Infected Macrophage Cells Research Articles

Antimicrobial Effect of Cannabidiol on Intracellular Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), has killed nearly one billion people during the last two centuries. Nowadays, TB remains a major global health problem ranked among the top 10 causes of death worldwide. One of the main challenges in developing new strategies to fight TB is focused on reducing the duration and complexity of drug regimens. Cannabidiol (CBD) is the main nonpsychoactive ingredient extracted from the Cannabis sativa L. plant, which has been shown to be biologically active against bacteria. The purpose of this work was to investigate the antimicrobial effect of CBD on M. tuberculosis intracellular infection. Materials and Methods: To assess the minimum inhibitory concentration (MIC) of CBD on mycobacterial strains, the MTT assay was performed on Mycobacterium smegmatis, and the Colony-Forming Unit (CFU) assay was conducted on MtbH37Rv. Additionally, the cytotoxic effect of CBD on THP-1 cells was assessed by MTT assay. Moreover, macrophages derived from the THP-1 cell were infected with MtbH37Rv (multiplicity of infection 1:10) to evaluate the intracellular activity of CBD by determining the CFU/mL. Results: Antimicrobial activity against M. smegmatis (MIC=100 μM) and MtbH37Rv (MIC=25 μM) cultures was exhibited by CBD. Furthermore, the effect of CBD was also evaluated on MtbH37Rv infected macrophage cells. Interestingly, a reduction in viable intracellular MtbH37Rv bacteria was observed after 24 h of treatment. Moreover, CBD exhibited a safe profile toward human THP-1 cells, since it showed no toxicity (CC50=1075 μM) at a concentration of antibacterial effect (selectivity index 43). Conclusion: These results extend the knowledge regarding the antimicrobial activity of CBD and demonstrate its ability to kill the human intracellular pathogen M. tuberculosis.

Streptococcal arginine deiminase system defences macrophage bactericidal effect mediated by XRE family protein XtrSs

ABSTRACT The arginine deiminase system (ADS) has been identified in various bacteria and functions to supplement energy production and enhance biological adaptability. The current understanding of the regulatory mechanism of ADS and its effect on bacterial pathogenesis is still limited. Here, we found that the XRE family transcriptional regulator XtrSs negatively affected Streptococcus suis virulence and significantly repressed ADS transcription when the bacteria were incubated in blood. Electrophoretic mobility shift (EMSA) and lacZ fusion assays further showed that XtrSs directly bind to the promoter of ArgR, an acknowledged positive regulator of bacterial ADS, to repress ArgR transcription. Moreover, we provided compelling evidence that S. suis could utilize arginine via ADS to adapt to acid stress, while ΔxtrSs enhanced this acid resistance by upregulating the ADS operon. Moreover, whole ADS-knockout S. suis increased arginine and antimicrobial NO in the infected macrophage cells, decreased intracellular survival, and even caused significant attenuation of bacterial virulence in a mouse infection model, while ΔxtrSs consistently presented the opposite results. Our experiments identified a novel ADS regulatory mechanism in S. suis, whereby XtrSs regulated ADS to modulate NO content in macrophages, promoting S. suis intracellular survival. Meanwhile, our findings provide a new perspective on how Streptococci evade the host’s innate immune system.

Immune-enhancing activity of Astragalus maximus extract for inhibiting the Toxoplasma gondii infection: experimental research.

This study aimed to evaluate the in-vitro anti-Toxoplasma effects and cytotoxicity effects of Astragalus maximus chloroformic extract (AMCE) on the T. gondii Rh strain. In-vitro effects of AMCE (2-64µg/ml) on tachyzoites were measured by MTT assay for 48h. The effects of AMCE on infectivity rate and intracellular parasites into macrophage cells (J774-A1) were evaluated. The Griess reaction assay and quantitative real-time PCR were used to determine the nitric oxide (NO) and the mRNA expression levels of IFN-γ and iNOS in infected J774-A1 macrophage cells. The mortality rate of the parasites significantly (P<0.001) increased in a dose-dependent manner with an IC50 value of 9.85μg/ml. The rate of infection and the mean number of intracellular tachyzoites in macrophage cells were significantly reduced (P<0.001) after exposure of the macrophage cells to AMCE. The mRNA expression levels of IFN-γ, iNOS, and NO production in macrophage cells after treatment with the AMCE were increased, especially at the concentration of ½ IC50 and IC50 (P<0.001). The findings of the current in vitro investigation revealed favorable anti-Toxoplasma effects of AMCE against tachyzoites and intracellular forms of T. gondii. Despite the fact that the accurate anti-Toxoplasma mechanisms of AMCE are not clear, our results showed that triggering NO production and cellular immunity can be considered as the main mechanisms of action of AMCE for controlling and eliminating T. gondii. However, further surveys are mandatory to assess the efficacy and safety of AMCE in an animal model and its accurate mechanisms of action.

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model.

Leishmaniasis is a zoonotic disease caused by an intracellular parasite from the genus Leishmania. Lack of safe and effective drugs has increasingly promoted researches into new drugs of natural origin to cure the disease. The study, therefore, aimed to investigate the anti-leishmanial effects of Lucilia sericata larval excretion/secretion (ES) in combination with Apis mellifera honey as a synergist on Leishmania major using an in vitro model. Various concentrations of honey and larval ES fractions were tested against promastigotes and intracellular amastigotes of L. major using macrophage J774A.1 cell line. The inhibitory effects and cytotoxicity of ES plus honey were evaluated using direct counting method and MTT assay. To assess the effects of larval ES plus honey on the amastigote form, the rate of macrophage infection and the number of amastigotes per infected macrophage cell were estimated. The 50% inhibitory concentration (IC50) values were 21.66 μg/ml, 43.25 60 μg/ml, 52.58 μg/ml, and 70.38 μg/ml for crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey alone, respectively. The IC50 for positive control (glucantime) was 27.03 μg/ml. There was a significant difference between viability percentages of promastigotes exposed to different doses of applied treatments compared to the negative control (p≤ 0.0001). Microscopic examination of amastigote forms revealed that dosages applied at 150 to 300 μg/ml significantly reduced the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Different doses of larval products plus honey did not show a significant toxic effect agaist macrophage J774 cells. The larval ES fractions of L. sericata in combination with A. mellifera honey acted synergistically against L. major.

In vitro Evaluation of the Potent Antileishmanial Activity of Ferula tabasensis Alone or in Combination with Shark Cartilage Extract Against the Standard Iranian Strain of Leishmania major (MRHO/IR/75/ER).

The available drugs for the treatment of leishmaniasis are highly toxic and extremely expensive, with low efficiency; therefore, the development of effective therapeutic compounds is essential. The present study aimed to explore the antileishmanial effects of ethyl acetate extract, methanol extract, and fractions 1-4 (F1-F4) of Ferula tabasensis, alone or in combination with shark cartilage extract (ShCE), on L. major in vitro. In this study, ethyl acetate, methanol, and n-hexane extracts were extracted from the aerial roots of F. tabasensis by the maceration method. The silica gel column chromatography was used to separate n-hexane extracts at varying polarities (F1-F4 fractions). Subsequently, the effects of extracts and fractions against promastigotes were assessed by the parasite counting method microscopic inhibition test and MTT assay. Besides, their effects on the infected macrophage cells and the number of amastigotes were investigated. Cytotoxicity was evaluated in non-infected J774A.1 macrophage cells. Finally, apoptosis induction of promastigotes, including infected and non-infected macrophages, was evaluated. The results indicated the highly potent activity of F. tabasensis extracts and F1-F4 fractions, alone or in combination with ShCE, against L. major promastigotes and amastigotes in a dose-dependent manner (P < 0.05). The F1 fraction and methanol extract showed markedly higher toxicity compared to the other extracts and fractions, with 50% inhibitory concentrations (IC50/72h) of 2.4 ± 0.29 and 2.9 ± 0.55 µg/mL against promastigotes and 1.79 ± 0.27 µg/mL and 1.39 ± 0.27 µg/mL against amastigotes (P < 0.001). Moreover, they had a high selectivity index (SI) due to the low toxicity of macrophages (P < 0.0001). The results of flow cytometry indicated that the percentages of apoptotic promastigote cells in contact with IC50 concentrations of F1 and methanol extract alone after 72 h were 43.83 and 43.93%, as well as 78.4%, and 65.45% for their combination with ShCE, respectively.Also, apoptosis of infected macrophages induced by F1 and methanol extracts was estimated at 68.5% and 83.7%, respectively. In this study, the F1 fraction and methanol extract of F. tabasensis showed potent efficacy against L. major, associated with low toxicity and apoptosis induction. Therefore, they can be promising therapeutic candidates in future animal and even human studies.

Leishmanicidal and immunomodulatory activities of the formononetin (a natural isoflavone) against Leishmania tropica

ObjectiveThis work aimed to examine the leishmanicidal, cellular mechanisms and cytotoxicity effects of formononetin (FMN), a natural isoflavone, against Leishmania tropica. We used the MTT assay to determine the leishmanicidal effects of FMN against promastigotes and its cytotoxicity effects on J774-A1 macrophage cells. The Griess reaction assay and quantitative real-time PCR were used to determine the nitric oxide (NO) and the mRNA expression levels of IFN-γ and iNOS in infected J774-A1 macrophage cells.ResultsFMN significantly (P < 0.001) decreased the viability and number of promastigotes and amastigotes forms. The 50% inhibitory concentrations value for FMN and glucantime was 9.3 and 14.3 µM for promastigote and amastigote, respectively. We found that the macrophages exposed with FMN especially at concentrations of 1/2 IC50 and IC50 significantly activated the NO release and the mRNA expression levels of IFN-γ, iNOS. The findings of the current research showed the favorable antileishmanial effects formononetin, a natural isoflavone, against various stages of L. tropica through inhibition of infectivity rate of macrophage cells and triggering the NO production and cellular immunity. However, supplementary works are essential to evaluate the ability and safety of FMN in animal model before use in the clinical phase.

Improved Subcutaneous Delivery of Ketoconazole Using EpiDerm and HSPiP Software-Based Simulations as Assessed by Cell Viability, Cellular Uptake, Permeation, and Hemolysis In Vitro Studies.

Ketoconazole (KETO) is the drug of choice to control local, systemic, and resistant types of fungal infections. Subcutaneous (sub-Q) delivery offers several benefits. The present study investigated the sub-Q delivery of KETO using HSPiP software based on optimized concentrations of dimethylacetamide (DMA) in binary solvents (DMA + water), in vitro cellular uptake (J774A.1) assays, cellular toxicity (L929), and in vitro hemolysis studies. Results showed that the estimated permeation coefficient (9.6 × 10-3 cm/h) and diffusion coefficient (3.9 × 10-3 cm2/h) of KETO (22.3 mg) in KF3 (300 mg of DMA + water) across EpiDerm were relatively higher as compared to the other formulations [KF1 (11.2 and 150 mg as KETO and DMA, respectively) and KF2 [(22.3 and 300 mg as KETO and DMA, respectively)] due to the increased content of DMA and KETO. HSPiP simulated and predicted the impact of constant and variable diffusion coefficients on the percent drug absorption across EpiDerm and the time needed to achieve equilibrium. The concentration-dependent diffusion coefficient fed into HSPiP predicted that the drug absorption and permeation values were linearly dependent on the square root of time. The HSPiP predicted permeation flux values from KF3, KF2, and KF1 across the EpiDerm were 4.07 × 10-6, 4.01 × 10-6, and 1.1 × 10-6 g/cm2/s, respectively, at respective D range values. The selected K30G (324 mOsm/Kg) showed an optimal pH (6.9) and minimum drug loss (0.01%) over a period of 1 month at room temperature. KG30 was found to be less toxic to normal L292 cells and caused maximum cytotoxicity to candida cells residing within infected macrophage cells (J774A.1 incubated for 24 h), which was attributed to the slow diffusion of K30G compared to DS (the drug solution with an equivalent concentration). KG30 elicited substantial internalization with candida albicans (MTCC 4748) compared to the control group (24 h). Lastly, in vitro hemolysis studies (1 and 5 μg/mL) corroborated the safety of K30G for sub-Q delivery. Therefore, this new formulation and approach for delivering KETO is a promising alternative to conventional products to control fungal infections and, thus, should be further studied in vivo.

Identification of pyrC gene as an immunosuppressive factor in Francisella novicida infection.

Francisella tularensis, a bacterial causative agent of the zoonosis tularemia, is highly pathogenic to humans. The pathogenicity of this bacterium is characterized by intracellular growth in immune cells, like macrophages, and host immune suppression. However, the detailed mechanism of immune suppression by F. tularensis is still unclear. To identify the key factors causing Francisella-mediated immunosuppression, large-scale screening using a transposon random mutant library containing 3552 mutant strains of F. tularensis subsp. novicida (F. novicida) was performed. Thirteen mutants that caused stronger tumor necrosis factor (TNF)-α production in infected U937 human macrophage cells than the wild-type F. novicida strain were isolated. Sequencing analysis of transposon insertion sites revealed 10 genes, including six novel genes, as immunosuppressive factors of Francisella. Among these, the relationship of the pyrC gene, which encodes dihydroorotase in the pyrimidine biosynthesis pathway, with Francisella-mediated immunosuppression was investigated. The pyrC deletion mutant strain (ΔpyrC) induced higher TNF-α production in U937 host cells than the wild-type F. novicida strain. The ΔpyrC mutant strain was also found to enhance host interleukin-1β and interferon (IFN)-β production. The heat-inactivated ΔpyrC mutant strain could not induce host TNF-α production. Moreover, the production of IFN-β resulting from ΔpyrC infection in U937 cells was repressed upon treatment with the stimulator of interferon genes (STING)-specific inhibitor, H-151. These results suggest that pyrC is related to the immunosuppressive activity and pathogenicity of Francisella via the STING pathway.

The attenuated Mycoplasma bovis strain promotes apoptosis of bovine macrophages by upregulation of CHOP expression.

Mycoplasma bovis (M. bovis) is one of the major pathogens in the bovine respiratory disease complex, which includes pneumonia, mastitis, and arthritis and causes a great economic loss in the cattle industry. In China, a live-attenuated vaccine strain M. bovis P150 was obtained by a continuous culture of the wild-type strain M. bovis HB0801 (P1) in vitro for 150 passages. Using the infected bovine macrophage cell line BoMac, this work attempted to investigate the mechanism of P150 attenuation and protective immune response. To begin, we show that M. bovis P150 effectively triggered cytotoxicity and apoptosis in BoMac, although with lower intracellular survival than P1. The transcriptomes of BoMac after infection with M. bovis strains P1 and P150 were sequenced, and bioinformatic analysis identified 233 differentially expressed genes (DEGs), with 185 upregulated and 48 downregulated. Further Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses revealed that the majority of the DEGs were linked to CHOP complex, MAP kinase phosphatase activity and were involved in the IL-17 signaling pathway in immune response, MAPK signaling pathway in signal transduction, and p53 signaling pathway in cell growth and death. Among them, the level of C/EBP homologous protein (CHOP) was significantly upregulated in P150-infected BoMac compared to P1-infected cells at different time points, along with its upstream and downstream genes phosphorylated-PERK, phosphorylated-EIF2α, ATF4, and GADD45A increased in the PERK-dependent ER stress response. The role of CHOP in apoptosis was further verified by M. bovis-induced siCHOP knockdown in BoMac cells. The results showed that CHOP knockdown enhanced P150-induced apoptosis and dramatically increased the M. bovis P1 and P150 intracellular survival, particularly for P150. These data suggest that P150 infection upregulates CHOP expression, which can increase apoptosis and mediate a crosstalk between ER stress and apoptosis during infection, and hence, contribute to high cytotoxicity and low intracellular survival.

Temporal profiling of newly translated proteome in Mycobacterium tuberculosis Infected macrophage cells

Temporal profiling of newly translated proteome in Mycobacterium tuberculosis Infected macrophage cells

In vitro anti-tuberculosis effect of probiotic Lacticaseibacillus rhamnosus PMC203 isolated from vaginal microbiota

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), poses a severe challenge for public health and remains the number one cause of death as a single infectious agent. There are 10 million active cases of TB per year with 1.5 million deaths, and 2–3 billion people are estimated to harbor latent M. tb infection. Moreover, the emergence of multi-drug-resistant (MDR), extremely-drug-resistant (XDR), and the recent totally drug-resistant (TDR) M. tb is becoming a global issue that has fueled the need to find new drugs different from existing regimens. In these circumstances, probiotics can be a potential choice, so we focused on developing them as an anti-tuberculosis drug candidate. Here, we report the anti-tubercular activities of Lacticaseibacillus rhamnosus PMC203 isolated from the vaginal microbiota of healthy women. PMC203 exhibited a promising intracellular killing effect against both drug-sensitive and resistant M. tb infected murine macrophage cell line RAW 264.7 without showing any cytotoxicity. Additionally, it also inhibited the growth of M. tb under broth culture medium. PMC203 did not cause weight change or specific clinical symptoms in a 2-week repeated oral administration toxicity test in a guinea pig model. Here, we also found that PMC203 induces autophagy in a dose dependent manner by increasing the signal of well-known autophagy gene markers, suggesting a possible intracellular killing mechanism.

Spatial resolution improved fluorescence lifetime imaging via deep learning.

We present a deep learning approach to obtain high-resolution (HR) fluorescence lifetime images from low-resolution (LR) images acquired from fluorescence lifetime imaging (FLIM) systems. We first proposed a theoretical method for training neural networks to generate massive semi-synthetic FLIM data with various cellular morphologies, a sizeable dynamic lifetime range, and complex decay components. We then developed a degrading model to obtain LR-HR pairs and created a hybrid neural network, the spatial resolution improved FLIM net (SRI-FLIMnet) to simultaneously estimate fluorescence lifetimes and realize the nonlinear transformation from LR to HR images. The evaluative results demonstrate SRI-FLIMnet's superior performance in reconstructing spatial information from limited pixel resolution. We also verified SRI-FLIMnet using experimental images of bacterial infected mouse raw macrophage cells. Results show that the proposed data generation method and SRI-FLIMnet efficiently achieve superior spatial resolution for FLIM applications. Our study provides a solution for fast obtaining HR FLIM images.

Tachyplesin and CyLoP-1 as efficient anti-mycobacterial peptides: A novel finding

Mycobacterium tuberculosis is an etiological agent of tuberculosis (TB) known to be a highly contagious disease and is the major cause of mortality from a single infectious agent worldwide. Emergence of multi-drug resistant and extremely drug resistant strains of M. tuberculosis has made TB management extremely challenging eliciting the urgent need for alternative therapeutics. Peptide based therapeutic strategies are an emerging area that can be employed as a prospective alternative to the currently existing therapeutic regime for TB treatment. Here, we are reporting the anti-mycobacterial activity of two peptides, Tachyplesin and CyLoP-1, derived from marine horseshoe crab and snake toxin respectively, with potent anti-mycobacterial activity against various mycobacterium species. Both the peptides exhibit appreciable antimicrobial and anti-biofilm activities against mycobacterium species with minimum cytotoxicity towards macrophage cells. They are also effective in eliminating mycobacterium cells from infected macrophage cells. Tachyplesin acts on mycobacterium cells in a lytic manner with outer membrane disruption confirmed by propidium iodide uptake with slight membrane depolarization and reactive oxygen species (ROS) production. CyLoP-1, on the other hand, does not rupture the mycobacterium cells even at high concentrations. It seems to follow intracellular pathway of killing mycobacterium cells by production of more ROS and membrane depolarization. Both the peptides do not lead to apoptotic way of mycobacterium cell death. These results suggest an effective peptide-based antimicrobial strategy for development of future anti-TB therapeutics.

Disease clearance of tuberculosis infection: An in-host continuous-time Markov chain model

The clearance of tuberculosis infection shows an elimination of infectious Mycobacterium tuberculosis (Mtb) pathogens and infected macrophage cells. The evidence shows the existence of individuals, who are still tested negative in tuberculin skin test after living with people with active tuberculosis for up to six months. Since the Mtb pathogen is spread from person to person through airborne particles, we build a continuous-time Markov chain (CTMC) model to describe the initial infection with small amount of inhaled bacteria. The CTMC model successfully simulates sample paths presenting disease clearance. We apply the theory of multitype branching processes to analytically approximate the probability of disease clearance. We also estimate the disease clearance time, which is as less than a month for R0∈[1,1.5]. Our results demonstrate that the host immune factors affect both the probability and the time of the disease clearance. These relationships are linked by the basic reproduction number R0. Our findings provide new mechanisms for disease prevention and therapy developments.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated “MKR superspreader”, and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR’s intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

The leishmanicidal effect of Lucilia sericata larval saliva and hemolymph on in vitro Leishmania tropica

BackgroundLeishmaniasis is a major parasitic disease worldwide, except in Australia and Antarctica, and it poses a significant public health problem. Due to the absence of safe and effective vaccines and drugs, researchers have begun an extensive search for new drugs. The aim of the current study was to investigate the in vitro leishmanicidal activity of larval saliva and hemolymph of Lucilia sericata on Leishmania tropica.MethodsThe effects of different concentrations of larval products on promastigotes and intracellular amastigotes of L. tropica were investigated using the mouse cell line J774A.1 and peritoneal macrophages as host cells. The 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct observation and counting method were used to assess the inhibitory effects and cell cytotoxicity of the larval products. The effects of larval products on the amastigote form of L. tropica were quantitatively estimated by calculating the rate of macrophage infection, number of amastigotes per infected macrophage cell, parasite load and survival index.ResultsThe 50% cytotoxicity concentration (CC50) value of both larval saliva and hemolymph was 750 µg/ml, and the 50% inhibitory concentration (IC50) values were 134 µg/ml and 60 µg/ml for larval saliva and larval hemolymph, respectively. The IC50 for Glucantime, used a positive control, was (11.65 µg/ml). Statistically significant differences in viability percentages of promastigotes were observed for different doses of both larval saliva and hemolymph when compared with the negative control (p ≤ 0.0001). Microscopic evaluation of the amastigote forms revealed that treatment with 150 µg/ml larval hemolymph and 450 µg/ml larval saliva significantly decreased the rate of macrophage infection and the number of amastigotes per infected macrophage cell.ConclusionLarval saliva and hemolymph of L. sericata have acceptable leishmanicidal properties against L. tropica.Graphical

Auranofin Has Advantages over First-Line Drugs in the Treatment of Severe Streptococcus suis Infections.

Streptococcal toxic shock-like syndrome (STSLS) likely occurs when an individual is infected with the Streptococcus suis (S. suis) epidemic strain and is characterized by a cytokine storm, multiple organ dysfunction syndrome (MODS) and a high incidence of mortality despite adequate treatment. A number of antibiotics exhibit excellent bactericidal effects in vivo, such as fluoroquinolones, aminoglycosides (gentamicin) and β-lactams (penicillin G, ceftiofur, or amoxicillin), but are less effective for treating STSLS. Therefore, there is an urgent need to identify new compounds that can reduce the damage caused by STSLS. In the present study, we identified auranofin, an orally bioavailable FDA-approved anti-rheumatic drug as a candidate repurposed drug to treat severe S. suis infections. Our results showed that auranofin can bind to the functional domain of bacterial thioredoxin reductase, decreasing the reducing redox-responsive capacity of target bacteria and allowing for the killing of S. suis cells. We also observed that auranofin has antibacterial activity against other gram-positive bacteria, such as multidrug resistant Streptococcus pneumoniae (MDRSP), Streptococcus agalactiae, and vancomycin-resistant strains of Staphylococcus aureus. Additionally, auranofin is capable of eradicating intracellular S.suis present inside infected macrophage cells. Mouse model experimental results showed that auranofin could effectively reduce the mortality of mice infected with S. suis. Compared to the ampicillin treatment group, the survival rate of mice in the auranofin treatment group in severely infected model mice was significantly improved. These results suggest that auranofin has the potential for use as an effective antibiotic against S. suis.

Comparison of transcriptional change of B. melitensis M5-90 after macrophage infection highlights the role of ribosome gene L31 in virulence

Brucella, a facultative intracellular bacterium, can survive and replicate in various cell types such as epithelial cell, fibroblasts and macrophage. Macrophage is the most important sites for the survival of Brucella in vivo. The mechanisms of pathogenesis are difficult to address, since the unknown virulence genes are still exist. RNA-seq is available to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Here we described and analyzed the transcriptional change of avirulent strain Brucella melitensis M5-90 (B. melitensis M5-90) during macrophage infection using RNA-seq technology. We detected 601 significant changed genes of which 428 were upregulated after infection. The upregulated gene L31 which involved in ribosome KEGG pathway was selected to illustrate its effect on virulence in a vaccine strain B. melitensis M5-90 and a virulent strain B. melitensis M28. Deletion of L31 significant attenuates the spleen colonization in model of M5-90 or M28 infection mouse at 7, 21 and 35 days post-infection (P < 0.05). We further examine the role of L31 in a macrophage cell infection model, and the result showed a significant reduction of intracellular M28ΔL31 cells at 48 h post-infection (P < 0.001). In total, our study provided a view of transcriptional landscape of B. melitensis M5-90 intracellular, and found L31 gene is required for the full virulence of B. melitensis.

Encephalitozoon intestinalis Infection Impacts the Expression of Apoptosis-Related Genes in U937 Macrophage Cells.

Encephalitozoon intestinalis affects many physiological processes of host cells to survive, proliferate, and spread to different regions within the body. In this study, the effects of the parasite on host cell apoptosis and proliferation were investigated. To determine the impact of the parasite on the host cell apoptosis, changes in the expression profile of genes were investigated with the qPCR array using the Human Apoptosis Panel in infected and non-infected macrophage cells. Also, the rate of apoptosis in the cells was determined by Giemsa staining method. Cell proliferation was determined by measuring the DNA concentration in infected and non-infected cells. The thirty-six of apoptosis-related genes were down-regulated, while 20 of apoptosis-related genes were up-regulated in infected cells compared to uninfected cells. However, there were no significant changes detected in 32 analyzed genes between infected and control groups. E. intestinalis was determined to decrease cell proliferation in U937 macrophage cells. Unexpectedly, Giemsa staining showed an increase in the rate of apoptosis in infected cells. Regulated genes after infection are involved in many different biological pathways and various components of the cell. This suggests that the parasite uses highly sophisticated ways to maintain the viability of the cell.

In vitro and in vivo antimicrobial activities of a novel piperazine-containing benzothiazinones candidate TZY-5-84 against Mycobacterium tuberculosis

A piperazine-containing benzothiazinones lead compound PBTZ169, served as DprE1 inhibitor, displays nanomolar bactericidal activity against Mycobacteria tuberculosis. Here, we systematically evaluate anti-tuberculosis activity of one of PBTZ169 analogues, TZY-5-84, in vitro and in vivo. The MIC value of TZY-5-84 against M. tuberculosis H37Rv ranged from 0.014 to 0.015 mg/L, lower than those of INH, RFP and BDQ. Five susceptible and thirteen drug-resistant clinical isolates were also susceptive to TZY-5-84. It had anti-tuberculosis activity against intracellular bacilli in infected macrophage model. It exhibited its activity in time-dependent manner and against intracellular bacilli in infected macrophage cells. However, the MIC of TZY-5-84 against three laboratory PBTZ169-induced resistant isolates increased four-fold increment compared to that of H37Rv. No antagonism was observed in any combination between TZY-5-84 and seven commonly used anti-tuberculosis drugs in an in vitro checkerboard assay. In murine infection model, TZY-5-84 at lower dosage (12.5 mg/kg) was found to be comparatively efficacious as PBTZ169 at 25 mg/kg. Our research suggests TZY-5-84 can be a promising preclinical candidate for further study on TB treatment.