Comparative Evaluation of the Antimicrobial and Mucus Induction Properties of Selected Bacillus Strains against Enterotoxigenic Escherichia coli.

Probiotics have been shown to bind to host receptors, which are important for pathogen adhesion and induce the host's production of defence factors. They can activate the goblet-cell-derived production of mucins, a major component of the mucus layer and a physical barrier participating in limiting the proximity of microorganisms to the epithelial layer. In the last decade, Bacillus spp. strains have gained interest in human and animal health due to their tolerance and stability under gastrointestinal tract conditions. Moreover, Bacillus spp. strains can also produce various antimicrobial peptides that can support their use as commercial probiotic supplements and functional foods. The present study aimed to evaluate and determine the ability of selected Bacillus spp. strains to inhibit the growth of enterotoxigenic Escherichia coli (ETEC) F4 and to reduce binding of ETEC F4 to HT29-16E (mucus-secreting and goblet-like) human intestinal cells. Moreover, mucus production in the HT29 cells in the presence of the Bacillus spp. strains was quantified by ELISA. Bacillus spp. strains (CHCC 15076, CHCC 15516, CHCC 15541, and CHCC 16872) significantly inhibited the growth of ETEC F4. Moreover, the ability of the probiotic Bacillus spp. strains to stimulate mucin release was highly strain dependent. The treatment with Bacillus subtilis CHCC 15541 resulted in a significant increase of both MUC2 and MUC3 in HT29-16E cells. Therefore, this strain could be an up-and-coming candidate for developing commercial probiotic supplements to prevent infections caused by ETEC F4 and, potentially, other pathogens.

The extracellular thioredoxin Etrx3 is required for macrophage infection in Rhodococcus equi.

Rhodococcus equi is an intracellular veterinary pathogen that is becoming resistant to current antibiotherapy. Genes involved in preserving redox homeostasis could be promising targets for the development of novel anti-infectives. Here, we studied the role of an extracellular thioredoxin (Etrx3/REQ_13520) in the resistance to phagocytosis. An etrx3-null mutant strain was unable to survive within macrophages, whereas the complementation with the etrx3 gene restored its intracellular survival rate. In addition, the deletion of etrx3 conferred to R. equi a high susceptibility to sodium hypochlorite. Our results suggest that Etrx3 is essential for the resistance of R. equi to specific oxidative agents.

Host-Targeted Therapeutics against Multidrug Resistant Intracellular Staphylococcus aureus.

Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of human cells. S. aureus has shown to rapidly overcome traditional antibiotherapy by developing multidrug resistance. Furthermore, intracellular S. aureus is protected from the last-resort antibiotics-vancomycin, daptomycin, and linezolid-as they are unable to achieve plasma concentrations sufficient for intracellular killing. Therefore, there is an urgent need to develop novel anti-infective therapies against S. aureus infections. Here, we review the current state of the field and highlight the exploitation of host-directed approaches as a promising strategy going forward.

Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi.

Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectives.

Identification of novel targets for host-directed therapeutics against intracellular Staphylococcus aureus.

During patient colonization, Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin. Hijacking specific host molecular factors and/or pathways is necessary for pathogens to successfully establish an intracellular infection. In this study, we employed an unbiased shRNA screening coupled with ultra-fast sequencing to screen 16,000 human genes during S. aureus infection and we identified several host genes important for this intracellular pathogen. In addition, we interrogated our screening results to find novel host-targeted therapeutics against intracellular S. aureus. We found that silencing the human gene TRAM2 resulted in a significant reduction of intracellular bacterial load while host cell viability was restored, showing its importance during intracellular infection. Furthermore, TRAM2 is an interactive partner of the endoplasmic reticulum SERCA pumps and treatment with the SERCA-inhibitor Thapsigargin halted intracellular MRSA survival. Our results suggest that Thapsigargin could be repurposed to tackle S. aureus host cell infection in combination with conventional antibiotics.

Intracellular Staphylococcus aureus Elicits the Production of Host Very Long-Chain Saturated Fatty Acids with Antimicrobial Activity.

As a facultative intracellular pathogen, Staphylococcus aureus is able to invade and proliferate within many types of mammalian cells. Intracellular bacterial replication relies on host nutrient supplies and, therefore, cell metabolism is closely bound to intracellular infection. Here, we investigated how S. aureus invasion affects the host membrane-bound fatty acids. We quantified the relative levels of fatty acids and their labelling pattern after intracellular infection by gas chromatography-mass spectrometry (GC-MS). Interestingly, we observed that the levels of three host fatty acids-docosanoic, eicosanoic and palmitic acids-were significantly increased in response to intracellular S. aureus infection. Accordingly, labelling carbon distribution was also affected in infected cells, in comparison to the uninfected control. In addition, treatment of HeLa cells with these three fatty acids showed a cytoprotective role by directly reducing S. aureus growth.

Host-directed kinase inhibitors act as novel therapies against intracellular Staphylococcus aureus.

Host-directed therapeutics are a promising anti-infective strategy against intracellular bacterial pathogens. Repurposing host-targeted drugs approved by the FDA in the US, the MHRA in the UK and/or regulatory equivalents in other countries, is particularly interesting because these drugs are commercially available, safe doses are documented and they have been already approved for other clinical purposes. In this study, we aimed to identify novel therapies against intracellular Staphylococcus aureus, an opportunistic pathogen that is able to exploit host molecular and metabolic pathways to support its own intracellular survival. We screened 133 host-targeting drugs and found three host-directed tyrosine kinase inhibitors (Ibrutinib, Dasatinib and Crizotinib) that substantially impaired intracellular bacterial survival. We found that Ibrutinib significantly increased host cell viability after S. aureus infection via inhibition of cell invasion and intracellular bacterial proliferation. Using phosphoproteomics data, we propose a putative mechanism of action of Ibrutinib involving several host factors, including EPHA2, C-JUN and NWASP. We confirmed the importance of EPHA2 for staphylococcal infection in an EPHA2-knock-out cell line. Our study serves as an important example of feasibility for identifying host-directed therapeutics as candidates for repurposing.

Acetaminophen cytotoxicity in HepG2 cells is associated with a decoupling of glycolysis from the TCA cycle, loss of NADPH production, and suppression of anabolism.

Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. We found that APAP exposure leads to limited cellular death and substantial growth inhibition. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. Co-treatment with a specific inhibitor of the lactate/H+ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth.

Intracellular Staphylococcus aureus Modulates Host Central Carbon Metabolism To Activate Autophagy.

Staphylococcus aureus is a facultative intracellular pathogen that invades and replicates within many types of phagocytic and nonphagocytic cells. During intracellular infection, S. aureus is capable of subverting xenophagy and escaping to the cytosol of the host cell. Furthermore, drug-induced autophagy facilitates the intracellular replication of S. aureus, but the reasons behind this are unclear. Here, we have studied the host central carbon metabolism during S. aureus intracellular infection. We found extensive metabolic rerouting and detected several distinct metabolic changes that suggested starvation-induced autophagic flux in infected cells. These changes included increased uptake but lower intracellular levels of glucose and low abundance of several essential amino acids, as well as markedly upregulated glutaminolysis. Furthermore, we show that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) phosphorylation levels are significantly increased in infected cells. Interestingly, while autophagy was activated in response to S. aureus invasion, most of the autophagosomes detected in infected cells did not contain bacteria, suggesting that S. aureus induces the autophagic flux during cell invasion for energy generation and nutrient scavenging. Accordingly, AMPK inhibition halted S. aureus intracellular proliferation.IMPORTANCEStaphylococcus aureus escapes from immune recognition by invading a wide range of human cells. Once the pathogen becomes intracellular, the most important last resort antibiotics are not effective. Therefore, novel anti-infective therapies against intracellular S. aureus are urgently needed. Here, we have studied the physiological changes induced in the host cells by S. aureus during its intracellular proliferation. This is important, because the pathogen exploits the host cell's metabolism for its own proliferation. We find that S. aureus severely depletes glucose and amino acid pools, which leads to increased breakdown of glutamine by the host cell in an attempt to meet its own metabolic needs. All of these metabolic changes activate autophagy in the host cell for nutrient scavenging and energy generation. The metabolic activation of autophagy could be used by the pathogen to sustain its own intracellular survival, making it an attractive target for novel anti-infectives.