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TCA cycle inactivation enhances stationary-phase survival. Single colonies of SA564 and SA564-acnA::ermB were inoculated into TSB, grown at 37°C, and aerated by being shaken at 225 rpm for up to 2 weeks. At 24-h intervals, aliquots were removed and CFU per milliliter were determined in quadruplicate. The data presented are averages and standard deviations.
Source publication
Staphylococcus aureus preferentially catabolizes glucose, generating pyruvate, which is subsequently oxidized to acetate under aerobic growth conditions. Catabolite repression of the tricarboxylic acid (TCA) cycle results in the accumulation of acetate. TCA cycle derepression coincides with exit from the exponential growth phase, the onset of aceta...
Citations
... The other type of SCVs, thymidine auxotrophs, are resistant to sulfa antibiotics (trimethoprim-sulfamethoxazole) as a result of disruption of the tetrahydrofolic acid pathway [143]. Thymidine auxotrophy results in reduced ClpC activity, which is needed to activate aconitase, an enzyme involved in the intraconversion of the tricarboxylic acids citrate, cis-aconitate, and isocitrate in the TCA cycle [144]. Hence the reduced TCA activity results in a SCV similar to menadione and hemin auxotrophs [143,145]. ...
Staphylococcus aureus bacteremia continues to be associated with significant morbidity and mortality, despite improvements in diagnostics and management. Persistent infections pose a major challenge to clinicians and have been consistently shown to increase the risk of mortality and other infectious complications. S. aureus, while typically not considered an intracellular pathogen, has been proven to utilize an intracellular niche, through several phenotypes including small colony variants, as a means for survival that has been linked to chronic, persistent, and recurrent infections. This intracellular persistence allows for protection from the host immune system and leads to reduced antibiotic efficacy through a variety of mechanisms. These include antimicrobial resistance, tolerance, and/or persistence in S. aureus that contribute to persistent bacteremia. This review will discuss the challenges associated with treating these complicated infections and the various methods that S. aureus uses to persist within the intracellular space.
... Collectively, these data suggest that Δagr increases respiration and fermentation to compensate for low metabolic efficiency. Consistent with this idea, agr deficiency also increases ATP-yielding carbon 'overflow' pathways, as evidenced by increased acetate production ( Figure 5B; Sadykov et al., 2013;Somerville et al., 2002b). The increase in accumulated acetate in the culture medium during exponential growth was largely consumed after 24 hr of growth ( Figure 5B). ...
The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H 2 O 2 , a crucial host defense against S. aureus . We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δ agr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δ agr strains to lethal H 2 O 2 doses. Increased survival of wild-type agr cells during H 2 O 2 exposure required sodA , which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δ agr cells from killing by H 2 O 2 . Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived ‘memory’ of agr -mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient ( Cybb −/− ) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.
... Modulation of TCA cycle activity may provide Staphylococcus with a survival advantage during infection (66,67). Inactivation of aconitase (CitB) leads to downregulation of the TCA cycle, thereby preventing maximal expression of virulence factors and altering the interaction between Staphylococcus and its host (68)(69)(70). Therefore, we hypothesize that Staphylococcus may exert its virulence effects by modulating the activity of the TCA cycle, ultimately contributing to the development of psoriatic skin lesions. ...
Background
Disturbed gut microbiota and associated metabolic dysfunction exist in Psoriasis. Despite the growing use of interleukin-17 inhibitor (anti-IL17) therapy, the effect of anti-IL17 on gut/skin microbiota function is not fully understood in patients with Psoriasis.
Objective
Therefore, we explored whether Psoriasis is associated with alterations in selected gut/skin microbiota in a study cohort, and a longitudinal cohort study to reveal the effects of IL-17A inhibitor treatment on gut microbiota in Psoriasis.
Methods
In a case-control study, 14 patients with Psoriasis and 10 age, sex and body mass index-matched Healthy Controls were recruited. Longitudinal mapping of the gut microbiome was performed using 16S rRNA gene sequencing. Mouse models were used to further study and validate the interrelationship between the skin microbiome and the gut microbiome in Psoriasis. PICRUST2 was applied to predict the function of the bacterial community.
Results
In Psoriasis patients, gut microbiota dysbiosis was present with increased heterogeneity: decreased Bacteroidota and increased Firmicutes as well as Actinobacteriota predominating in Psoriasis. Escherichia-Shigella enrichment was associated with reduction in serum levels of total bile acid and markers in Apoptotic pathways. After IL-17A inhibitor treatment in Psoriasis patients, longitudinal studies observed a trend toward a normal distribution of the gut microbiome and modulation of apoptosis-related metabolic pathways. Results from a mouse model showed dysregulation of the skin microbiota in Psoriasis characterized by Staphylococcus colonization.
Conclusion
The psoriatic gut/skin microbiota exhibits loss of community stability and pathogen enrichment. IL-17A inhibitors restore microbiota homeostasis and metabolic pathways, reduce pro-inflammatory cytokine expression, and alleviate symptoms in patients with Psoriasis.
... Acetyl-CoA carboxylase carboxyltransferase (Q2FXM7) was elevated in both stress conditions to initiate type II fatty acid biosynthesis. Under laboratory conditions and exponential growth, TCA cycle enzymes are often repressed or dispensable for viability (22). Here in the growth-arrested persistent populations, most enzymes of the TCA cycle were, as expected, repressed. ...
Bacteria possess the ability to enter a growth-arrested state known as persistence in order to survive antibiotic exposure. Clinically, persisters are regarded as the main causative agents for chronic and recurrent infectious diseases. To combat this antibiotic-tolerant population, a better understanding of the molecular physiology of persisters is required. In this study, we collected samples at different stages of the biphasic kill curve to reveal the dynamics of the cellular molecular changes that occur in the process of persister formation. After exposure to antibiotics with different modes of action, namely, vancomycin and enrofloxacin, similar persister levels were obtained. Both shared and distinct stress responses were enriched for the respective persister populations. However, the dynamics of the presence of proteins linked to the persister phenotype throughout the biphasic kill curve and the molecular profiles in a stable persistent population did show large differences, depending on the antibiotic used. This suggests that persisters at the molecular level are highly stress specific, emphasizing the importance of characterizing persisters generated under different stress conditions. Additionally, although generated persisters exhibited cross-tolerance toward tested antibiotics, combined therapies were demonstrated to be a promising approach to reduce persister levels. In conclusion, this investigation sheds light on the stress-specific nature of persisters, highlighting the necessity of tailored treatment approaches and the potential of combined therapy.
... An excess amount of acetate excretes into the culture medium until the concentration of glucose decreases to a level at which it can no longer sustain rapid growth. The departure from the exponential phase of growth increases the utilization of acetate in energy generation and biofilm formation [32][33][34][35][36][37]. In the absence of oxygen, S. aureus does not induce the full tricarboxylic acid (TCA) cycle [8,29,33,34,38]; thus, ATP must come from substrate phosphorylation of acetate and acetyl phosphate (acP) via the Pta-AckA pathway [33,39,40]. ...
... During growth under aerobic conditions, the end product of the glycolytic pathway, pyruvate, is decarboxylated to acetyl-coenzyme A. This acetyl-coenzyme A is converted to acetyl phosphate that is used to produce ATP and acetate and excess acetate is excreted into the culture medium during the exponential phase of growth, yet during the stationary phase of growth, when nutrient availability is minimal, S. aureus utilizes accumulated acetate as a carbon source via acetate kinase (ackA) [33,36,37]. In this process, acetate kinase is a key enzyme responsible for the dephosphorylation of acetyl phosphate with the concomitant production of acetate and ATP during anaerobic growth [33,36,64,65]; the present study results concur with these findings. ...
Background
Staphylococcus aureus spreads its infections through biofilms. This usually happens in the stationary phase of S. aureus growth where it utilizes accumulated acetate as a carbon source via the phosphotrans-acetylase-acetate kinase (Pta-Ack) pathway. In which acetate kinase (ackA) catalyzes the substrate-level phosphorylation, a vital secondary energy-yielding pathway that promotes biofilms formation aids bacterium survival in hostile environments. In this study, we describe the cloning, sequencing, and expression of S. aureus ackA gene. The expression analysis of ackA gene in methicillin-resistant strains of S. aureus (MRSA) correlates with ackA activity and biofilm units. The uniqueness of ackA was analyzed by using in silico methods.
Results
Elevated ackA gene expression was observed in MRSA strains, which correlates with increased ackA activity and biofilm units, explaining ackA role in MRSA growth and pathogenicity. The pure recombinant acetate kinase showed a molecular weight of 44 kDa, with enzyme activity of 3.35 ± 0.05 μM/ml/min. The presence of ACKA-1, ACKA-2 sites, one ATP, and five serine/threonine-protein kinase sites in the ackA gene (KC954623.1) indicated that acetyl phosphate production is strongly controlled. The comparative structural analysis of S. aureus ackA with ackA structures of Mycobacterium avium (3P4I) and Salmonella typhimurium (3SLC) exhibited variations as indicated by the RMSD values 1.877 Å and 2.141 Å respectively, explaining why ackA functions are differently placed in bacteria, concurring its involvement in S. aureus pathogenesis.
Conclusions
Overall findings of this study highlight the correlation of ackA expression profoundly increases survival capacity through biofilm formation, which is a pathogenic factor in MRSA and plays a pivotal role in infection spreading.
... A smaller proportion of knockouts showed positive fold change in abundance from the initial transposon mutant pools after macrophage invasion, indicating that spontaneous chromosomal mutations in S. aureus can also enhance this virulence phenotype (53) and may facilitate adaptation in chronic infection. Collectively, physiological and metabolic functions consistently contributing to macrophage invasion in both strains (Fig. 3) may reflect the association between intracellular pathogenesis and the SCV phenotype (7)(8)(9)(10)(11), as disruption of many such pathways has been identified in SCVs (54)(55)(56)(57). ...
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 .
... Under laboratory conditions and exponential growth, TCA cycle enzymes are often repressed or dispensable for viability [20]. Here in the growth arrested persistent populations, most enzymes of the TCA cycle were, as expected, repressed. ...
Bacteria possess the ability to enter a growth arrested state known as persistence in order to survive antibiotic exposure. Clinically, persisters are regarded as the main causative agents for chronic and recurrent infectious diseases. To combat this antibiotic-tolerant population, a better understanding of the molecular physiology of persisters is required. In this study, we collected samples at different stages of the biphasic kill curve to reveal the dynamics of the cellular molecular changes that occur in the process of persister formation. After exposure to antibiotics with different modes of action, namely vancomycin and enrofloxacin, similar persister levels were obtained. Both shared and distinct stress responses were enriched for the respective persister populations. However, the dynamics of the presence of proteins linked to the persister phenotype throughout the biphasic kill curve and the molecular profiles in a stable persistent population did show large differences depending on the antibiotic used. This suggests that persisters at the molecular level are highly stress specific, emphasizing the importance of characterizing persisters generated under different stress conditions. Additionally, although generated persisters exhibited cross-tolerance toward tested antibiotics, combined therapies were demonstrated to be a promising approach to reduce persister levels. In conclusion, this investigation sheds light on the stress-specific nature of persisters, highlighting the necessity of tailored treatment approaches and the potential of combined therapy.
Importance
By monitoring proteome and metabolites during Staphylococcus aureus persister formation under vancomycin and enrofloxacin exposure, we revealed the dynamic information of the molecular physiology of persister formation upon exposure to two different antibiotics with different modes of action. The data shows that cells that phenotypically are similarly classified as persisters, do have several molecular characteristics in common but, remarkably so, differ substantially in a significant number of other aspects of their molecular makeup. These contrasts provided valuable insights into persister eradication, which holds considerable clinical relevance.
... Collectively, these data suggest that Δagr increases respiration and aerobic fermentation to compensate for low metabolic efficiency. Consistent with this idea, agr deficiency also increases ATP-yielding carbon "overflow" pathways, as evidenced by increased acetate production ( Figure 5B) (21,22). The increase in accumulated acetate in the culture medium during exponential growth was largely consumed after 20 h of growth ( Figure 5B). ...
The agr, a quorum-sensing regulator of virulence, primes Staphylococcus aureus for tolerance to lethal oxidative stress, a critical component of host and antimicrobial defenses. Knowledge of factors that govern tolerance has advanced considerably in recent years. For example, studies have been centered on how a deficiency of specific metabolic features, such as aerobic respiration, affects tolerance. How defense against ROS and metabolism changes integrate with the virulence regulatory network to promote S. aureus pathogenesis is less well characterized. We report, for the first time, how the highly flexible components of metabolism involved in tolerance link to the virulence regulatory network to help S. aureus cause invasive infection. Second, in contrast to the kinetics of classical quorum-sensing, agr-mediated tolerance was long-lived, occurring throughout the growth cycle. Experiments in mice demonstrate how this uncoupling of agr-mediated tolerance from bacterial population density anticipates increases in exogenous oxidative stress expected during S. aureus-host interactions, thereby contributing to virulence. The striking existence of “memory” to withstand lethal stress suggest a critical feature of the agr system not previously appreciated. Third, agr activity induced profound metabolic alterations that mitigate endogenous bacterial oxidative stress, thereby explaining agr-mediated tolerance to lethal reactive oxygen species. Tolerance is associated with persistent S. aureus infection and poor outcome, and agr activity is a clinically relevant marker that varies widely among S. aureus isolates. Thus, understanding agr’s role in the biology of endogenous ROS and tolerance should inform development of personalized approaches for patient prognostication and therapy.
... Iron is needed for several pathways, including metabolic ones, in S. aureus. There have been studies showing that in the ironrestricted host environment, S. aureus has reduced virulence due to low expression of specific virulence factors (including haemolysin, capsule, Staphylococcal Protein A (spa), biofilm formation and surface proteins such as Emp) [17,18]. Intriguingly, recent work using an antibiotic-resistant (to ciprofloxacin) strain revealed that under iron restriction, this strain actually increased its virulence, upregulating various virulence factors, as well as amino acid synthesis and energy storage [19]. ...
The ability of Staphylococcus aureus to colonise different niches across the human body is linked to an adaptable metabolic capability, as well as its ability to persist within specific tissues despite adverse conditions. In many cases, as S. aureus proliferates within an anatomical niche, there is an associated pathology. The immune response, together with medical interventions such as antibiotics, often removes the S. aureus cells that are causing this disease. However, a common issue in S. aureus infections is a relapse of disease. Within infected tissue, S. aureus exists as a population of cells, and it adopts a diversity of cell types. In evolutionary biology, the concept of “bet-hedging” has established that even in positive conditions, there are members that arise within a population that would be present as non-beneficial, but if those conditions change, these traits could allow survival. For S. aureus, some of these cells within an infection have a reduced fitness, are not rapidly proliferating or are the cause of an active host response and disease, but these do remain even after the disease seems to have been cleared. This is true for persistence against immune responses but also as a continual presence in spite of antibiotic treatment. We propose that the constant arousal of suboptimal populations at any timepoint is a key strategy for S. aureus long-term infection and survival. Thus, understanding the molecular basis for this feature could be instrumental to combat persistent infections.
... Стафилококки -условно-патогенные представители нормальной микробиоты организма человека, могут представлять серьезную угрозу как возбудители оппортунистических инфекций человека и животных (Миронов и др., 2000, 2011). Метаболически стафилококки реагируют на лимит железа в питательной среде стимуляцией гликолиза и снижением интенсивности реакций цикла трикарбоновых кислот; крайнее уменьшение концентрации железа в питательной среде ведет к инактивации цепи переноса электронов (Somerville et al., 2002). ...
Представлены данные о роли железа в регуляции вирулентности и межмикробных взаимодействий условно-патогенных микроорганизмов. Охарактеризованы способы получения железа стафилококками, кандидами, синегнойной палочкой. Продемонстрировано влияние железосодержащих
субстратов – лактоферрина, гема, гемоглобина на регуляцию вирулентности условно-патогенных микроорганизмов. Выявлены возможные механизмы объединения микроорганизмов в ассоциации с целью удовлетворения своих потребностей в железе. Описаны способы получения железа и его влияние на биологические свойства молочнокислых бактерий. Показано, что появление в
окружающей среде лактоферрина, гема, гемоглобина увеличивает антагонистическую активность некоторых молочнокислых бактерий в отношении ассоциативной микробиоты.
