue specific expression patterns are observed for different family members in mature leaves, apical inflorescence, bracts, fruit skin and fruit pulp suggesting possible roles other than flowering. This is the first exhaustive study of the COL genes belonging to Group I of banana.

Cyanobacterial heat-shock response: role and regulation of molecular chaperones.

Cyanobacteria constitute a morphologically diverse group of oxygenic photoautotrophic microbes which range from unicellular to multicellular, and non-nitrogen-fixing to nitrogen-fixing types. Sustained long-term exposure to changing environmental conditions, during their three billion years of evolution, has presumably led to their adaptation to diverse ecological niches. The ability to maintain protein conformational homeostasis (folding-misfolding-refolding or aggregation-degradation) by molecular chaperones holds the key to the stress adaptability of cyanobacteria. Although cyanobacteria possess several genes encoding DnaK and DnaJ family proteins, these are not the most abundant heat-shock proteins (Hsps), as is the case in other bacteria. Instead, the Hsp60 family of proteins, comprising two phylogenetically conserved proteins, and small Hsps are more abundant during heat stress. The contribution of the Hsp100 (ClpB) family of proteins and of small Hsps in the unicellular cyanobacteria (Synechocystis and Synechococcus) as well as that of Hsp60 proteins in the filamentous cyanobacteria (Anabaena) to thermotolerance has been elucidated. The regulation of chaperone genes by several cis-elements and trans-acting factors has also been well documented. Recent studies have demonstrated novel transcriptional and translational (mRNA secondary structure) regulatory mechanisms in unicellular cyanobacteria. This article provides an insight into the heat-shock response: its organization, and ecophysiological regulation and role of molecular chaperones, in unicellular and filamentous nitrogen-fixing cyanobacterial strains.

Repeated Exposure of Vancomycin to Vancomycin-Susceptible Staphylococcus aureus (VSSA) Parent Emerged VISA and VRSA Strains with Enhanced Virulence Potentials.

The emergence of resistance against the last-resort antibiotic vancomycin in staphylococcal infections is a serious concern for human health. Although various drug-resistant pathogens of diverse genetic backgrounds show higher virulence potential, the underlying mechanism behind this is not yet clear due to variability in their genetic dispositions. In this study, we investigated the correlation between resistance and virulence in adaptively evolved isogenic strains. The vancomycin-susceptible Staphylococcus aureus USA300 was exposed to various concentrations of vancomycin repeatedly as a mimic of the clinical regimen to obtain mutation(s)-accrued-clonally-selected (MACS) strains. The phenotypic analyses followed by expression of the representative genes responsible for virulence and resistance of MACS strains were investigated. MACS strains obtained under 2 and 8 µg/ml vancomycin, named Van2 and Van8, respectively; showed enhanced vancomycin minimal inhibitory concentrations (MIC) to 4 and 16 µg/ml, respectively. The cell adhesion and invasion of MACS strains increased in proportion to their MICs. The correlation between resistance and virulence potential was partially explained by the differential expression of genes known to be involved in both virulence and resistance in MACS strains compared to parent S. aureus USA300. Repeated treatment of vancomycin against vancomycin-susceptible S. aureus (VSSA) leads to the emergence of vancomycin-resistant strains with variable levels of enhanced virulence potentials.

Anaerobic benzene oxidation via phenol in Geobacter metallireducens.

Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance. Therefore, benzene metabolism was investigated in Geobacter metallireducens, the only genetically tractable organism known to anaerobically degrade benzene. Trace amounts (<0.5 µM) of phenol accumulated in cultures of Geobacter metallireducens anaerobically oxidizing benzene to carbon dioxide with the reduction of Fe(III). Phenol was not detected in cell-free controls or in Fe(II)- and benzene-containing cultures of Geobacter sulfurreducens, a Geobacter species that cannot metabolize benzene. The phenol produced in G. metallireducens cultures was labeled with (18)O during growth in H2(18)O, as expected for anaerobic conversion of benzene to phenol. Analysis of whole-genome gene expression patterns indicated that genes for phenol metabolism were upregulated during growth on benzene but that genes for benzoate or toluene metabolism were not, further suggesting that phenol was an intermediate in benzene metabolism. Deletion of the genes for PpsA or PpcB, subunits of two enzymes specifically required for the metabolism of phenol, removed the capacity for benzene metabolism. These results demonstrate that benzene hydroxylation to phenol is an alternative to carboxylation for anaerobic benzene activation and suggest that this may be an important metabolic route for benzene removal in petroleum-contaminated groundwaters, in which Geobacter species are considered to play an important role in anaerobic benzene degradation.

Sensor histidine kinases kdpD and aauS regulate biofilm and virulence in Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen that utilizes two-component systems (TCSs) to sense pathophysiological signals and coordinate virulence. P. aeruginosa contains 64 sensor histidine kinases (HKs) and 72 response regulators (RRs) that play important roles in metabolism, bacterial physiology, and virulence. However, the role of some TCSs in virulence remains uncharacterized. In this study, we evaluated the virulence potential of some uncharacterized sensor HK and RR knockouts in P. aeruginosa using a Galleria mellonella infection model. Furthermore, we demonstrated that KdpD and AauS HKs regulate virulence by affecting P. aeruginosa biofilm formation and motility. Both ΔkdpD and ΔaauS showed reduced biofilm and motility which were confirmed by restored phenotypes upon complementation. Moreover, ΔkdpD and ΔaauS exhibited increased survival of HeLa cells and G. mellonella during in vivo infection. Altered expression of the transcriptional regulators anR and lasR, along with the virulence genes lasA, pelA, cupA, pqsA, pqsB, pqsC, and pqsD in the mutant strains elucidated the mechanism by which ΔkdpD and ΔaauS affect virulence. These findings confirm that kdpD and aauS play important roles in P. aeruginosa pathogenesis by regulating biofilm formation and motility.

Polyaniline-based highly sensitive microbial biosensor for selective detection of lindane.

A highly sensitive, selective, and rapid, whole-cell-based electrochemical biosensor was developed for detection of the persistent organochlorine pesticide γ-hexachlorocyclohexane (γ-HCH), commonly known as lindane. The gene linA2 encoding the enzyme γ-hexachlorocyclohexane (HCH) dehydrochlorinase (LinA2), involved in the initial steps of lindane (γ-HCH) biotransformation, was cloned and overexpressed in Escherichia coli . The lindane-biodegrading E. coli cells were immobilized on polyaniline film. The rapid and selective degradation of lindane and concomitant generation of hydrochloric acid by the recombinant E. coli cells in the microenvironment of polyaniline led to a change in its conductivity, which was monitored by pulsed amperometry. The biosensor could detect lindane in the part-per-trillion concentration range with a linear response from 2 to 45 ppt. The sensor was found to be selective to all the isomers of hexachlorocyclohexane (HCH) and to pentachlorocyclohexane (PCCH) but did not respond to other aliphatic and aromatic chlorides or to the end product of lindane degradation, i.e., trichlorobenzene (TCB). The sensor also did not respond to other commonly used organochlorine pesticides like DDT and DDE. On the basis of experimental results, a rationale has been proposed for the excellent sensitivity of polyaniline as a pH sensor for detection of H(+) ions released in its microenvironment.

Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential.

Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields.

Genome-Wide Analysis of Staphylococcus aureus Sequence Type 72 Isolates Provides Insights Into Resistance Against Antimicrobial Agents and Virulence Potential.

Staphylococcus aureus sequence type 72 (ST72) is a major community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) that has rapidly entered the hospital setting in Korea, causing mild superficial skin wounds to severe bloodstream infections. In this study, we sequenced and analyzed the genomes of one methicillin-resistant human isolate and one methicillin-sensitive human isolate of ST72 from Korea, K07-204 and K07-561, respectively. We used a subtractive genomics approach to compare these two isolates to other 27 ST72 isolates to investigate antimicrobial resistance (AMR) and virulence potential. Furthermore, we validated genotypic differences by phenotypic characteristics analysis. Comparative and subtractive genomics analysis revealed that K07-204 contains methicillin (mecA), ampicillin (blaZ), erythromycin (ermC), aminoglycoside (aadD), and tetracycline (tet38, tetracycline efflux pump) resistance genes while K07-561 has ampicillin (blaZ) and tetracycline (tet38) resistance genes. In addition to antibiotics, K07-204 was reported to show resistance to lysostaphin treatment. K07-204 also has additional virulence genes (adsA, aur, hysA, icaABCDR, lip, lukD, sdrC, and sdrE) compared to K07-561, which may explain the differential virulence potential of these human isolates of ST72. Unexpectedly, the virulence potential of K07-561 was higher in an in vivo wax-worm infection model than that of K07-204, putatively due to the presence of a 20-fold higher staphyloxanthin concentration than K07-204. Comprehensive genomic analysis of these two human isolates, with 27 ST72 isolates, and S. aureus USA300 (ST8) suggested that acquisition of both virulence and antibiotics resistance genes by ST72 isolates might have facilitated their adaptation from a community to a hospital setting where the selective pressure imposed by antibiotics selects for more resistant and virulent isolates. Taken together, the results of the current study provide insight into the genotypic and phenotypic features of various ST72 clones across the globe, delivering more options for developing therapeutics and rapid molecular diagnostic tools to detect resistant bacteria.

Draft Genome Sequences of Lysostaphin-Resistant (K07-204) and Lysostaphin-Susceptible (K07-561) Staphylococcus aureus Sequence Type 72 Strains Isolated from Patients in South Korea.

Methicillin-resistant Staphylococcus aureus sequence type 72 (ST72) is prevalent in South Korea and has shown resistance to multiple antimicrobials. ST72 isolates display different levels of resistance to the antistaphylococcal lysostaphin. Draft genome sequencing of ST72 human isolates exhibiting lysostaphin resistance or susceptibility was performed to better understand the mechanism of lysostaphin resistance using subtractive genomics.

Overexpression of the groESL operon enhances the heat and salinity stress tolerance of the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC7120.

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial P(psbA1) promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses.

Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase in Staphylococcus aureus.

Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of ΔmtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.IMPORTANCE Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.

An integrative expression vector for strain improvement and environmental applications of the nitrogen fixing cyanobacterium, Anabaena sp. strain PCC7120.

An integrative expression vector, incorporating a cassette for genomic integration and expression of gene of interest into Anabaena sp. strain PCC7120 was constructed. The cassette comprised of the following elements: (a) an intergenic non-coding region from Anabaena to facilitate its genomic integration (b) a strong functional PpsbA1 promoter from Anabaena for desired gene expression and (c) neomycin phosphotransferase gene with its own promoter for the selection of transformants. The cassette was cloned in the plasmid pBluescript II SK (+), which served as a suicide vector for Anabaena. The resultant plasmid designated as pFPN, when transferred into Anabaena by electrotransformation, integrated the cassette into Anabaena genome at the defined location. The vector was evaluated by cloning, transfer, integration and expression of the native hetR gene encoding a regulator of heterocyst differentiation. Formation of multiple heterocysts and enhanced nitrogenase activity of constitutively expressing hetR transformants of Anabaena established the utility of pFPN for basic molecular biology research and strain improvement. A transgene phoN encoding a non-specific acid phosphatase from Salmonella typhi was also transferred into Anabaena using pFPN. Strong constitutive expression of PhoN from PpsbA1 facilitated easier and sensitive visual screening of PhoN for tracking of transgenic Anabaena. The vector pFPN offers a stable integrative expression system for environmental application of genetically modified Anabaena strains.

Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus.

Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct P rtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

In silico analysis and experimental validation of lipoprotein and novel Tat signal peptides processing in Anabaena sp. PCC7120.

Signal peptide (SP) plays a pivotal role in protein translocation. Lipoprotein- and twin arginine translocase (Tat) dependent signal peptides were studied in All3087, a homolog of competence protein of Synechocystis PCC6803 and in two putative alkaline phosphatases (ALPs, Alr2234 and Alr4976), respectively. In silico analysis of All3087 is shown to possess the characteristics feature of competence proteins such as helix-hairpin-helix, N and C-terminal HKD endonuclease domain, calcium binding domain and N-terminal lipoprotein signal peptide. The SP recognition-cleavage site in All3087 was predicted (AIA-AC) using SignalP while further in-depth analysis using Pred-Lipo and WebLogo analysis for consensus sequence showed it as IAA-C. Activities of putative ALPs were confirmed by heterologous overexpression, activity assessment and zymogram analysis. ALP activity in Anabaena remains cell bound in log-phase, but during late log/stationary phase, an enhanced ALP activity was detected in extracellular milieu. The enhancement of ALP activity during stationary phase was not only due to inorganic phosphate limitation but also contributed by the presence of novel bipartite Tat-SP. The Tat signal transported the folded active ALPs to the membrane, followed by anchoring into the membrane and successive cleavage enabling transportation of the ALPs to the extracellular milieu, because of bipartite architecture and processing of transit Tat-SP.