scholarly journals Heme-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

2021 ◽  
Author(s):  
Izabela Z Batko ◽  
Ronald S Flannagan ◽  
Veronica Guariglia-Oropeza ◽  
Jessica R Sheldon ◽  
David E Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Iron Acquisition ◽  
Iron Starvation ◽  
Minimal Growth ◽  
Atp Production ◽  
Small Colony ◽  
Sense And Respond ◽  
Outstanding Question

The ability to acquire iron is essential for Staphylococcus aureus to cause infection. Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they too acquire iron. How SCVs obtain iron remains unknown and so here we addressed this outstanding question by creating a stable hemB mutant in S. aureus USA300 strain LAC. The mutant, auxotrophic for hemin, was assessed for its ability to grow under iron-restriction and with various iron sources. The hemB SCV utilizes exogenously supplied heme but was attenuated for growth under conditions of iron starvation. RNA-seq analyses showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrates limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine, which enabled the SCV to sustain only minimal growth in iron deplete media. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo. Indeed, the administration of Desferal failed to promote hemB bacterial growth in vivo, in contrast to WT, in every organ analyzed except for the murine kidney where growth was enhanced. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased heme availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that heme is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes.

Hemin-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

2021 ◽  
Author(s):  
Izabela Z. Batko ◽  
Ronald S. Flannagan ◽  
Veronica Guariglia-Oropeza ◽  
Jessica R. Sheldon ◽  
David E. Heinrichs
Keyword(s):  
Iron Acquisition ◽  
Recurrent Infection ◽  
Nutritional Requirement ◽  
Iron Starvation ◽  
Small Colony Variants ◽  
Atp Production ◽  
Small Colony ◽  
Sense And Respond

Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they acquire iron, yet how SCVs obtain iron remains unknown. To address this, we created a stable hemB mutant in S. aureus USA300 strain LAC. The hemB SCV utilized exogenously supplied hemin but was attenuated for growth under conditions of iron starvation. RNA-seq showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrated limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo , as administration of Desferal failed to promote hemB bacterial growth in every organ analyzed except for the kidneys. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased hemin availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that hemin is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes. Importance S. aureus small colony variants (SCVs) are associated with chronic recurrent infection and worsened clinical outcome. SCVs persist within the host despite administration of antibiotics. This study yields insight into how S. aureus SCVs acquire iron which, during infection of a host, is a difficult-to-acquire metal nutrient. Under hemin-limited conditions, hemB S. aureus is impaired for siderophore-dependent growth and, in agreement, murine infection indicates that hemin-deficient SCVs meet their nutritional requirement for iron through utilization of hemin. Importantly, we demonstrate that hemB SCVs rely upon hemin as a nutrient to promote siderophore utilization. Therefore, perturbation of heme biosynthesis and/or utilization represents a viable to strategy to mitigate the ability of SCV bacteria to acquire siderophore-bound iron during infection.

scholarly journals Staphylococcus aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture

2005 ◽  
Vol 187 (2) ◽  
pp. 554-566 ◽  
Author(s):  
Lauren M. Mashburn ◽  
Amy M. Jett ◽  
Darrin R. Akins ◽  
Marvin Whiteley
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Iron Acquisition ◽  
Low Oxygen ◽  
Dialysis Membrane ◽  
Opportunistic Human Pathogen ◽  
The Impact

ABSTRACT Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.

scholarly journals Mechanism of Action of a Prostaglandin E2 Receptor Antagonist with antimicrobial activity against Staphylococcus aureus

2020 ◽  
Author(s):  
Elizabeth A. Lilly ◽  
Mélanie A. C. Ikeh ◽  
Paul L. Fidel ◽  
Mairi C. Noverr
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Receptor Antagonist ◽  
Mechanism Of Action ◽  
Atp Production ◽  
Direct Growth ◽  
Prostaglandin E2 Receptor ◽  
Intra Abdominal Infection

AbstractOur laboratory recently reported that the EP4 receptor antagonist, L-161,982, had direct growth-inhibitory effects on Staphylococcus aureus in vitro and in vivo, reducing microbial burden and providing significant protection against lethality in models of S. aureus monomicrobial and polymicrobial intra-abdominal infection. This antimicrobial activity was observed with both methicillin-sensitive and methicillin-resistant S. aureus (MRSA), as well as other Gram-positive bacteria. The antimicrobial activity of L-161,982 was independent of EP4 receptor inhibitory activity. In this study, we investigated the mechanism of action (MOA) of L-161,982, which contains a sulfonamide functional group. However, results demonstrate L-161,982 does not affect folate synthesis (sulfonamide MOA), oxidative stress, or membrane permeability. Instead, our results suggest that the inhibitor works via effects on inhibition of the electron transport chain (ETC). Similar to other ETC inhibitors, L-161,982 exposure results in a small colony size variant phenotype and inhibition of pigmentation, as well as significantly reduced hemolytic activity, and ATP production. In addition, L-161,982 potentiated the antimicrobial activity of another ETC inhibitor and inhibition was partially rescued by supplementation with nutrients required for ETC auxotrophs. Taken together, these findings demonstrate that L-161,982 exerts antimicrobial activity against MRSA via inhibition the ETC, representing a new member of a potentially novel antimicrobial drug class.

SC5005 dissipates the membrane potential to kill Staphylococcus aureus persisters without detectable resistance

2021 ◽  
Author(s):  
Chieh-Hsien Lu ◽  
Chung-Wai Shiau ◽  
Yung-Chi Chang ◽  
Hsiu-Ni Kung ◽  
Jui-Ching Wu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mode Of Action ◽  
Cytoplasmic Membrane ◽  
Resistance Development ◽  
Antibiotic Resistant ◽  
Atp Production ◽  
The Past ◽  
Ht 29

Abstract Objectives In the past few decades, multiple-antibiotic-resistant Staphylococcus aureus has emerged and quickly spread in hospitals and communities worldwide. Additionally, the formation of antibiotic-tolerant persisters and biofilms further reduces treatment efficacy. Previously, we identified a sorafenib derivative, SC5005, with bactericidal activity against MRSA in vitro and in vivo. Here, we sought to elucidate the resistance status, mode of action and anti-persister activity of this compound. Methods The propensity of S. aureus to develop SC5005 resistance was evaluated by assessment of spontaneous resistance and by multi-passage selection. The mode of action of SC5005 was investigated using macromolecular synthesis, LIVE/DEAD and ATPlite assays and DiOC2(3) staining. The effect of SC5005 on the mammalian cytoplasmic membrane was measured using haemolytic and lactate dehydrogenase (LDH) assays and flow cytometry. Results SC5005 depolarized and permeabilized the bacterial cytoplasmic membrane, leading to reduced ATP production. Because of this mode of action, no resistance of S. aureus to SC5005 was observed after constant exposure to sub-lethal concentrations for 200 passages. The membrane-perturbing activity of SC5005 was specific to bacteria, as no significant haemolysis or release of LDH from human HT-29 cells was detected. Additionally, compared with other bactericidal antibiotics, SC5005 exhibited superior activity in eradicating both planktonic and biofilm-embedded S. aureus persisters. Conclusions Because of its low propensity for resistance development and potent persister-eradicating activity, SC5005 is a promising lead compound for developing new therapies for biofilm-related infections caused by S. aureus.

scholarly journals Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander L. Greninger ◽  
Amin Addetia ◽  
Yue Tao ◽  
Amanda Adler ◽  
Xuan Qin
Keyword(s):  
Iron Acquisition ◽  
Culture Conditions ◽  
Single Nucleotide Variants ◽  
Genetic Rescue ◽  
Small Colony Variant ◽  
E Coli ◽  
Small Colony ◽  
Oxidative Respiration

AbstractIsolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.

scholarly journals Thymidine-Dependent Staphylococcus aureus Small-Colony Variants Are Induced by Trimethoprim-Sulfamethoxazole (SXT) and Have Increased Fitness during SXT Challenge

2015 ◽  
Vol 59 (12) ◽  
pp. 7265-7272 ◽  
Author(s):  
Andre Kriegeskorte ◽  
Nicola Ivan Lorè ◽  
Alessandra Bragonzi ◽  
Camilla Riva ◽  
Marco Kelkenberg ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Short Term ◽  
Small Colony Variants ◽  
Content Type ◽  
Short Term Exposure ◽  
Small Colony ◽  
Selection Of

ABSTRACTTrimethoprim-sulfamethoxazole (SXT) is a possible alternative for the treatment of community- and hospital-acquired methicillin-resistantStaphylococcus aureus(MRSA) due to the susceptibility of most MRSA strains to the drug. However, after long-term treatment with SXT, thymidine-dependent (TD) SXT-resistant small-colony variants (SCVs) emerge. In TD-SCVs, mutations of thymidylate synthase ([TS]thyA) occur. Until now, it has never been systematically investigated that SXT is triggering the induction and/or selection of TD-SCVs. In our study, we performed induction, reversion, and competition experimentsin vitroandin vivousing a chronic mouse pneumonia model to determine the impact of SXT on the emergence of TD-SCVs. SCVs were characterized by light and transmission electron microscopy (TEM) and auxotrophism testing. Short-term exposure ofS. aureusto SXT induced the TD-SCV phenotype inS. aureusSH1000, while selection of TD-SCVs withthyAmutations occurred after long-term exposure. In reversion experiments with clinical and laboratory TD-SCVs, all revertants carried compensating mutations at the initially identified mutation site. Competition experimentsin vitroandin vivorevealed a survival and growth advantage of the ΔthyAmutant under low-thymidine availability and SXT exposure although this advantage was less profoundin vivo. Our results show that SXT induces the TD-SCV phenotype after short-term exposure, while long-term exposure selects forthyAmutations, which provide an advantage for TD-SCVs under specified conditions. Thus, our results further an understanding of the dynamic processes occurring during SXT exposure with induction and selection ofS. aureusTD-SCVs.

scholarly journals Group V Phospholipase A2 Mediates Endothelial Dysfunction and Acute Lung Injury Caused by Methicillin-Resistant Staphylococcus aureus

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1731
Author(s):  
Yu Maw Htwe ◽  
Huashan Wang ◽  
Patrick Belvitch ◽  
Lucille Meliton ◽  
Mounica Bandela ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Acute Lung Injury ◽  
Endothelial Dysfunction ◽  
Lung Injury ◽  
Phospholipase A2 ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Group V ◽  
Methicillin Resistant

Lung endothelial dysfunction is a key feature of acute lung injury (ALI) and clinical acute respiratory distress syndrome (ARDS). Previous studies have identified the lipid-generating enzyme, group V phospholipase A2 (gVPLA2), as a mediator of lung endothelial barrier disruption and inflammation. The current study aimed to determine the role of gVPLA2 in mediating lung endothelial responses to methicillin-resistant Staphylococcus aureus (MRSA, USA300 strain), a major cause of ALI/ARDS. In vitro studies assessed the effects of gVPLA2 inhibition on lung endothelial cell (EC) permeability after exposure to heat-killed (HK) MRSA. In vivo studies assessed the effects of intratracheal live or HK-MRSA on multiple indices of ALI in wild-type (WT) and gVPLA2-deficient (KO) mice. In vitro, HK-MRSA increased gVPLA2 expression and permeability in human lung EC. Inhibition of gVPLA2 with either the PLA2 inhibitor, LY311727, or with a specific monoclonal antibody, attenuated the barrier disruption caused by HK-MRSA. LY311727 also reduced HK-MRSA-induced permeability in mouse lung EC isolated from WT but not gVPLA2-KO mice. In vivo, live MRSA caused significantly less ALI in gVPLA2 KO mice compared to WT, findings confirmed by intravital microscopy assessment in HK-MRSA-treated mice. After targeted delivery of gVPLA2 plasmid to lung endothelium using ACE antibody-conjugated liposomes, MRSA-induced ALI was significantly increased in gVPLA2-KO mice, indicating that lung endothelial expression of gVPLA2 is critical in vivo. In summary, these results demonstrate an important role for gVPLA2 in mediating MRSA-induced lung EC permeability and ALI. Thus, gVPLA2 may represent a novel therapeutic target in ALI/ARDS caused by bacterial infection.

scholarly journals In Vitro Antibacterial Effect of the Methanolic Extract of the Korean Soybean Fermented Product Doenjang against Staphylococcus aureus

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2319
Author(s):  
Klara Lalouckova ◽  
Lucie Mala ◽  
Petr Marsik ◽  
Eva Skrivanova
Keyword(s):  
Staphylococcus Aureus ◽  
High Performance ◽  
Methanolic Extract ◽  
Bioactive Substances ◽  
Microdilution Method ◽  
Antibacterial Compound ◽  
Fermented Product ◽  
Broth Microdilution Method

Ultra-high performance liquid chromatography/mass spectrometry showed soyasaponin I and the isoflavones daidzein, genistein, and glycitein to be the main components of the methanolic extract of the Korean soybean fermented product doenjang, which is known to be a rich source of naturally occurring bioactive substances, at average contents of 515.40, 236.30, 131.23, and 29.00 ng/mg, respectively. The antimicrobial activity of the methanolic extract of doenjang against nine Staphylococcusaureus strains was determined in vitro by the broth microdilution method to investigate its potential to serve as an alternative antibacterial compound. The results suggest that the extract is an effective antistaphylococcal agent at concentrations of 2048–4096 µg/mL. Moreover, the tested extract also showed the ability to inhibit the growth of both methicillin-sensitive and methicillin-resistant animal and clinical S. aureus isolates. The growth kinetics of the chosen strains of S. aureus at the minimum inhibitory concentration of the methanolic extract of doenjang support the idea that the tested extract acts as an antibacterial compound. To the best of our knowledge, this is the first report on the antistaphylococcal action of the methanolic extract of doenjang thus, additional studies including in vivo testing are necessary to confirm this hypothesis.

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