The sirtuins SIRT3 and SIRT5 are the main mitochondrial lysine deacetylase and desuccinylase, respectively. SIRT3 and SIRT5 regulate metabolism and redox homeostasis and have been involved in ...age-associated metabolic, neurologic and oncologic diseases. We have previously shown that single deficiency in either SIRT3 or SIRT5 had no impact on host defenses in a large panel of preclinical models of sepsis. However, SIRT3 and SIRT5 may compensate each other considering that they share subcellular location and targets. Here, we generated a SIRT3/5 double knockout mouse line. SIRT3/5 deficient mice multiplied and developed without abnormalities. Hematopoiesis and immune cell development were largely unaffected in SIRT3/5 deficient mice. Whole blood, macrophages and neutrophils from SIRT3/5 deficient mice displayed enhanced inflammatory and bactericidal responses. In agreement, SIRT3/5 deficient mice showed somewhat improved resistance to
infection. Overall, the double deficiency in SIRT3 and SIRT5 has rather subtle impacts on immune cell development and anti-microbial host defenses unseen in single deficient mice, indicating a certain degree of overlap between SIRT3 and SIRT5. These data support the assumption that therapies directed against mitochondrial sirtuins, at least SIRT3 and SIRT5, should not impair antibacterial host defenses.
Sirtuin 2 (SIRT2) and SIRT3 are cytoplasmic and mitochondrial NAD-dependent deacetylases. SIRT2 and SIRT3 target proteins involved in metabolic, proliferation and inflammation pathways and have been ...implicated in the pathogenesis of neurodegenerative, metabolic and oncologic disorders. Both pro- and anti-inflammatory effects have been attributed to SIRT2 and SIRT3, and single deficiency in SIRT2 or SIRT3 had minor or no impact on antimicrobial innate immune responses. Here, we generated a SIRT2/3 double deficient mouse line to study the interactions between SIRT2 and SIRT3. SIRT2/3
mice developed normally and showed subtle alterations of immune cell populations in the bone marrow, thymus, spleen, blood and peritoneal cavity that contained notably more anti-inflammatory B-1a cells and less NK cells.
, SIRT2/3
macrophages favored fatty acid oxidation (FAO) over glycolysis and produced increased levels of both proinflammatory and anti-inflammatory cytokines. In line with metabolic adaptation and increased numbers of peritoneal B-1a cells, SIRT2/3
mice were robustly protected from endotoxemia. Yet, SIRT2/3 double deficiency did not modify endotoxin tolerance. Overall, these data suggest that sirtuins can act in concert or compensate each other for certain immune functions, a parameter to be considered for drug development. Moreover, inhibitors targeting multiple sirtuins developed for clinical purposes may be useful to treat inflammatory diseases.
Trained immunity refers to the ability of the innate immune system exposed to a first challenge to provide an enhanced response to a secondary homologous or heterologous challenge. We reported that ...training induced with β-glucan one week before infection confers protection against a broad-spectrum of lethal bacterial infections. Whether this protection persists over time is unknown. To tackle this question, we analyzed the immune status and the response to
(
) of mice trained 9 weeks before analysis. The induction of trained immunity increased bone marrow myelopoiesis and blood counts of Ly6C
inflammatory monocytes and polymorphonuclear neutrophils (PMNs).
, whole blood, PMNs and monocytes from trained mice produced increased levels of cytokines in response to microbial products and limited the growth of
.
, following challenge with
, peripheral blood leukocytes were massively depleted in control mice but largely preserved in trained mice. PMNs were reduced also in the spleen from control mice, and increased in the spleen of trained mice. In transwell experiments, PMNs from trained mice showed increased spontaneous migration and CXCL2/MIP2α-induced chemotaxis, suggesting that training promotes the migration of PMNs in peripheral organs targeted by
. Trained PMNs and monocytes had higher glycolytic activity and mitochondrial respiration than control cells when exposed to
. Bacterial burden and dissemination in blood, spleen and liver as well as systemic cytokines and inflammation (multiplex bead assay and bioluminescence imaging) were reduced in trained mice. In full agreement with these results, mice trained 9 weeks before infection were powerfully protected from lethal listeriosis. Altogether, these data suggest that training increases the generation and the antimicrobial activity of PMNs and monocytes, which may confer prolonged protection from lethal bacterial infection.
Sirtuin 3 (SIRT3) is the main mitochondrial deacetylase. SIRT3 regulates cell metabolism and redox homeostasis, and protects from aging and age-associated pathologies. SIRT3 may drive both oncogenic ...and tumor-suppressive effects. SIRT3 deficiency has been reported to promote chronic inflammation-related disorders, but whether SIRT3 impacts on innate immune responses and host defenses against infections remains essentially unknown. This aspect is of primary importance considering the great interest in developing SIRT3-targeted therapies. Using SIRT3 knockout mice, we show that SIRT3 deficiency does not affect immune cell development and microbial ligand-induced proliferation and cytokine production by splenocytes, macrophages and dendritic cells. Going well along with these observations, SIRT3 deficiency has no major impact on cytokine production, bacterial burden and survival of mice subjected to endotoxemia, Escherichia coli peritonitis, Klebsiella pneumoniae pneumonia, listeriosis and candidiasis of diverse severity. These data suggest that SIRT3 is not critical to fight infections and support the safety of SIRT3-directed therapies based on SIRT3 activators or inhibitors for treating metabolic, oncologic and neurodegenerative diseases without putting patients at risk of infection.
Sirtuin 2 (SIRT2) is one of the seven members of the family of NAD
-dependent histone deacetylases. Sirtuins target histones and non-histone proteins according to their subcellular localization, ...influencing various biological processes. SIRT2 resides mainly in the cytoplasm and regulates cytoskeleton dynamics, cell cycle, and metabolic pathways. As such, SIRT2 has been implicated in the pathogenesis of neurodegenerative, metabolic, oncologic, and chronic inflammatory disorders. This motivated the development of SIRT2-directed therapies for clinical purposes. However, the impact of SIRT2 on antimicrobial host defense is largely unknown. Here, we address this question using SIRT2 knockout mice. We show that SIRT2 is the most highly expressed sirtuin in myeloid cells, especially macrophages. SIRT2 deficiency does not affect immune cell development and marginally impacts on intracellular signaling and cytokine production by splenocytes and macrophages. However, SIRT2 deficiency enhances bacterial phagocytosis by macrophages. In line with these observations, in preclinical models, SIRT2 deficiency increases survival of mice with chronic staphylococcal infection, while having no effect on the course of toxic shock syndrome toxin-1, LPS or TNF-induced shock, fulminant
peritonitis, sub-lethal
pneumonia, and chronic candidiasis. Altogether, these data support the safety profile of SIRT2 inhibitors under clinical development in terms of susceptibility to infections.
Sirtuin 5 (SIRT5) is a member of the family of NAD
-dependent lysine/histone deacetylases. SIRT5 resides mainly in the mitochondria where it catalyzes deacetylation, demalonylation, desuccinylation, ...and deglutarylation of lysine to regulate metabolic and oxidative stress response pathways. Pharmacologic inhibitors of SIRT5 are under development for oncologic conditions, but nothing is known about the impact of SIRT5 on antimicrobial innate immune defenses. Using SIRT5 knockout mice, we show that SIRT5 deficiency does not affect immune cell development, cytokine production and proliferation by macrophages and splenocytes exposed to microbial and immunological stimuli. Moreover, preclinical models suggest that SIRT5 deficiency does not worsen endotoxemia,
and
pneumonia,
peritonitis, listeriosis, and staphylococcal infection. Altogether, these data support the safety profile in terms of susceptibility to infections of SIRT5 inhibitors under development.
The rapid emergence of antibiotic-resistant bacteria poses a serious threat to public health worldwide. Antimicrobial peptides (AMPs) are promising antibiotic alternatives; however, little is known ...about bacterial mechanisms of AMP resistance and the interplay between AMP resistance and the bacterial response to other antimicrobials. In this study, we identified Escherichia coli mutants resistant to the TAT-RasGAP317-326 antimicrobial peptide and found that resistant bacteria show collateral sensitivity to other AMPs and antibacterial agents. We determined that resistance to TAT-RasGAP317-326 peptide arises through mutations in the histidine kinase EnvZ, a member of the EnvZ/OmpR two-component system responsible for osmoregulation in E. coli. In particular, we found that TAT-RasGAP317-326 binding and entry is compromised in E. coli peptide-resistant mutants. We showed that peptide resistance is associated with transcriptional regulation of a number of pathways and EnvZ-mediated resistance is dependent on the OmpR response regulator but is independent of the OmpC and OmpF outer membrane porins. Our findings provide insight into the bacterial mechanisms of TAT-RasGAP317-326 resistance and demonstrate that resistance to this AMP is associated with collateral sensitivity to other antibacterial agents. IMPORTANCE Antimicrobial peptides (AMP) are promising alternatives to classical antibiotics in the fight against antibiotic resistance. Resistance toward antimicrobial peptides can occur, but little is known about the mechanisms driving this phenomenon. Moreover, there is limited knowledge on how AMP resistance relates to the bacterial response to other antimicrobial agents. Here, we address these questions in the context of the antimicrobial peptide TAT-RasGAP317-326. We show that resistant Escherichia coli strains can be selected and do not show resistance to other antimicrobial agents. Resistance is caused by a mutation in a regulatory pathway, which lowers binding and entry of the peptide in E. coli. Our results highlight a mechanism of resistance that is specific to TAT-RasGAP317-326. Further research is required to characterize these mechanisms and to evaluate the potential of antimicrobial combinations to curb the development of antimicrobial resistance.
Antibiotic resistance is an increasing threat for public health, underscoring the need for new antibacterial agents. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics. ...TAT-RasGAP317-326 is a recently described AMP effective against a broad range of bacteria, but little is known about the conditions that may influence its activity. Using RNA-sequencing and screening of mutant libraries, we show that Escherichia coli and Pseudomonas aeruginosa respond to TAT-RasGAP317-326 by regulating metabolic and stress response pathways, possibly implicating two-component systems. Our results also indicate that bacterial surface properties, in particular integrity of the lipopolysaccharide layer, influence peptide binding and entry. Finally, we found differences between bacterial species with respect to their rate of resistance emergence against this peptide. Our findings provide the basis for future investigation on the mode of action of TAT-RasGAP317-326, which may help developing antimicrobial treatments based on this peptide.
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•Antimicrobial activity of TAT-RasGAP317-327 is affected by medium composition•Peptide exposure induces metabolic and stress responses•Cell surface modifications influence peptide antimicrobial activity•Resistance to TAT-RasGAP317-327 peptide does not lead to multidrug resistance
Biochemistry; Peptides; Microbiology
Abstract
Background
The innate immune system recalls a challenge to adapt to a secondary challenge, a phenomenon called trained immunity. Training involves cellular metabolic, epigenetic and ...functional reprogramming, but how broadly trained immunity protects from infections is unknown. For the first time, we addressed whether trained immunity provides protection in a large panel of preclinical models of infections.
Methods
Mice were trained and subjected to systemic infections, peritonitis, enteritis, and pneumonia induced by Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Citrobacter rodentium, and Pseudomonas aeruginosa. Bacteria, cytokines, leukocytes, and hematopoietic precursors were quantified in blood, bone marrow, and organs. The role of monocytes/macrophages, granulocytes, and interleukin 1 signaling was investigated using depletion or blocking approaches.
Results
Induction of trained immunity protected mice in all preclinical models, including when training and infection were initiated in distant organs. Trained immunity increased bone marrow hematopoietic progenitors, blood Ly6Chigh inflammatory monocytes and granulocytes, and sustained blood antimicrobial responses. Monocytes/macrophages and interleukin 1 signaling were required to protect trained mice from listeriosis. Trained mice were efficiently protected from peritonitis and listeriosis for up to 5 weeks.
Conclusions
Trained immunity confers broad-spectrum protection against lethal bacterial infections. These observations support the development of trained immunity-based strategies to improve host defenses.
We used preclinical models to demonstrate that trained immunity confers broad-spectrum protection against bacterial infections. Trained immunity increased myeloid progenitors and circulating inflammatory monocytes and neutrophils, and depletion or neutralization of monocytes/macrophages and interleukin 1 signaling impaired trained immunity-mediated protection.
Lactobacillus algidus is a meat spoilage bacterium often dominating the bacterial communities on chilled, packaged meat. Yet, L. algidus strains are rarely recovered from meat, and only few studies ...have focused on this species. The main reason limiting detailed studies on L. algidus is related to its poor growth on the media routinely used for culturing food spoilage bacteria. Thus, our study sought to develop reliable culture media for L. algidus to enable its recovery from meat, and to allow subculturing and phenotypic analyses of the strains. We assessed the growth of meat-derived L. algidus strains on common culture media and their modifications, and explored the suitability of potential media for the recovery of L. algidus from meat. Moreover, we determined whether 12 meat-derived L. algidus strains selected from our culture collection produce biogenic amines that may compromise safety or quality of meat, and finally, sequenced de novo and annotated the genomes of two meat-derived L. algidus strains to uncover genes and metabolic pathways relevant for phenotypic traits observed. MRS agar supplemented with complex substances (peptone, meat and yeast extract, liver digest) supported the growth of L. algidus, and allowed the recovery of new L. algidus isolates from meat. However, most strains grew poorly on standard MRS agar and on general-purpose media. In MRS broth, most strains grew well but a subset of strains required supplementation of MRS broth with additional cysteine. Supplementation of MRS broth with catalase allowed growth in aerated cultures suggesting that the strains produced hydrogen peroxide when grown aerobically. The strains tested (n = 12) produced ornithine from arginine and putrescine from agmatine, and two strains produced tyramine from tyrosine. Our findings reveal that L. algidus populations are underestimated if routine culture protocols are applied, and prompt concerns that L. algidus may generate tyramine or putrescine in meat or fermented meat products.
•MRS fortified with complex substances supports the growth of Lactobacillus algidus.•Most strains grew poorly on standard MRS and on general-purpose media.•Catalase in MRS broth allowed growth in aerated cultures.•L. algidus produced ornithine, putrescine and tyramine in vitro.•Genome analysis supported results obtained for biogenic amine production.
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