Search

During infection, the host employs nutritional immunity to restrict access to iron. Staphylococcus lugdunensis has been recognized for its ability to utilize host-derived heme to overcome iron restriction. However, the mechanism behind this process involves the release of hemoglobin from erythrocytes, and the hemolytic factors of S. lugdunensis remain poorly understood. S. lugdunensis encodes four phenol-soluble modulins (PSMs), short peptides with hemolytic activity. The peptides SLUSH A, SLUSH B, and SLUSH C are β-type PSMs, and OrfX is an α-type PSM. Our study shows the SLUSH locus to be essential for the hemolytic phenotype of S. lugdunensis. All four peptides individually exhibited hemolytic activity against human and sheep erythrocytes, but synergism with sphingomyelinase was observed exclusively against sheep erythrocytes. Furthermore, our findings demonstrate that SLUSH is crucial for allowing the utilization of erythrocytes as the sole source of nutritional iron and confirm the transcriptional regulation of SLUSH by Agr. Additionally, our study reveals that SLUSH peptides stimulate the human immune system. Our analysis identifies SLUSH as a pivotal hemolytic factor of S. lugdunensis and demonstrates its concerted action with heme acquisition systems to overcome iron limitation in the presence of host erythrocytes., (© 2024 Federation of American Societies for Experimental Biology.)

The Staphylococcus sp. are a dominant part of the human skin microbiome and present across the body. Staphylococcus epidermidis is a ubiquitous skin commensal, while S. aureus is thought to colonize at least 30% of the population. S. aureus are not only colonizers but a leading cause of skin and soft tissue infections and a critical healthcare concern. To understand how healthy human skin may differentiate commensal bacteria, such as S. epidermidis, from the potential pathogen methicillin-resistant S. aureus (MRSA), we use ex vivo human skin models that allow us to study this host-bacterial interaction in the most clinically relevant environment. Our work highlights the role of the outer stratum corneum as a protective physical barrier against invasion by colonizing Staphylococci. We show how the structural cells of the skin can internalize and respond to different Staphylococci with increasing sensitivity. In intact human skin, a discriminatory IL-1β response was identified, while disruption of the protective stratum corneum triggered an increased and more diverse immune response. We identified and localized tissue resident Langerhans cells (LCs) as a potential source of IL-1β and go on to show a dose-dependent response of MUTZ-LCs to S. aureus but not S. epidermidis. This suggests an important role of LCs in sensing and discriminating between bacteria in healthy human skin, particularly in intact skin and provides a detailed snapshot of how human skin differentiates between friend and potential foe. With the rise in antibiotic resistance, understanding the innate immune response of healthy skin may help us find ways to enhance or manipulate these natural defenses to prevent invasive infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Macrophage metabolic plasticity is central to inflammatory programming, yet mechanisms of coordinating metabolic and inflammatory programs during infection are poorly defined. Here, we show that type I interferon (IFN) temporally guides metabolic control of inflammation during methicillin-resistant Staphylococcus aureus (MRSA) infection. We find that staggered Toll-like receptor and type I IFN signaling in macrophages permit a transient energetic state of combined oxidative phosphorylation (OXPHOS) and aerobic glycolysis followed by inducible nitric oxide synthase (iNOS)-mediated OXPHOS disruption. This disruption promotes type I IFN, suppressing other pro-inflammatory cytokines, notably interleukin-1β. Upon infection, iNOS expression peaks at 24 h, followed by lactate-driven Nos2 repression via histone lactylation. Type I IFN pre-conditioning prolongs infection-induced iNOS expression, amplifying type I IFN. Cutaneous MRSA infection in mice constitutively expressing epidermal type I IFN results in elevated iNOS levels, impaired wound healing, vasculopathy, and lung infection. Thus, kinetically regulated type I IFN signaling coordinates immunometabolic checkpoints that control infection-induced inflammation., Competing Interests: Declaration of interests C.A.L. has received consulting fees from Astellas Pharmaceuticals, Odyssey Therapeutics, and T-Knife Therapeutics and is an inventor on patents pertaining to Kras-regulated metabolic pathways, redox control pathways in pancreatic cancer, and targeting the GOT1 pathway as a therapeutic approach (US patent no. 2015126580-A1, May 7, 2015; US patent no. 20190136238, May 9, 2019; and international patent no. WO2013177426-A2, April 23, 2015). J.M.K. has received grant support from Q32 Bio, Celgene/BMS, Ventus Therapeutics, ROME Therapeutics, and Janssen. J.M.K. has served on advisory boards for AstraZeneca, Eli Lilly, GlaxoSmithKline, Gilead, Bristol Myers Squibb, Avion Pharmaceuticals, Provention Bio, Aurinia Pharmaceuticals, Ventus Therapeutics, and ROME Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ability of Staphylococcus aureus (S. aureus) to survive within macrophages is a critical strategy for immune evasion, contributing to the pathogenesis and progression of osteomyelitis. However, the underlying mechanisms remain poorly characterized. This study discovered that inhibiting the MEK1/2 pathway reduced bacterial load and mitigated bone destruction in a mouse model of S. aureus osteomyelitis. Histological staining revealed increased phosphorylated MEK1/2 levels in bone marrow macrophages surrounding abscess in the mouse model of S. aureus osteomyelitis. Activation of MEK1/2 pathway and its roles in impairing macrophage bactericidal function were confirmed in primary mouse bone marrow-derived macrophages (BMDMs). Transcriptome analysis and in vitro experiments demonstrated that S. aureus activates the MEK1/2 pathway through EGFR signaling. Moreover, we found that excessive activation of EGFR-MEK1/2 cascade downregulates mitochondrial reactive oxygen species (mtROS) levels by suppressing Chek2 expression, thereby impairing macrophage bactericidal function. Furthermore, pharmacological inhibition of EGFR signaling prevented upregulation of phosphorylated MEK1/2 and restored Chek2 expression in macrophages, significantly enhancing S. aureus clearance and improving bone microstructure in vivo. These findings highlight the critical role of the EGFR-MEK1/2 cascade in host immune defense against S. aureus, suggesting that S. aureus may reduce mtROS levels by overactivating the EGFR-MEK1/2 cascade, thereby suppressing macrophage bactericidal function. Therefore, combining EGFR-MEK1/2 pathway blockade with antibiotics could represent an effective therapeutic approach for the treatment of S. aureus osteomyelitis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Serious Staphylococcus aureus (SA) infection is one of the most life-threatening diseases. Interferon-induced protein 35 (IFP35) is a pleiotropic factor that participates in multiple biological functions, however, its biological role in SA infection is not fully understood. Ferroptosis is a new type of regulated cell death driven by the accretion of free iron and toxic lipid peroxides and plays critical roles in tissue damage. Whether ferroptosis is involved in SA-induced immunopathology and its regulatory mechanisms remain unknown., Objectives: We aimed to determine the role and underlying mechanisms of IFP35 in SA-induced lung infections., Methods: SA infection models were established using wild-type (WT) and IFP35 knockout (Ifp35 -/- ) mice or macrophages. Histological analysis was performed to assess lung injury. Quantitative real-time PCR, western blotting, flow cytometry, and confocal microscopy were performed to detect ferroptosis. Co-IP and immunofluorescence were used to elucidate the molecular regulatory mechanisms., Results: We found that IFP35 levels increased in the macrophages and lung tissue of SA-infected mice. IFP35 deficiency protected against SA-induced lung damage in mice. Moreover, ferroptosis occurred and contributed to lung injury after SA infection, which was ameliorated by IFP35 deficiency. Mechanically, IFP35 facilitated the ubiquitination and degradation of nuclear factor E2-related factor 2 (Nrf2), aggravating SA-induced ferroptosis and lung injury., Conclusions: Our data demonstrate that IFP35 promotes ferroptosis by facilitating the ubiquitination and degradation of Nrf2 to exacerbate SA infection. Targeting IFP35 may be a promising approach for treating infectious diseases caused by SA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Intracellular pathogens including Staphylococcus aureus contribute to the non-healing phenotype of chronic wounds. Lactobacilli, well known as beneficial bacteria, are also reported to modulate the immune system, yet their role in cutaneous immunity remains largely unknown. We explored the therapeutic potential of bacteria-free postbiotics, bioactive lysates of lactobacilli, to reduce intracellular S. aureus colonization and promote healing. Fourteen postbiotics derived from various lactobacilli species were screened, and Latilactobacillus curvatus BGMK2-41 was selected for further analysis based on the most efficient ability to reduce intracellular infection by S. aureus diabetic foot ulcer clinical isolate and S. aureus USA300. Treatment of both infected keratinocytes in vitro and infected human skin ex vivo with BGMK2-41 postbiotic cleared S. aureus. Keratinocytes treated in vitro with BGMK2-41 upregulated expression of antimicrobial response genes, of which DEFB4, ANG, and RNASE7 were also found upregulated in treated ex vivo human skin together with CAMP exclusively upregulated ex vivo. Furthermore, BGMK2-41 postbiotic treatment has a multifaceted impact on the wound healing process. Treatment of keratinocytes stimulated cell migration and the expression of tight junction proteins, while in ex vivo human skin BGMK2-41 increased expression of anti-inflammatory cytokine IL-10, promoted re-epithelialization, and restored the epidermal barrier via upregulation of tight junction proteins. Together, this provides a potential therapeutic approach for persistent intracellular S. aureus infections., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Craniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored., Methods: A mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity., Results: TNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected., Conclusion: These findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection., (© 2024. The Author(s).)

Methicillin-resistant Staphylococcus aureus (MRSA) infection, a major cause of hospital- and community-acquired pneumonia, still has a high mortality rate. Extracellular vesicles (EVs), as crucial mediators of intercellular communication, have a significant impact on infectious diseases. However, the role of EVs from alveolar macrophages (AMs) in MRSA pneumonia remains unclear. We report that AMs phagocytose MRSA and release more EVs in mice with MRSA pneumonia. EVs from AMs harboring phagocytosed MRSA exhibit significant proinflammatory effects and induce necroptosis by delivering tumor necrosis factor α (TNF-α) and miR-146a-5p. Mechanically, the upregulated miR-146a-5p in these EVs enhances the phosphorylation of RIPK1, RIPK3, and MLKL by targeting TNF receptor-associated factor 6 (TRAF6), thereby promoting TNF-α-induced necroptosis. The combination of a TNF-α antagonist and an miR-146a-5p antagomir effectively improves the outcomes of mice with MRSA pneumonia. Overall, we reveal the pronecrotic effect of EVs from MRSA-infected AMs and provide a promising target for the prevention and treatment of MRSA pneumonia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Skin/soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) pose a major healthcare burden. Distinct inflammatory and resolution phases comprise the host immune response to SSTIs. Resolution is a myeloid PPARγ-dependent anti-inflammatory phase that is essential for the clearance of MRSA. However, the signals activating PPARγ to induce resolution remain unknown. Here, we demonstrate that myeloid glucose transporter 1 (GLUT-1) is essential for the onset of resolution. MRSA-challenged macrophages are unsuccessful in generating an oxidative burst or immune radicals in the absence of GLUT-1 due to a reduction in the cellular NADPH pool. This translates in vivo as a significant reduction in lipid peroxidation products required for the activation of PPARγ in MRSA-infected mice lacking myeloid GLUT-1. Chemical induction of PPARγ during infection circumvents this GLUT-1 requirement and improves resolution. Thus, GLUT-1-dependent oxidative burst is essential for the activation of PPARγ and subsequent resolution of SSTIs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Age-associated impairments in innate immunity are believed to be a causative factor responsible for severe pathogenesis of Staphylococcus aureus (S. aureus) infection in the bone tissue. However, the basis for age-associated decline in innate immune response upon S. aureus infection remains poorly understood., Results: Our transcriptional data (GEO: GSE166522) from a mouse model of S. aureus osteomyelitis show up-regulated CXCL9 and CXCL10 (CXCL9/10), which is further confirmed in vitro and in vivo by the present study. Notably, monocytes are a main source for CXCL9/10 production in bone marrow upon S. aureus challenge, but this response declines in middle-aged mice. Interestingly, conditional medium of bone marrow monocytes from middle-aged mice has a strikingly decreased effect on bactericidal functions of neutrophils and macrophages compares with that from young mice. We further show that activation of CXCL9/10-CXCR3 axis between monocytes and macrophages/neutrophils promotes the bactericidal function of the cells, whereas blocking the axis impairs such function. Importantly, treatment with either exogenous CXCL9 or CXCL10 in a middle-aged mice model enhances, while pharmacological inhibition of CXCR3 in young mice model impairs, bacterial clearance and bone marrow structure., Conclusions: These findings demonstrate that bone marrow monocytes act as a critical promotor of innate immune response via the CXLCL9/10-CXCR3 axis upon S. aureus infection, and that the increased susceptibility to S. aureus infection in skeleton in an aged host may be largely attributable to the declined induction of CXCR9/10 in monocytes., (© 2024. The Author(s).)

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host., (© 2024. The Author(s).)

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection., (© 2024. The Author(s).)

Osteomyelitis is an invasive bone infection that can lead to severe pain and even disability, posing a challenge for orthopedic surgery. Naringin can reduce bone-related inflammatory conditions. This study aimed to elucidate the function and mechanism of naringin in a Staphylococcus aureus-induced mouse model of osteomyelitis. Femurs of S. aureus-infected mice were collected after naringin administration and subjected to microcomputed tomography to analyze cortical bone destruction and bone loss. Bacterial growth in femurs was also assessed. Proinflammatory cytokine levels in mouse femurs were measured using enzyme-linked immunosorbent assays. Pathological changes and bone resorption were analyzed using hematoxylin and eosin staining and tartrate-resistant acid phosphatase staining, respectively. Quantitative reverse transcription polymerase chain reaction and western blot analysis were used to quantify the messenger RNA and protein expression of osteogenic differentiation-associated genes in the femurs. The viability of human bone marrow-derived stem cells (hBMSCs) was determined using cell counting kit-8. Alizarin Red S staining and alkaline phosphatase staining were performed to assess the formation of mineralization nodules and bone formation in vitro. Notch signaling-related protein levels in femur tissues and hBMSCs were assessed using western blot analysis. Experimental results revealed that naringin alleviated S. aureus-induced cortical bone destruction and bone loss in mice by increasing the bone volume/total volume ratio. Naringin suppressed S. aureus-induced bacterial growth and inflammation in femurs. Moreover, it alleviated histopathological changes, inhibited bone resorption, and increased the expression of osteogenic markers in osteomyelitic mice. It increased the viability of hBMSCs and promoted their differentiation and bone mineralization in vitro. Furthermore, naringin activated Notch signaling by upregulating the protein levels of Notch1, Jagged1, and Hes1 in the femurs of model mice and S. aureus-stimulated hBMSCs. In conclusion, naringin reduces bacterial growth, inflammation, and bone resorption while upregulating the expression of osteogenic markers in S. aureus-infected mice and hBMSCs by activating Notch signaling., (© 2024 Wiley Periodicals LLC.)

Endometritis is one of the important causes of infertility. Puerarin (PU) can inhibit oxidative stress and reduce inflammation; however, it is unclear whether PU has a protective effect on the endometritis. In our study, we used Staphylococcus aureus to induce mouse endometritis. The PU group (100 mg/kg PU) and the S. aureus + PU group received daily intraperitoneal injection of PU (25, 50 or 100 mg/kg PU). The results showed that S. aureus significantly increased the levels of MPO, TNF-α, IL-1β and IL-6 in uterine tissue, and increased the expression of p-p65 and p-IκBα proteins in uterine tissue to induce endometritis in mice (p < 0.05). Furthermore, it has been found that S. aureus promotes the occurrence of ferroptosis by reducing GSH and ATP content, increasing MDA and iron content and reducing GPX4 and SLC7A11 protein expression levels (p < 0.05). S. aureus significantly increase the expression of NLRP3, ASC, caspase-1 and P2X7 proteins in uterine tissue (p < 0.05). However, PU obviously reduced the inflammatory response and reversed the changes of ferroptosis and the expression of P2X7 receptor/NLRP3 pathway associated proteins of the uterus induced by S. aureus (p < 0.05). Taken together, these findings emphasize the protective effect of PU on endometritis by regulating the P2X7 receptor/NLRP3 signalling pathway and inhibiting ferroptosis., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting the growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed the production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets the virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite Staphylococcus aureus (S. aureus) being a highly studied zoontic bacterium, its enteropathogenicity remains elusive. Herein, our findings demonstrated that S. aureus infection led to the accumulation of lipid droplets (LDs) in intestinal epithelial cells, accompanied by marked elevation inflammatory response that ultimately decreases intracellular bacterial load. The aforestated phenomenon may be partly attributed to the up-regulation of hypoxia-inducible lipid droplet-associated protein (HILPDA) and the concomitant down-regulation of cystathionine β-synthase (CBS) protein. Moreover, S. aureus infection up-regulated the expression of HILPDA, thereby promoting LDs accumulation, and down-regulated that of CBS, consequently inhibiting microsomal triglyceride transfer protein (MTTP) expression. This process may suppress the transport of LDs to the extracellular environment, further contributing to the formation of intracellular LDs. In summary, the results of this study provide significant insights into the intricate mechanisms through which the host organism combats pathogens and maintains the balance of sulfur and lipid metabolism. These findings not only enhance our understanding of the host's defense mechanisms but also offer promising avenues for the development of novel strategies to combat intestinal infectious diseases., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Staphylococcus aureus osteomyelitis leads to extensive bone destruction. Osteoclasts are bone resorbing cells that are often increased in bone infected with S. aureus. The cytokine RANKL is essential for osteoclast formation under physiological conditions but in vitro evidence suggests that inflammatory cytokines may by-pass the requirement for RANKL. The goal of this study was to determine whether RANKL-dependent osteoclast formation is essential for the bone loss that occurs in a murine model of S. aureus osteomyelitis. To this end, humanized-RANKL mice were infected by direct inoculation of S. aureus into a unicortical defect in the femur. Mice were treated with vehicle or denosumab, a human monoclonal antibody that inhibits RANKL, both before and during a 14-day infection period. The severe cortical bone destruction caused by infection was completely prevented by denosumab administration even though the bacterial burden in the femur was not affected. Osteoclasts were abundant near the inoculation site in vehicle-treated mice but absent in denosumab-treated mice. In situ hybridization demonstrated that S. aureus infection potently stimulated RANKL expression in bone marrow stromal cells. The extensive reactive bone formation that occurs in this osteomyelitis model was also reduced by denosumab administration. Lastly, there was a notable lack of osteoblasts near the infection site suggesting that the normal coupling of bone formation to bone resorption was disrupted by S. aureus infection. These results demonstrate that RANKL-mediated osteoclast formation is required for the bone loss that occurs in S. aureus infection and suggest that disruption of the coupling of bone formation to bone resorption may also contribute to bone loss in this condition., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Published by Elsevier Inc.)

Staphylococcus aureus ( S. aureus ) can evade antibiotics and host immune defenses by persisting within infected cells. Here, we demonstrate that in infected host cells, S. aureus type VII secretion system (T7SS) extracellular protein B (EsxB) interacts with the stimulator of interferon genes (STING) protein and suppresses the inflammatory defense mechanism of macrophages during early infection. The binding of EsxB with STING disrupts the K48-linked ubiquitination of EsxB at lysine 33, thereby preventing EsxB degradation. Furthermore, EsxB-STING binding appears to interrupt the interaction of 2 vital regulatory proteins with STING: aspartate-histidine-histidine-cysteine domain-containing protein 3 (DHHC3) and TNF receptor-associated factor 6. This persistent dual suppression of STING interactions deregulates intracellular proinflammatory pathways in macrophages, inhibiting STING's palmitoylation at cysteine 91 and its K63-linked ubiquitination at lysine 83. These findings uncover an immune-evasion mechanism by S. aureus T7SS during intracellular macrophage infection, which has implications for developing effective immunomodulators to combat S. aureus infections., Competing Interests: Competing interests statement:The authors declare no competing interest.

Disseminated intravascular coagulation (DIC) is considered to be the most common and lethal complication of sepsis. NLR-family pyrin domain-containing-3 (NLRP3) inflammasome plays an important role in host defense against microbial pathogens, and its deregulation may cause coagulation cascade and should be strictly managed. Here, we identified the deubiquitinase YOD1, which played a vital role in regulating coagulation in a NLRP3 inflammasome-dependent manner in sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA). YOD1 interacted with NLRP3 to remove K33-linked ubiquitination of NLRP3 based on its deubiquitinating enzyme activity and specifically inhibited expression of NLRP3 as well as activation of NLRP3 inflammasome. Deficiency of YOD1 expression enhanced NLRP3 inflammasome activation and coagulation both in vitro and in vivo. In addition, pharmacological inhibition of the NLRP3 effectively improved coagulation and alleviated organ injury in Yod1 -/- mice infected with MRSA. Thus, our study reported that YOD1 is a key regulator of coagulation during MRSA infection, and provided YOD1 as a potential therapeutic target for the treatment of NLRP3 inflammasome-related diseases, especially MRSA sepsis-induced DIC., (© 2024. The Author(s).)

The utilization of ATP within cells plays a fundamental role in cellular processes that are essential for the regulation of host-pathogen dynamics and the subsequent immune response. This study focuses on ATP-binding proteins to dissect the complex interplay between Staphylococcus aureus and human cells, particularly macrophages (THP-1) and keratinocytes (HaCaT), during an intracellular infection. A snapshot of the various protein activity and function is provided using a desthiobiotin-ATP probe, which targets ATP-interacting proteins. In S. aureus , we observe enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when located inside human cells. Additionally, the direct profiling of the protein activity revealed specific adaptations of S. aureus to the keratinocytes and macrophages. Mapping the differentially activated proteins to biochemical pathways in the human cells with intracellular bacteria revealed cell-type-specific adaptations to bacterial challenges where THP-1 cells prioritized immune defenses, autophagic cell death, and inflammation. In contrast, HaCaT cells emphasized barrier integrity and immune activation. We also observe bacterial modulation of host processes and metabolic shifts. These findings offer valuable insights into the dynamics of S. aureus -host cell interactions, shedding light on modulating host immune responses to S. aureus , which could involve developing immunomodulatory therapies., Importance: This study uses a chemoproteomic approach to target active ATP-interacting proteins and examines the dynamic proteomic interactions between Staphylococcus aureus and human cell lines THP-1 and HaCaT. It uncovers the distinct responses of macrophages and keratinocytes during bacterial infection. S. aureus demonstrated a tailored response to the intracellular environment of each cell type and adaptation during exposure to professional and non-professional phagocytes. It also highlights strategies employed by S. aureus to persist within host cells. This study offers significant insights into the human cell response to S. aureus infection, illuminating the complex proteomic shifts that underlie the defense mechanisms of macrophages and keratinocytes. Notably, the study underscores the nuanced interplay between the host's metabolic reprogramming and immune strategy, suggesting potential therapeutic targets for enhancing host defense and inhibiting bacterial survival. The findings enhance our understanding of host-pathogen interactions and can inform the development of targeted therapies against S. aureus infections., Competing Interests: The authors declare no conflict of interest.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection., (© 2024. The Author(s).)

Staphylococcus aureus ( S. aureus ) persistence in macrophages, potentially a reservoir for recurrence of chronic osteomyelitis, contributes to resistance and failure in treatment. As the mechanisms underlying survival of S. aureus in macrophages remain largely unknown, there has been no treatment approved. Here, in a mouse model of S. aureus osteomyelitis, we identified significantly up-regulated expression of SLC7A11 in both transcriptomes and translatomes of CD11b + F4/80 + macrophages, and validated a predominant distribution of SLC7A11 in F4/80 + cells around the S. aureus abscess. Importantly, pharmacological inhibition or genetic knockout of SLC7A11 promoted the bactericidal function of macrophages, reduced bacterial burden in the bone and improved bone structure in mice with S. aureus osteomyelitis. Mechanistically, aberrantly expressed SLC7A11 down-regulated the level of intracellular ROS and reduced lipid peroxidation, contributing to the impaired bactericidal function of macrophages. Interestingly, blocking SLC7A11 further activated expression of PD-L1 via the ROS-NF-κB axis, and a combination therapy of targeting both SLC7A11 and PD-L1 significantly enhanced the efficacy of clearing S. aureus in vitro and in vivo . Our findings suggest that targeting both SLC7A11 and PD-L1 is a promising therapeutic approach to reprogram the bactericidal function of macrophages and promote bacterial clearance in S. aureus osteomyelitis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Mastitis is a disease characterized by congestion, swelling, and inflammation of the mammary gland and usually caused by infection with pathogenic microorganisms. Furthermore, the development of mastitis is closely linked to the exogenous pathway of the gastrointestinal tract. However, the regulatory mechanisms governing the gut-metabolism-mammary axis remain incompletely understood. The present study revealed alterations in the gut microbiota of mastitis rats characterized by an increased abundance of the Proteobacteria phylum. Plasma analysis revealed significantly higher levels of L-isoleucine and cholic acid along with 7-ketodeoxycholic acid. Mammary tissue showed elevated levels of arachidonic acid metabolites and norlithocholic acid. Proteomic analysis showed increased levels of IFIH1, Tnfaip8l2, IRGM, and IRF5 in mastitis rats, which suggests that mastitis triggers an inflammatory response and immune stress. Follistatin (Fst) and progesterone receptor (Pgr) were significantly downregulated, raising the risk of breast cancer. Extracellular matrix (ECM) receptors and focal adhesion signaling pathways were downregulated, while blood-milk barrier integrity was disrupted. Analysis of protein-metabolic network regulation revealed that necroptosis, protein digestion and absorption, and arachidonic acid metabolism were the principal regulatory pathways involved in the development of mastitis. In short, the onset of mastitis leads to changes in the microbiota and alterations in the metabolic profiles of various biological samples, including colonic contents, plasma, and mammary tissue. Key manifestations include disturbances in bile acid metabolism, amino acid metabolism, and arachidonic acid metabolism. At the same time, the integrity of the blood-milk barrier is compromised while inflammation is promoted, thereby reducing cell adhesion in the mammary glands. These findings contribute to a more comprehensive understanding of the metabolic status of mastitis and provide new insights into its impact on the immune system., (© 2024 Federation of American Societies for Experimental Biology.)

The exacerbation of pneumonia in children with human adenovirus type 3 (HAdV-3E) is secondary to a Staphylococcus aureus ( S. aureus ) infection. The influence of host-pathogen interactions on disease progression remains unclear. It is important to note that S. aureus infections following an HAdV-3E infection are frequently observed in clinical settings, yet the underlying susceptibility mechanisms are not fully understood. This study utilized an A549 cell model to investigate secondary infection with S. aureus following an HAdV-3E infection. The findings suggest that HAdV-3E exacerbates the S. aureus infection by intensifying lung epithelial cell damage. The results highlight the role of HAdV-3E in enhancing the interferon signaling pathway through RIG-I ( DDX58 ), resulting in the increased expression of interferon-stimulating factors like MX1 , RSAD2 , and USP18 . The increase in interferon-stimulating factors inhibits the NF-κB and MAPK/P38 pro-inflammatory signaling pathways. These findings reveal new mechanisms of action for HAdV-3E and S. aureus in secondary infections, enhancing our comprehension of pathogenesis.

Staphylococcus aureus is one of the leading causes of hospital-acquired infections, many of which begin following attachment and accumulation on indwelling medical devices or diseased tissue. These infections are often linked to the establishment of biofilms, but another often overlooked key characteristic allowing S. aureus to establish persistent infection is the formation of planktonic aggregates. Such aggregates are physiologically similar to biofilms and protect pathogens from innate immune clearance and increase antibiotic tolerance. The cell-wall-associated protein SasG has been implicated in biofilm formation via mechanisms of intercellular aggregation but the mechanism in the context of disease is largely unknown. We have previously shown that the expression of cell-wall-anchored proteins involved in biofilm formation is controlled by the ArlRS-MgrA regulatory cascade. In this work, we demonstrate that the ArlRS two-component system controls aggregation, by repressing the expression of sasG by activation of the global regulator MgrA. We also demonstrate that SasG must be proteolytically processed by a non-staphylococcal protease to induce aggregation and that strains expressing functional full-length sasG aggregate significantly upon proteolysis by a mucosal-derived host protease found in human saliva. We used fractionation and N-terminal sequencing to demonstrate that human trypsin within saliva cleaves within the A domain of SasG to expose the B domain and induce aggregation. Finally, we demonstrated that SasG is involved in virulence during mouse lung infection. Together, our data point to SasG, its processing by host proteases, and SasG-driven aggregation as important elements of S. aureus adaptation to the host environment.IMPORTANCEHere, we demonstrate that the Staphylococcus aureus surface protein SasG is important for cell-cell aggregation in the presence of host proteases. We show that the ArlRS two-component regulatory system controls SasG levels through the cytoplasmic regulator MgrA. We identified human trypsin as the dominant protease triggering SasG-dependent aggregation and demonstrated that SasG is important for S. aureus lung infection. The discovery that host proteases can induce S. aureus aggregation contributes to our understanding of how this pathogen establishes persistent infections. The observations in this study demonstrate the need to strengthen our knowledge of S. aureus surface adhesin function and processing, regulation of adhesin expression, and the mechanisms that promote biofilm formation to develop strategies for preventing chronic infections., Competing Interests: The authors declare no conflict of interest.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE -dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage- Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange., Competing Interests: The authors declare no conflict of interest.

Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis., (© 2023 International Federation of Cell Biology.)

Staphylococcus aureus , a bacterium found on human skin, produces toxins and various virulence factors that can lead to skin infections such as atopic dermatitis. These toxins and virulence factors are carried in membrane vesicles (MVs), composed of the bacterium's own cell membranes, and are expected to reach host target cells in a concentrated form, inducing inflammation. This study investigated the effects of two polyphenols, (-)-epigallocatechin gallate (EGCG) and nobiletin (NOL), on the expression of S. aureus virulence factors and the inflammation induced by MVs. The study found that EGCG alone decreased the production of Staphylococcal Enterotoxin A (SEA), while both EGCG and NOL reduced biofilm formation and the expression of virulence factor-related genes. When S. aureus was cultured in a broth supplemented with these polyphenols, the resulting MVs showed a reduction in SEA content and several cargo proteins. These MVs also exhibited decreased levels of inflammation-related gene expression in immortalized human keratinocytes. These results suggest that EGCG and NOL are expected to inhibit inflammation in the skin by altering the properties of MVs derived from S. aureus .

Staphylococcus aureus skin colonization and eosinophil infiltration are associated with many inflammatory skin disorders, including atopic dermatitis, bullous pemphigoid, Netherton's syndrome, and prurigo nodularis. However, whether there is a relationship between S. aureus and eosinophils and how this interaction influences skin inflammation is largely undefined. We show in a preclinical mouse model that S. aureus epicutaneous exposure induced eosinophil-recruiting chemokines and eosinophil infiltration into the skin. Remarkably, we found that eosinophils had a comparable contribution to the skin inflammation as T cells, in a manner dependent on eosinophil-derived IL-17A and IL-17F production. Importantly, IL-36R signaling induced CCL7-mediated eosinophil recruitment to the inflamed skin. Last, S. aureus proteases induced IL-36α expression in keratinocytes, which promoted infiltration of IL-17-producing eosinophils. Collectively, we uncovered a mechanism for S. aureus proteases to trigger eosinophil-mediated skin inflammation, which has implications in the pathogenesis of inflammatory skin diseases., Competing Interests: Competing interests statement:N.K.A. has received previous grant support from Pfizer and Boehringer Ingelheim and was a paid consultant for Janssen Pharmaceuticals. The remaining authors state no conflict of interest.

Importance: Staphylococcus aureus uses numerous strategies to survive and persist in the intracellular environment of professional phagocytes, including modulation of the SUMOylation process. This study aims to understand how S. aureus alters host SUMOylation to enhance its intracellular survival in professional phagocytes. Our results indicate that S. aureus strain Newman utilizes PtpA-driven phosphorylation to decrease the amount of SUMOylated proteins in murine macrophages to facilitate its survival in this immune cell type., Competing Interests: The authors declare no conflict of interest.

Importance: Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3', 5'-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus infection is associated with increased levels of neutrophil extracellular traps (NETs) and von Willebrand factor (VWF), and with reduced activity of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Peptidylarginine deiminase 4 (PAD4) contributes to NET formation and inactivates ADAMTS13 in vitro . The role of PADs in the dynamics of NETs, VWF and ADAMTS13 has not yet been studied. We thus aimed to assess the longitudinal evolution of NETs, PADs, VWF and ADAMTS13 activity in S. aureus infection. Plasma samples from S. aureus bacteraemia patients were longitudinally collected and analysed for NETs, PAD4/PAD2, VWF and ADAMTS13 activity. Correlation analyses with clinical data were performed. Recombinant PAD4 and S. aureus were assessed in vitro for their potential to modulate ADAMTS13 activity. Sixty-seven patients were included. Plasma levels of NETs, VWF, PAD4 and PAD2 were increased and ADAMTS13 activity was decreased. Levels of PADs were negatively correlated with ADAMTS13 activity. NETs were positively correlated with PADs, and negatively with ADAMTS13 activity. In vitro , recombinant PAD4 but not S. aureus reduced ADAMTS13 activity in plasma. Levels of PAD4 and PAD2 correlate with reduced ADAMTS13 activity, with neutrophils as the likely source of PAD activity in S. aureus bacteraemia. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Staphylococcus aureus strains are a great contributor to both hospital acquired infections as well as community acquired infections. The objective of the present investigation was to compare potential differences in cytoplasmic amino acid levels between clinical and ATCC 29213 strains of S. aureus. The two strains were grown under ideal conditions to mid-exponential and stationary growth phases, after which they were harvested to analyze their amino acid profiles. Initially, the amino acid patterns of both strains were compared at the mid-exponential phase when grown in controlled conditions. At the mid-exponential phase, both strains shared common features in cytoplasmic amino acid levels, with glutamic acid, aspartic acid, proline, and alanine identified as key amino acids. However, the concentration profiles of seven amino acids exhibited major variances between the strains, even though the total cytoplasmic levels of amino acids did not alter significantly. At the stationary phase, the magnitudes of the amino acids abundant in the mid-exponential phase were altered. Aspartic acid became the most abundant amino acid in both strains accounting for 44% and 59% of the total amino acids in the clinical and ATCC 29213 strains, respectively. Lysine was the second most abundant amino acid in both strains, accounting for 16% of the total cytoplasmic amino acids, followed by glutamic acid, the concentration of which was significantly higher in the clinical strain than in the ATCC 29213 strain. Interestingly, histidine was clearly present in the clinical strain but was virtually lacking in the ATCC 29213 strain. This study reveals the dynamic diversity of amino acid levels among strains, which is an essential step toward illustrating the variability in S. aureus cytoplasmic amino acid profiles and could be significant in explaining variances among strains of S. aureus.

Importance: Miscommunication of antiviral and antibacterial immune signals drives worsened morbidity and mortality during respiratory viral-bacterial coinfections. Extracellular vesicles (EVs) are a form of intercellular communication with broad implications during infection, and here we show that epithelium-derived EVs released during the antiviral response impair the antibacterial activity of macrophages, an innate immune cell crucial for bacterial control in the airway. Macrophages exposed to antiviral EVs display reduced clearance of Staphylococcus aureus as well as altered inflammatory signaling and anti-inflammatory metabolic reprogramming, thus revealing EVs as a source of dysregulated epithelium-macrophage crosstalk during coinfection. As effective epithelium-macrophage communication is critical in mounting an appropriate immune response, this novel observation of epithelium-macrophage crosstalk shaping macrophage metabolism and antimicrobial function provides exciting new insight and improves our understanding of immune dysfunction during respiratory coinfections., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both S. aureus strains, with most ( n = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups ( n = 237) of clonally related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater in vivo purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo ., Competing Interests: This work was supported by grants from Vertex Pharmaceuticals (to S.J.S.).

Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. β-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with β-lactam resistance. In this study, structure-defined β-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the β-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of β-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that β-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Previously considered as an exclusive extracellular bacterium, Staphylococcus aureus has been shown to be able to invade many cells in vitro and in humans. Once inside the host cell, both cytosolic and endosome-associated S. aureus strongly induce macroautophagy/autophagy. Whether autophagy fosters S. aureus intracellular survival or clearance remains unclear. The YAP1-TEAD axis regulates the expression of target genes controlling the cell fate ( e.g ., proliferation, migration, cell cycle …). Growing evidence indicates that YAP1-TEAD also regulates autophagy and lysosomal pathways. Recently we showed that the YAP1-TEAD axis promotes autophagy and lysosome biogenesis to restrict S. aureus intracellular replication. We also discovered that the C3 exoenzyme-like EDIN-B toxin produced by the pathogenic S. aureus ST80 strain inhibits YAP1 nuclear translocation resulting in a strong increase of intracellular S. aureus burden.

Staphylococcus epidermidis (S. epidermidis) is the most common nosocomial pathogen in preterm infants and associated with increased risk of cognitive delay, however, underlying mechanisms are unknown. We employed morphological, transcriptomic and physiological methods to extensively characterize microglia in the immature hippocampus following S. epidermidis infection. 3D morphological analysis revealed activation of microglia after S. epidermidis. Differential expression combined with network analysis identified NOD-receptor signaling and trans-endothelial leukocyte trafficking as major mechanisms in microglia. In support, active caspase-1 was increased in the hippocampus and using the LysM-eGFP knock-in transgenic mouse, we demonstrate infiltration of leukocytes to the brain together with disruption of the blood-brain barrier. Our findings identify activation of microglia inflammasome as a major mechanism underlying neuroinflammation following infection. The results demonstrate that neonatal S. epidermidis infection share analogies with S. aureus and neurological diseases, suggesting a previously unrecognized important role in neurodevelopmental disorders in preterm born children., (© 2023 The Authors. GLIA published by Wiley Periodicals LLC.)

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of complicated urinary tract infection (UTI) associated with the use of indwelling urinary catheters. Previous reports have revealed host and pathogen effectors critical for MRSA uropathogenesis. Here, we sought to determine the significance of specific metabolic pathways during MRSA UTI. First, we identified four mutants from the Nebraska transposon mutant library in the MRSA JE2 background that grew normally in rich medium but displayed significantly reduced growth in pooled human urine (HU). This prompted us to transduce the uropathogenic MRSA 1369 strain with the transposon mutants in sucD and fumC (tricarboxylic acid [TCA] cycle), mtlD (mannitol metabolism), and lpdA (pyruvate oxidation). Notably, sucD , fumC , and mtlD were also significantly upregulated in the MRSA 1369 strain upon exposure to HU. Compared to the WT, the MRSA 1369 lpdA mutant was significantly defective for (i) growth in HU, and (ii) colonization of the urinary tract and dissemination to the kidneys and the spleen in the mouse model of catheter-associated UTI (CAUTI), which may be attributed to its increased membrane hydrophobicity and higher susceptibility to killing by human blood. In contrast to their counterparts in the JE2 background, the sucD , fumC , and mtlD mutants in the MRSA 1369 background grew normally in HU; however, they displayed significant fitness defects in the CAUTI mouse model. Overall, identification of novel metabolic pathways important for the urinary fitness and survival of MRSA can be used for the development of novel therapeutics. IMPORTANCE While Staphylococcus aureus has historically not been considered a uropathogen, S. aureus urinary tract infection (UTI) is clinically significant in certain patient populations, including those with chronic indwelling urinary catheters. Moreover, most S. aureus strains causing catheter-associated UTI (CAUTI) are methicillin-resistant S. aureus (MRSA). MRSA is difficult to treat due to limited treatment options and the potential to deteriorate into life-threatening bacteremia, urosepsis, and shock. In this study, we found that pathways involved in pyruvate oxidation, TCA cycle, and mannitol metabolism are important for MRSA fitness and survival in the urinary tract. Improved understanding of the metabolic needs of MRSA in the urinary tract may help us develop novel inhibitors of MRSA metabolism that can be used to treat MRSA-CAUTI more effectively., Competing Interests: The authors declare no conflict of interest.

α-Hemolysin (Hla) is a potent pore-forming toxin (PFT) produced by Staphylococcus aureus that exacerbates the pathogenesis of S. aureus enterotoxicity and plays a role in population food poisoning. Hla lyses cells by binding to host cell membranes and oligomerizing to form heptameric structures, thereby disrupting the cell barrier. Although the broad bactericidal effect of electron beam irradiation (EBI) has been demonstrated whether it has a damaging or degrading effect on Hla's remains unknown. In this study, EBI was found to have the effect of altering the secondary structure of Hla proteins, verifying that the damaging effect of EBI-treated Hla on intestinal and skin epithelial cell barriers was significantly reduced. It was noted by hemolysis and protein interactions that EBI treatment significantly disrupted the binding of Hla to its high-affinity receptor, but did not affect the binding between Hla monomers to form heptamers. Thus, EBI can effectively reduce the threat of Hla to food safety., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is an important cause of chronic infections resulting from the failure of the host to eliminate the pathogen. Effective S. aureus clearance requires CD4+ T cell-mediated immunity. We previously showed that myeloid-derived suppressor cells (MDSC) expand during staphylococcal infections and support infection chronicity by inhibiting CD4+ T cell responses. The aim of this study was to elucidate the mechanisms underlying the suppressive effect exerted by MDSC on CD4+ T cells during chronic S. aureus infection. It is well known that activated CD4+ T cells undergo metabolic reprogramming from oxidative metabolism to aerobic glycolysis to meet their increased bioenergetic requirements. In this process, pyruvate is largely transformed into lactate by lactate dehydrogenase with the concomitant regeneration of NAD+, which is necessary for continued glycolysis. The by-product lactate needs to be excreted to maintain the glycolytic flux. Using SCENITH (single-cell energetic metabolism by profiling translation inhibition), we demonstrated here that MDSC inhibit CD4+ T cell responses by interfering with their metabolic activity. MDSC are highly glycolytic and excrete large amount of lactate in the local environment that alters the transmembrane concentration gradient and prevent removal of lactate by activated CD4+ T. Accumulation of endogenous lactate impedes the regeneration of NAD+, inhibit NAD-dependent glycolytic enzymes and stop glycolysis. Together, the results of this study have uncovered a role for metabolism on MDSC suppression of CD4+ T cell responses. Thus, reestablishment of their metabolic activity may represent a mean to improve the functionality of CD4+ T cells during chronic S. aureus infection., (© 2023. The Author(s).)

Overexpression of Staphylococcus aureus mediated CXCL8/CXCR1 axis is a major cause of sepsis and severe inflammatory diseases. This chemokine acts conjointly with various pro-inflammatory and anti-inflammatory cytokines that govern the severity of inflammation. The effects of different combinations of exogenous cytokines on CXCR1 expression in macrophages remain undetermined. Exogenous cytokine and anti-inflammatory cytokine therapy had been used to modulate CXCL8 and CXCR1 expression in peritoneal macrophages. Male Swiss albino mice were inoculated with live S. aureus (10 6 cells/ mouse) for the development of infection. Exogenous cytokines (TNF-α, IL-12, IFN-γ and IL-10) were administered intraperitoneally (single or combination) 24 h post S. aureus infection. The mice were sacrificed and peritoneal macrophages were isolated three days post infection. CXCL8, IL-12, IL-10 secretion, ROS generation and the bacterial phagocytic process had been evaluated. Western blot was used to study the expressions of TNFR1, IL-1R, CXCR1 and NF-κB. TNF-α, IL-12 and IFN-γ treatments aggravated CXCL8 and CXCR1 expression in the macrophages of infected mice. TNF-α + IFN-γ treatment was a major inducer of nitric oxide release and mediated maximum bacterial killing. IL-12 + TNF-α treatment was most potent in increasing ROS, CXCL8/CXCR1 expression through increased levels of TNFR1, IL-1R and NF-κB activation. IL-10 reversed the effects of exogenous cytokines but also impaired the bacterial clearance phenomenon in peritoneal lavage. Treatment with IL-12 + TNF-α + IL-10 was most effective in ameliorating oxidative stress, reduced CXCL8 release and expression levels of TNFR1, IL-1R, and NF-κB. Concludingly, IL-12 + TNF-α + IL-10 treatment mitigated CXCL8/CXCR1 expression and inflammatory signalling via downregulation of TNFR1-IL-1R-NF-κB pathway in peritoneal macrophages and inflammatory sequelae during S. aureus infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

To effectively prevent and control bovine mastitis, farmers and their advisors need to take infection pathways and durations into account. Still, studies exploring both aspects through molecular epidemiology with sampling of entire dairy cow herds over longer periods are scarce. Therefore, quarter foremilk samples were collected at 14-d intervals from all lactating dairy cows (n = 263) over 18 wk in one commercial dairy herd. Quarters were considered infected with Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae when ≥100 cfu/mL of the respective pathogen was detected, or with Staphylococcus epidermidis or Staphylococcus haemolyticus when ≥500 cfu/mL of the respective pathogen was detected. All isolates of the mentioned species underwent randomly amplified polymorphic DNA (RAPD)-PCR to explore strain diversity and to distinguish ongoing from new infections. Survival analysis was used to estimate infection durations. Five different strains of Staph. aureus were isolated, and the most prevalent strain caused more than 80% of all Staph. aureus infections (n = 46). In contrast, 46 Staph. epidermidis and 69 Staph. haemolyticus strains were isolated, and none of these caused infections in more than 2 different quarters. The 3 most dominant strains of Strep. dysgalactiae (7 strains) and Strep. uberis (18 strains) caused 81% of 33 and 49% of 37 infections in total, respectively. The estimated median infection duration for Staph. aureus was 80 d, and that for Staph. epidermidis and Staph. haemolyticus was 28 and 22 d, respectively. The probability of remaining infected with Strep. dysgalactiae or Strep. uberis for more than 84 and 70 d was 58.7 and 53.5%, respectively. Staphylococcus epidermidis and Staph. haemolyticus were not transmitted contagiously and the average infection durations were short, which brings into question whether antimicrobial treatment of intramammary infections with these organisms is justified. In contrast, infections with the other 3 pathogens lasted longer and largely originated from contagious transmission., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Currently, there is no effective therapy for Staphylococcus aureus-induced osteomyelitis. It is widely recognized that the inflammatory microenvironment around abscess plays an essential role in protracting the course of S. aureus-induced osteomyelitis. In this study, we found TWIST1 was highly expressed in macrophages around abscesses but less related to local S. aureus in the later stages of Staphylococcus aureus-infected osteomyelitis. Mouse bone marrow macrophages show apoptosis and elevated TWIST1 expression when treated with the inflammatory medium. Knockdown of TWIST1 induced macrophage apoptosis, impaired the bacteria phagocytosis/killing abilities, and promoted cell apoptosis markers expression in inflammatory microenvironment stimulation. Furthermore, inflammatory microenvironments were responsible for inducing calcium overload in macrophage mitochondrial while calcium overload inhibition significantly rescued macrophage apoptosis, bacteria phagocytosis/killing abilities and improved the mice's antimicrobial ability. Our findings indicated that TWIST1 is a crucial molecule that protects macrophages from calcium overload induced by inflammatory microenvironments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Early and accurate diagnosis of infection-induced osteomyelitis, which often involves increased PD-L1 expression, is crucial for better treatment outcomes. Radiolabeled anti-PD-L1 nuclear imaging allows for sensitive and non-invasive whole-body assessments of PD-L1 expression. This study aimed to compare the efficacy of 18 F-FDG and an 18 F-labeled PD-L1-binding peptide probe ( 18 F-PD-L1P) in PET imaging of implant-associated Staphylococcus aureus osteomyelitis (IAOM)., Methods: In this study, we synthesized an anti-PD-L1 probe and compared its efficacy with 18 F-FDG and 18 F-PD-L1P in PET imaging of implant-associated Staphylococcus aureus osteomyelitis (IAOM). The %ID/g ratios (i.e., radioactivity ratios between the infected and non-infected sides) of both probes were evaluated for sensitivity and accuracy in post-infected 7-day tibias and post-infected 21 days, and the intensity of 18 F-PD-L1P uptake was compared with pathological changes measured by PD-L1 immunohistochemistry (IHC)., Results: Compared with 18 F-FDG, 18 F-PDL1P demonstrated higher %ID/g ratios for both post-infected 7-day tibias (P=0.001) and post-infected 21 days (P=0.028). The intensity of 18 F-PD-L1P uptake reflected the pathological changes of osteomyelitic bones. In comparison to 18 F-FDG, 18 F-PDL1P provides earlier and more sensitive detection of osteomyelitis caused by S. aureus., Conclusion: Our findings suggest that the 18 F-PDL1P probe is a promising tool for the early and accurate detection of osteomyelitis caused by S. aureus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ren, Ma, Fu, Hu, Liang, Yu and Tang.)

Background: Secoeudesma sesquiterpenes lactone A (SESLA) is a sesquiterpene derived from Inula japonica Thunb. and is known to possess many pharmacological properties, e.g. anti-tumor and anti-inflammatory activities. However, the immunomodulatory role of SESLA in gram-positive (G + ) bacterial infection is not clear., Materials and Methods: To set up a G + bacterial infection model in vitro , we carried out a bacterial mimic (PGN or Pam3CSK4) or Methicillin-resistant Staphylococcus aureus (MRSA) stimulated experiment using macrophages or dendritic cells (DCs). ELISA and qPCR were performed to measure the expression of inflammatory cytokines. Flow cytometry was used to detect the expression of MHC II and co-stimulatory molecules on the surface of DCs. The network pharmacology was used to identify the molecular mechanism and potential targets of SESLA that are predicted to be involved in the MRSA-elicited inflammation. Western blot and dual luciferase reporter assay were adopted to certify possible molecular mechanism of SESLA., Results: This study demonstrated that SESLA treatment significantly reduced the levels of inflammatory cytokines stimulated by PGN, Pam3CSK4 or even MRSA in vitro , and it also reduced PGN-induced expression of MHC II and co-stimulatory molecules on the surface of DCs. Mechanistically, the inhibition of IκBα phosphorylation and the suppression of T cells activation could account for its anti-inflammatory activity., Conclusion: The present study validated the notable anti-inflammatory activity of SESLA and discovered its previously uncharacterized immunoregulatory role and the underlying mechanism in G + bacterial infections. Overall, SESLA has a potential to be an antibiotic adjuvant for the treatment of G + bacterial infections.

Peptidoglycan hydrolases, or autolysins, play a critical role in cell wall remodeling and degradation, facilitating bacterial growth, cell division, and cell separation. In Staphylococcus aureus, the so-called "major" autolysin, Atl, has long been associated with host adhesion; however, the molecular basis underlying this phenomenon remains understudied. To investigate, we used the type V glycopeptide antibiotic complestatin, which binds to peptidoglycan and blocks the activity of autolysins, as a chemical probe of autolysin function. We also generated a chromosomally encoded, catalytically inactive variant of the Atl enzyme. Autolysin-mediated peptidoglycan hydrolysis, in particular Atl-mediated daughter cell separation, was shown to be critical for maintaining optimal surface levels of S. aureus cell wall-anchored proteins, including the fibronectin-binding proteins (FnBPs) and protein A (Spa). As such, disrupting autolysin function reduced the affinity of S. aureus for host cell ligands, and negatively impacted early stages of bacterial colonization in a systemic model of S. aureus infection. Phenotypic studies revealed that Spa was sequestered at the septum of complestatin-treated cells, highlighting that autolysins are required to liberate Spa during cell division. In summary, we reveal the hydrolytic activities of autolysins are associated with the surface display of S. aureus cell wall-anchored proteins. We demonstrate that by blocking autolysin function, type V glycopeptide antibiotics are promising antivirulence agents for the development of strategies to control S. aureus infections.

Staphylococcus aureus infections of bone tissue are associated with inflammatory bone loss. Resident bone cells, including osteoblasts and osteoclasts, can perceive S. aureus and produce an array of inflammatory and pro-osteoclastogenic mediators, thereby contributing to such damage. The neuropeptide substance P (SP) has been shown to exacerbate microbially induced inflammation at sites such as the gut and the brain and has previously been shown to affect bone cell differentiation and activity. Here we demonstrate that the interaction of SP with its high affinity receptor, neurokinin-1 receptor (NK-1R), expressed on murine osteoblasts and osteoclasts, augments the inflammatory responses of these cells to S. aureus challenge. Additionally, SP alters the production of pro- and anti-osteoclastogenic factors by bacterially challenged bone cells and their proteolytic functions in a manner that would be anticipated to exacerbate inflammatory bone loss at sites of infection. Furthermore, we have demonstrated that the clinically approved NK-1R antagonist, aprepitant, attenuates local inflammatory and pro-osteoclastogenic mediator expression in an in vivo mouse model of post-traumatic staphylococcal osteomyelitis. Taken together, these results indicate that SP/NK-1R interactions could play a significant role in the initiation and/or progression of damaging inflammation in S. aureus bone infections and suggest that the repurposing of currently approved NK-1R antagonists might represent a promising new adjunct therapy for such conditions., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)