Yersinia pseudotuberculosis doxycycline tolerance strategies include modulating expression of genes involved in cell permeability and tRNA modifications

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by TusB, and the resulting impacts on translational machinery, may play an important role in promoting tolerance. We believe this is the first observation of bactericidal activity of doxycycline, which was revealed by reversing tusB downregulation.

Doxycycline promotes proteasome fitness in the central nervous system

Several studies reported that mitochondrial stress induces cytosolic proteostasis in yeast and C. elegans. Notably, inhibition of mitochondrial translation with doxcycyline decreases the toxicity of β-amyloid aggregates, in a C. elegans. However, how mitochondrial stress activates cytosolic proteostasis remains unclear. Further whether doxycycline has this effect in mammals and in disease relevant tissues also remains unclear. We show here that doxycycline treatment in mice drastically reduces the accumulation of proteins destined for degradation by the proteasome in a CNS region-specific manner. This effect is associated with the activation of the ERα axis of the mitochondrial unfolded protein response (UPRmt), in both males and females. However, sexually dimorphic mechanisms of proteasome activation were observed. Doxycycline also activates the proteasome in fission yeast, where ERα is not expressed. Rather, the ancient ERα-coactivator Mms19 regulates this response in yeast. Our results suggest that the UPRmt initiates a conserved mitochondria-to-cytosol stress signal, resulting in proteasome activation, and that this signal has adapted during evolution, in a sex and tissue specific-manner. Therefore, while our results support the use of doxycycline in the prevention of proteopathic diseases, they also indicate that sex is an important variable to consider in the design of future clinical trials using doxycycline.

Doxycycline Interferes with Zika Virus Serine Protease and Inhibits Virus Replication in Human Skin Fibroblasts

Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (−7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.

Endoplasmic reticulum−mitochondria coupling increases during doxycycline-induced mitochondrial stress in HeLa cells

Subcellular organelles communicate with each other to regulate function and coordinate responses to changing cellular conditions. The physical-functional coupling of the endoplasmic reticulum (ER) with mitochondria allows for the direct transfer of Ca2+ between organelles and is an important avenue for rapidly increasing mitochondrial metabolic activity. As such, increasing ER−mitochondrial coupling can boost the generation of ATP that is needed to restore homeostasis in the face of cellular stress. The mitochondrial unfolded protein response (mtUPR) is activated by the accumulation of unfolded proteins in mitochondria. Retrograde signaling from mitochondria to the nucleus promotes mtUPR transcriptional responses aimed at restoring protein homeostasis. It is currently unknown whether the changes in mitochondrial−ER coupling also play a role during mtUPR stress. We hypothesized that mitochondrial stress favors an expansion of functional contacts between mitochondria and ER, thereby increasing mitochondrial metabolism as part of a protective response. Hela cells were treated with doxycycline, an antibiotic that inhibits the translation of mitochondrial-encoded proteins to create protein disequilibrium. Treatment with doxycycline decreased the abundance of mitochondrial encoded proteins while increasing expression of CHOP, C/EBPβ, ClpP, and mtHsp60, markers of the mtUPR. There was no change in either mitophagic activity or cell viability. Furthermore, ER UPR was not activated, suggesting focused activation of the mtUPR. Within 2 h of doxycycline treatment, there was a significant increase in physical contacts between mitochondria and ER that was distributed throughout the cell, along with an increase in the kinetics of mitochondrial Ca2+ uptake. This was followed by the rise in the rate of oxygen consumption at 4 h, indicating a boost in mitochondrial metabolic activity. In conclusion, an early phase of the response to doxycycline-induced mitochondrial stress is an increase in mitochondrial−ER coupling that potentiates mitochondrial metabolic activity as a means to support subsequent steps in the mtUPR pathway and sustain cellular adaptation.

The effect of doxycycline and collagenase A on the differentiation of mesenchymal stem cells towards a nucleus pulposus phenotype

Intervertebral disc (IVD) degeneration costs the healthcare system billions of dollars annually and leads to reduced quality of life. Current treatments are invasive and primarily focus on symptom relief rather than repair. This study aimed to facilitate the development of an injectable therapy using chondrogenically differentiated mesenchymal stem cells (MSCs) in the absence of collagen II deposition. Briefly, pelleted MSCs were cultivated in chondrogenic medium and were supplemented with collagenase A or doxycycline in order to inhibit collagen assembly. Results indicated that collagenase A and doxycycline treatment had no negative effects on DNA or proteoglycan content. Collagenase A at all concentrations affected collagen content, as did doxycycline at low concentrations. Furthermore, preliminary gene expression studies for nucleus pulposus markers showed that collagenase A and doxycycline may have some effect on terminal differentiation of MSCs in chondrogenic medium. Overall, the findings suggest that collagenase A and doxycycline supplementation can be used to inhibit collagen formation, thereby facilitating the further development of an injectable therapy for IVD repair.

The effect of doxycycline and collagenase A on the differentiation of mesenchymal stem cells towards a nucleus pulposus phenotype

Intervertebral disc (IVD) degeneration costs the healthcare system billions of dollars annually and leads to reduced quality of life. Current treatments are invasive and primarily focus on symptom relief rather than repair. This study aimed to facilitate the development of an injectable therapy using chondrogenically differentiated mesenchymal stem cells (MSCs) in the absence of collagen II deposition. Briefly, pelleted MSCs were cultivated in chondrogenic medium and were supplemented with collagenase A or doxycycline in order to inhibit collagen assembly. Results indicated that collagenase A and doxycycline treatment had no negative effects on DNA or proteoglycan content. Collagenase A at all concentrations affected collagen content, as did doxycycline at low concentrations. Furthermore, preliminary gene expression studies for nucleus pulposus markers showed that collagenase A and doxycycline may have some effect on terminal differentiation of MSCs in chondrogenic medium. Overall, the findings suggest that collagenase A and doxycycline supplementation can be used to inhibit collagen formation, thereby facilitating the further development of an injectable therapy for IVD repair.

Perinatal exposure to tetracycline contributes to lasting developmental effects on offspring

Background For more than 30 years, the tetracycline on/off system of inducible gene expression has been leveraged to study disease mechanisms across many research areas, especially that of metabolism and neuroscience. This system requires acute or chronic exposure to tetracycline derivatives, such as doxycycline, to manipulate gene expression in a temporal and tissue-specific manner, with exposure often being restricted to gestational and early developmental windows. Despite evidence showing that early life antibiotic exposure has adverse effects on gut microbiota, metabolism, physiology, immunity and behavior, little is known regarding the lasting impact of doxycycline treatment on relevant outcomes in experimental offspring. Results To examine the hypothesis that early life doxycycline exposure produces effects on offspring growth, behavior, and gut microbiota, we employed the most commonly used method for tetracycline on/off system by administering a low dose of doxycycline (0.5 mg/ml) in the drinking water to C57Bl/6J and C57BL/6J:129S1/SvImJ dams from embryonic day 15.5 to postnatal day 28. Developmental exposure to low dose doxycycline resulted in significant alterations to growth trajectories and body weight in both strains, which persisted beyond cessation of doxycycline exposure. Developmental doxycycline exposure influenced offspring bacterial community assembly in a temporal and sex-specific manner. Further, gut microbiota composition failed to recover by adulthood, suggesting a lasting imprint of developmental antibiotic exposure. Conclusions Our results demonstrated that early life doxycycline exposure shifts the homeostatic baseline of prior exposed animals that may subsequently impact responses to experimental manipulations. These results highlight the gut microbiota as an important factor to consider in systems requiring methods of chronic antibiotic administration during pregnancy and critical periods of postnatal development.

Effect of Doxycycline on Survival in Abdominal Aortic Aneurysms in a Mouse Model

Background. Currently, there is no reliable nonsurgical treatment for abdominal aortic aneurysm (AAA). This study, therefore, investigates if doxycycline reduces AAA growth and the number of rupture-related deaths in a murine ApoE−/− model of AAA and whether gadofosveset trisodium-based MRI differs between animals with and without doxycycline treatment. Methods. Nine ApoE−/− mice were implanted with osmotic minipumps continuously releasing angiotensin II and treated with doxycycline (30 mg/kg/d) in parallel. After four weeks, MRI was performed at 3T with a clinical dose of the albumin-binding probe gadofosveset (0.03 mmol/kg). Results were compared with previously published wild-type control animals and with previously studied ApoE−/− animals without doxycycline treatment. Differences in mortality were also investigated between these groups. Results. In a previous study, we found that approximately 25% of angiotensin II-infused ApoE−/− mice died, whereas in the present study, only one out of 9 angiotensin II-infused and doxycycline-treated ApoE−/− mice (11.1%) died within 4 weeks. Furthermore, doxycycline-treated ApoE−/− mice showed significantly lower contrast-to-noise (CNR) values ( p = 0.017 ) in MRI compared to ApoE−/− mice without doxycycline treatment. In vivo measurements of relative signal enhancement (CNR) correlated significantly with ex vivo measurements of albumin staining (R2 = 0.58). In addition, a strong visual colocalization of albumin-positive areas in the fluorescence albumin staining with gadolinium distribution in LA-ICP-MS was shown. However, no significant difference in aneurysm size was observed after doxycycline treatment. Conclusion. The present experimental in vivo study suggests that doxycycline treatment may reduce rupture-related deaths in AAA by slowing endothelial damage without reversing aneurysm growth.