Cajanin stilbene acid: A direct inhibitor of colistin resistance protein MCR-1 that restores the efficacy of polymyxin B against resistant Gram-negative bacteria.

BACKGROUND: The resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli). PURPOSE: In this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery. STUDY DESIGN AND METHODS: Checkerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments. RESULTS: Here, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 µg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18­hydroxyl oxygens of CSA were the key sites binding to MCR-1. CONCLUSION: CSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein.

DosR Regulates the Transcription of the Arginine Biosynthesis Gene Cluster by Binding to the Regulatory Sequences in Mycobacterium bovis Bacille Calmette-Guerin.

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.

Characterization of a novel Mycobacterium tuberculosis serine protease (Rv3194c) activity and pathogenicity.

Mycobacterium tuberculosis (MTB) serine proteases are important pathogen-associated virulence factors that are involved in the invasion, bacterial persistence, and degradation of host defense factors. The current study identified and characterized a novel serine protease, Rv3194c, of MTB. A heterologous Rv3194c protein, purified from Escherichia coli, possessed proteolytic activity that could hydrolyze bovine serum albumin (BSA), milk, casein, and gelatin at an optimal temperature of 40 °C and a pH of 8.0. Furthermore, the divalent metal ions Ca2+ and Mn2+ increased the activity of Rv3194c. Betulinic acid, a Traditional Chinese Medicine (TCM) monomer; PMSF, a chemical inhibitor; and the Roche inhibitor cocktail inhibited proteolytic activity. Site-directed mutagenesis demonstrated that D308 and particularly S309 play a crucial role in the catalytic activity of Rv3194c protease. The cellular assays revealed that Rv3194c inhibits THP1-derived macrophage migration. Moreover, Rv3194c degraded the complement components, C3b and C5a, causing inhibition of phagocytosis and chemotaxis. In mice, Rv3194c enhanced the persistence of Mycobacterium smegmatis (Ms) in the lung, induced lung lesions, and promoted the release of inflammatory cytokines. The results of this study indicate that Rv3194c may play an important role in the pathogenicity of mycobacteria.

Streptococcus suis sortase A is Ca2+ independent and is inhibited by acteoside, isoquercitrin and baicalin.

Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 µM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 µM, 100.0 ± 1.3 µM and 85.4 ± 1.5 µM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.

New Targets and Cofactors for the Transcription Factor LrpA from Mycobacterium tuberculosis.

Rv3291c (MtbLrpA), a transcriptional regulator, belongs to the leucine-responsive regulatory protein (Lrp) family and is thought to play an important role in Mycobacterium tuberculosis persistence. In this study, we verified 17 novel potential binding sites for MtbLrpA by in vitro binding assays on the basis of previous predictions from an in silico analysis and bacterial one-hybrid (BIH) reporter system. Amino acids, such as tyrosine, phenylalanine, tryptophan, and histidine, strongly affect the binding affinity of MtbLrpA, and vitamins, including B1, B3, B6, VC, B7, B9, B12, VA, and VK3, also decrease MtbLrpA binding affinity. This is the first report regarding that an Lrp-like protein can sense vitamins as an environmental signal. Vitamin supplementation to the environment can change the expression level of the target genes, which provides a potential mechanism for tuberculosis supplementary treatment with vitamins.

Recombinant human lactoferrin enhances the efficacy of triple therapy in mice infected with Helicobacter pylori.

Helicobacter pylori (H. pylori) is a life-threatening pathogen which causes chronic gastritis, gastric ulcers and even stomach cancer. Treatment normally involves bacterial eradication; however, this type of treatment only has a rate of effectiveness of <80%. Thus, it is a matter of some urgency to develop new therapeutic strategies. Lactoferrin, a member of the transferrin family of iron-binding proteins, has been proven to be effective in removing a vast range of pathogens, including H. pylori. In the present study, we examined the effectiveness of recombinant human lactoferrin (rhLf) isolated from transgenic goats as a treatment for H. pylori in vitro and in vivo. For the in vivo experiments, BALB/c mice received an intragastric administration of 0.1 ml of a suspension of H. pylori. The mice were then divided into 4 groups: group A, treated with saline; group B, treated with 1.5 g of rhLF; group C, treated with the standard triple therapy regimen; and group D, treated with the standard triple therapy regimen plus.5 g of rhLF. Following sacrifice, the stomach tissues of the mice were histologically examined for the presence of bacteria. For the in vitro experiments, the bacteria were cultured in BHI broth and RT-qPCR and western blot analysis were carried out to determine the mRNA and protein levels of virulence factors (CagA and VacA) in the cultures. Our results revealed that rhLf not only inhibited the growth of H. pylori, but also suppressed the expression of two major virulence factors. Moreover, rhLf markedly increased bacterial eradication and effectively reduced the inflammatory response when combined with the standard triple therapy regimen. These results provide evidence supporting the use of rhLF as an adjuvant to traditional therapeutic strategies in the treatment of H. pylori.

An inhibitor of cathepsin K, icariin suppresses cartilage and bone degradation in mice of collagen-induced arthritis.

The collagenase cathepsin K has been shown important in the pathogenesis of rheumatoid arthritis (RA). Icariin is the major pharmacologically active flavonol diglycoside of Herba Epimedii, an herb used in Chinese traditional medicine to treat arthritis. We investigated whether icariin can inhibit the protease activity of cathepsin K and its effects on a murine model of collagen-induced arthritis (CIA). Six-week old female BALB/C mice were immunized with type II collagen and treated with vehicle alone icariin (25mg/kg) for 21 days; a control remained untreated. Serum concentrations of type I collagen C-terminal telopeptide (CTX-I) and cartilage oligomeric matrix protein (COMP) and urinary concentrations of deoxypyridinoline (DPD) were measured, and disease severity was assessed. Compared with immunized, untreated mice, immunized icariin-treated mice had significantly lower urinary DPD (~25%, p<0.01) and serum COMP (~11.9%, p<0.01) concentrations, with serum CTX-1 (RatLaps) concentrations being significantly lower in immunized, icariin treated mice than in immunized, vehicle treated (p<0.01) and non-immunized (p<0.005) mice. Icariin also reduced the clinical signs of arthritis. Icariin inhibited cathpesin K activity in vitro and was effective in a mouse model of CIA similar to human RA, suggesting that this agent may have promise in the treatment of patients with RA.

In vitro synergistic interactions of oleanolic acid in combination with isoniazid, rifampicin or ethambutol against Mycobacterium tuberculosis.

Reports have shown that oleanolic acid (OA), a triterpenoid, exists widely in food, medicinal herbs and other plants, and that it has antimycobacterial activity against the Mycobacterium tuberculosis strain H37Rv (ATCC 27294). In this study it was found that OA had antimycobacterial properties against eight clinical isolates of M. tuberculosis and that the MICs of OA against drug-sensitive and drug-resistant isolates were 50-100 and 100-200 microg ml(-1), respectively. The combination of OA with isoniazid (INH), rifampicin (RMP) or ethambutol (EMB) showed favourable synergistic antimycobacterial effects against six drug-resistant strains, with fractional inhibitory concentration indices of 0.121-0.347, 0.113-0.168 and 0.093-0.266, respectively. The combination treatments of OA/INH, OA/RMP and OA/EMB displayed either a synergistic interaction or did not show any interaction against two drug-sensitive strains. No antagonism resulting from the OA/INH, OA/RMP or OA/EMB combination was observed for any of the strains tested. OA exhibited a relatively low cytotoxicity in Vero cells. These results indicate that OA may serve as a promising lead compound for future antimycobacterial drug development.