scholarly journals The general stress response of Staphylococcus aureus promotes tolerance of antibiotics and survival in whole human blood

Microbiology ◽  
2020 ◽  
Vol 166 (11) ◽  
pp. 1088-1094 ◽  
Author(s):  
Nisha Ranganathan ◽  
Rebecca Johnson ◽  
Andrew M. Edwards
Keyword(s):  
Staphylococcus Aureus ◽  
Stress Response ◽  
Human Blood ◽  
Chronic Infection ◽  
Type Species ◽  
Content Type ◽  
General Stress Response ◽  
Link Type ◽  
General Stress ◽  
Immune Defences

Staphylococcus aureus is a frequent cause of invasive human infections such as bacteraemia and infective endocarditis. These infections frequently relapse or become chronic, suggesting that the pathogen has mechanisms to tolerate the twin threats of therapeutic antibiotics and host immunity. The general stress response of S. aureus is regulated by the alternative sigma factor B (σB) and provides protection from multiple stresses including oxidative, acidic and heat. σB also contributes to virulence, intracellular persistence and chronic infection. However, the protective effect of σB on bacterial survival during exposure to antibiotics or host immune defences is poorly characterized. We found that σB promotes the survival of S. aureus exposed to the antibiotics gentamicin, ciprofloxacin, vancomycin and daptomycin, but not oxacillin or clindamycin. We also found that σB promoted staphylococcal survival in whole human blood, most likely via its contribution to oxidative stress resistance. Therefore, we conclude that the general stress response of S. aureus may contribute to the development of chronic infection by conferring tolerance to both antibiotics and host immune defences.

scholarly journals WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

2016 ◽  
Vol 198 (8) ◽  
pp. 1281-1293 ◽  
Author(s):  
Julien Herrou ◽  
Daniel M. Czyż ◽  
Jonathan W. Willett ◽  
Hye-Sook Kim ◽  
Gekleng Chhor ◽  
...  
Keyword(s):  
Stress Response ◽  
Mouse Model ◽  
Binding Site ◽  
Brucella Abortus ◽  
Chronic Infection ◽  
Content Type ◽  
General Stress Response ◽  
General Stress

ABSTRACTThe general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family.IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We presentin vitroandin vivofunctional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activityin vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

scholarly journals Thermal and Nutritional Regulation of Ribosome Hibernation inStaphylococcus aureus

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Arnab Basu ◽  
Kathryn E. Shields ◽  
Christopher S. Eickhoff ◽  
Daniel F. Hoft ◽  
Mee-Ngan F. Yap
Keyword(s):  
Staphylococcus Aureus ◽  
Transcription Factor ◽  
Stress Response ◽  
Nutrient Sensing ◽  
Dependent Manner ◽  
Nutritional Regulation ◽  
Content Type ◽  
General Stress Response ◽  
Regulatory Pathways ◽  
General Stress

ABSTRACTThe translationally silent 100S ribosome is a poorly understood form of the dimeric 70S complex that is ubiquitously found in all bacterial phyla. The elimination of the hibernating 100S ribosome leads to translational derepression, ribosome instability, antibiotic sensitivity, and biofilm defects in some bacteria. InFirmicutes, such as the opportunistic pathogenStaphylococcus aureus, a 190-amino acid protein calledhibernating-promotingfactor (HPF) dimerizes and conjoins two 70S ribosomes through a direct interaction between the HPF homodimer, with each HPF monomer tethered on an individual 70S complex. While the formation of the 100S ribosome in gammaproteobacteria and cyanobacteria is exclusively induced during postexponential growth phase and darkness, respectively, the 100S ribosomes inFirmicutesare constitutively produced from the lag-logarithmic phase through the post-stationary phase. Very little is known about the regulatory pathways that controlhpfexpression and 100S ribosome abundance. Here, we show that a general stress response (GSR) sigma factor (SigB) and a GTP-sensing transcription factor (CodY) integrate nutrient and thermal signals to regulatehpfsynthesis inS. aureus, resulting in an enhanced virulence of the pathogen in a mouse model of septicemic infection. CodY-dependent regulation ofhpfis strain specific. An epistasis analysis further demonstrated that CodY functions upstream of the GSR pathway in a condition-dependent manner. The results reveal an important link betweenS. aureusstress physiology, ribosome metabolism, and infection biology.IMPORTANCEThe dimerization of 70S ribosomes (100S complex) plays an important role in translational regulation and infectivity of the major human pathogenStaphylococcus aureus. Although the dimerizing factor HPF has been characterized biochemically, the pathways that regulate 100S ribosome abundance remain elusive. We identified a metabolite- and nutrient-sensing transcription factor, CodY, that serves both as an activator and a repressor ofhpfexpression in nutrient- and temperature-dependent manners. Furthermore, CodY-mediated activation ofhpfmasks a secondaryhpftranscript derived from a general stress response SigB promoter. CodY and SigB regulate a repertoire of virulence genes. The unexpected link between ribosome homeostasis and the two master virulence regulators provides new opportunities for alternative druggable sites.

Impact of pH on growth of Staphylococcus epidermidis and Staphylococcus aureus in vitro

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Vidula Iyer ◽  
Janhavi Raut ◽  
Anindya Dasgupta
Keyword(s):  
Staphylococcus Aureus ◽  
Growth Kinetics ◽  
Staphylococcus Epidermidis ◽  
Type Species ◽  
Ph Dependence ◽  
Skin Microbiome ◽  
Content Type ◽  
Link Type ◽  
Kinetics Of

The pH of skin is critical for skin health and resilience and plays a key role in controlling the skin microbiome. It has been well reported that under dysbiotic conditions such as atopic dermatitis (AD), eczema, etc. there are significant aberrations of skin pH, along with a higher level of Staphylococcus aureus compared to the commensal Staphylococcus epidermidis on skin. To understand the effect of pH on the relative growth of S. epidermidis and S. aureus , we carried out simple in vitro growth kinetic studies of the individual microbes under varying pH conditions. We demonstrated that the growth kinetics of S. epidermidis is relatively insensitive to pH within the range of 5–7, while S. aureus shows a stronger pH dependence in that range. Gompertz’s model was used to fit the pH dependence of the growth kinetics of the two bacteria and showed that the equilibrium bacterial count of S. aureus was the more sensitive parameter. The switch in growth rate happens at a pH of 6.5–7. Our studies are in line with the general hypothesis that keeping the skin pH within an acidic range is advantageous in terms of keeping the skin microbiome in balance and maintaining healthy skin.

scholarly journals Fatal exudative dermatitis in island populations of red squirrels (Sciurus vulgaris): spillover of a virulent Staphylococcus aureus clone (ST49) from reservoir hosts

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Kay Fountain ◽  
Tiffany Blackett ◽  
Helen Butler ◽  
Catherine Carchedi ◽  
Anna-Katarina Schilling ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Species ◽  
Small Ruminants ◽  
Sciurus Vulgaris ◽  
Red Squirrels ◽  
Content Type ◽  
Island Populations ◽  
Link Type ◽  
Bacterial Cell Membrane ◽  
Reservoir Hosts

Fatal exudative dermatitis (FED) is a significant cause of death of red squirrels (Sciurus vulgaris) on the island of Jersey in the Channel Islands where it is associated with a virulent clone of Staphylococcus aureus, ST49. S. aureus ST49 has been found in other hosts such as small mammals, pigs and humans, but the dynamics of carriage and disease of this clone, or any other lineage in red squirrels, is currently unknown. We used whole-genome sequencing to characterize 228 isolates from healthy red squirrels on Jersey, the Isle of Arran (Scotland) and Brownsea Island (England), from red squirrels showing signs of FED on Jersey and the Isle of Wight (England) and a small number of isolates from other hosts. S. aureus was frequently carried by red squirrels on the Isle of Arran with strains typically associated with small ruminants predominating. For the Brownsea carriage, S. aureus was less frequent and involved strains associated with birds, small ruminants and humans, while for the Jersey carriage S. aureus was rare but ST49 predominated in diseased squirrels. By combining our data with publicly available sequences, we show that the S. aureus carriage in red squirrels largely reflects frequent but facile acquisitions of strains carried by other hosts sharing their habitat (‘spillover’), possibly including, in the case of ST188, humans. Genome-wide association analysis of the ruminant lineage ST133 revealed variants in a small number of mostly bacterial-cell-membrane-associated genes that were statistically associated with squirrel isolates from the Isle of Arran, raising the possibility of specific adaptation to red squirrels in this lineage. In contrast there is little evidence that ST49 is a common carriage isolate of red squirrels and infection from reservoir hosts such as bank voles or rats, is likely to be driving the emergence of FED in red squirrels.

Detection and partial characterization of extracellular inducers of persistence in Staphylococcus epidermidis and Staphylococcus aureus

2021 ◽  
Vol 70 (6) ◽  
Author(s):  
Elyse C. Curry ◽  
Ryan G. Hart ◽  
Danni Y. Habtu ◽  
Neal R. Chamberlain
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Type Species ◽  
Proteinase K ◽  
Partial Characterization ◽  
Content Type ◽  
Link Type ◽  
Inducing Factors

Introduction. This study describes the identification and partial characterization of persistence-inducing factors (PIFs) from staphylococci. Hypothesis/Gap Statement. Increases in persisters during mid-log phase growth indicate that quorum-sensing factors might be produced by staphylococci. Aim. To identify and partially characterize PIFs from Staphylococcus epidermidis RP62A and Staphylococcus aureus SH1000. Methodology. Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCFs) from S. epidermidis and S. aureus were obtained at various OD600s and following incubation at 16 h. The CCFs were used to develop a PIF assay. The PIF assay was used to partially characterize PIF from S. epidermidis and S. aureus for sizing of PIF activity, temperature and protease sensitivity and inter-species communications. Results. The optimal OD600s for S. epidermidis and S. aureus PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis ’ PIF activity was decreased by storage at 4 oC but not at 20 oC (16 h), 37 oC (1 h) or 100 oC (15 min). S. aureus ’ PIF activity was decreased following storage at 4 oC (2 weeks) and after boiling at 100 oC for 5 min but not after incubation at 37 oC (1 h). PIF activity from both species went through a 3000 molecular weight cutoff ultrafilter. Proteinase K treatment of S. aureus PIF decreased activity but did not decrease the PIF activity of S. epidermidis . PIF from S. epidermidis did not increase persisters when used to treat S. aureus cells and nor did PIF from S. aureus increase persisters when used to treat S. epidermidis cells. Conclusions. Attempts to discover PIFs for staphylococci were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species produce extracellular, low-molecular-weight inducers of persistence when assayed using an OD600 -based PIF assay.

Staphylococcus singaporensis sp. nov., a new member of the Staphylococcus aureus complex, isolated from human clinical specimens

2021 ◽  
Vol 71 (10) ◽  
Author(s):  
Ka Lip Chew ◽  
Sophie Octavia ◽  
Deborah Lai ◽  
Raymond T. P. Lin ◽  
Jeanette W. P. Teo
Keyword(s):  
Staphylococcus Aureus ◽  
Type Species ◽  
Clinical Laboratory ◽  
Core Gene ◽  
Separate Species ◽  
New Members ◽  
Content Type ◽  
Link Type ◽  
Flight Mass Spectrometry ◽  
Genotypic Analysis

Staphylococcus argenteus and Staphylococcus schweitzeri are the newest members of the Staphylococcus aureus complex. The number of clinical reports attributed to these new S. aureus complex members is limited. In a retrospective clinical laboratory study conducted over a 4-month period investigating the prevalence of S. argenteus and S. schweitzeri , a total of 43 isolates were selected. Phylogeny based on core-gene multilocus sequence typing (MLST) analysis confirmed that 37 were S. argenteus but a genetically distinct clade of six isolates was identified. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses further supported the classification of these six isolates as a separate species. When compared to S. aureus complex reference genomes, the ANI values were ≤94 % and the dDDH values were <53 %. Based on the seven-gene S. aureus MLST scheme, the six isolates belong to five novel allelic profiles (ST6105, ST6106, ST6107, ST6108 and ST109). Their clinical infection features were similar to S. aureus . Skin and soft tissue infections presented in four out of the six cases. Routine clinical diagnostic identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and biochemical profiling does not differentiate these new members from the rest of the complex. Genotypic analysis suggests that the six isolates belong to a novel species, Staphylococcus singaporensis sp. nov. with isolate SS21T (=DSM 111408T=NCTC14419T) designated as the type strain.

scholarly journals Exposure of Bacillus subtilis to Low Pressure (5 Kilopascals) Induces Several Global Regulons, Including Those Involved in the SigB-Mediated General Stress Response

2014 ◽  
Vol 80 (16) ◽  
pp. 4788-4794 ◽  
Author(s):  
Samantha M. Waters ◽  
José A. Robles-Martínez ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
High Pressures ◽  
Low Pressure ◽  
Bacterial Cells ◽  
Content Type ◽  
General Stress Response ◽  
Cellular Processes ◽  
General Stress ◽  
Microbial Responses

ABSTRACTStudies of how microorganisms respond to pressure have been limited mostly to the extreme high pressures of the deep sea (i.e., the piezosphere). In contrast, despite the fact that the growth of most bacteria is inhibited at pressures below ∼2.5 kPa, little is known of microbial responses to low pressure (LP). To study the global LP response, we performed transcription microarrays onBacillus subtiliscells grown under normal atmospheric pressure (∼101 kPa) and a nearly inhibitory LP (5 kPa), equivalent to the pressure found at an altitude of ∼20 km. Microarray analysis revealed altered levels of 363 transcripts belonging to several global regulons (AbrB, CcpA, CodY, Fur, IolR, ResD, Rok, SigH, Spo0A). Notably, the highest number of upregulated genes, 86, belonged to the SigB-mediated general stress response (GSR) regulon. Upregulation of the GSR by LP was confirmed by monitoring the expression of the SigB-dependentctc-lacZreporter fusion. Measuring transcriptome changes resulting from exposure of bacterial cells to LP reveals insights into cellular processes that may respond to LP exposure.

scholarly journals Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1

2015 ◽  
Vol 197 (8) ◽  
pp. 1466-1477 ◽  
Author(s):  
Andreas Kaczmarczyk ◽  
Ramon Hochstrasser ◽  
Julia A. Vorholt ◽  
Anne Francez-Charlot
Keyword(s):  
Stress Response ◽  
Heat Shock ◽  
Experimental Evidence ◽  
Histidine Kinase ◽  
Response Regulators ◽  
Catalytic Core ◽  
Content Type ◽  
General Stress Response ◽  
General Stress ◽  
Kinase Pathway

ABSTRACTThe general stress response (GSR) allows bacteria to monitor and defend against a broad set of unrelated, adverse environmental conditions. InAlphaproteobacteria, the key step in GSR activation is phosphorylation of the response regulator PhyR. InSphingomonas melonisFr1, seven PhyR-activating kinases (Paks), PakA to PakG, are thought to directly phosphorylate PhyR under different stress conditions, but the nature of the activating signals remains obscure. PakF, a major sensor of NaCl and heat shock, lacks a putative sensor domain but instead harbors a single receiver (REC) domain (PakFREC) N-terminal to its kinase catalytic core. Such kinases are called “hybrid response regulators” (HRRs). How HRRs are able to perceive signals in the absence of a true sensor domain has remained largely unexplored. In the present work, we show that stresses are actually sensed by another kinase, KipF (kinase of PakF), which phosphorylates PakFRECand thereby activates PakF. KipF is a predicted transmembrane kinase, harboring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic kinase catalytic core. We demonstrate that KipF senses different salts through its CHASE3 domain but is not a sensor of general osmotic stress. While salt sensing depends on the CHASE3 domain, heat shock sensing does not, suggesting that these stresses are perceived by different mechanisms. In summary, our results establish a two-tiered histidine kinase pathway involved in activation of the GSR inS. melonisFr1 and provide the first experimental evidence for the so far uncharacterized CHASE3 domain as a salt sensor.IMPORTANCEHybrid response regulators (HRRs) represent a particular class of histidine kinases harboring an N-terminal receiver (REC) domain instead of a true sensor domain. This suggests that the actual input for HRRs may be phosphorylation of the REC domain. In the present study, we addressed this question by using the HRR PakF. Our results suggest that PakF is activated through phosphorylation of its REC domain and that this is achieved by another kinase, KipF. KipF senses heat shock and salt stress, with the latter requiring the periplasmic CHASE3 domain. This work not only suggests that HRRs work in two-tiered histidine kinase pathways but also provides the first experimental evidence for a role of the so far uncharacterized CHASE3 domain in salt sensing.

scholarly journals A Mutant RNA Polymerase Activates the General Stress Response, Enabling Escherichia coli Adaptation to Late Prolonged Stationary Phase

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Pabitra Nandy ◽  
Savita Chib ◽  
Aswin Seshasayee
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Slow Growth ◽  
Content Type ◽  
General Stress Response ◽  
E Coli ◽  
General Stress ◽  
Small Colony

ABSTRACT Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after 3 weeks of maintaining an E. coli K-12 population in stationary phase. This variant displayed a small colony phenotype and slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC(A494V) mutation confers the slow growth and small colony phenotype on this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary-phase sigma factor σS. The RpoC(A494V) mutation upregulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly or via mutations such as RpoC(A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase. IMPORTANCE An important general mechanism of a bacterium’s adaptation to its environment involves adjusting the balance between growing fast and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favor of the general stress response about 2 weeks later. This can be established by direct mutations in the master regulator of the general stress response or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance although the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

scholarly journals rpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1160-1170 ◽  
Author(s):  
Laura Cutugno ◽  
Jennifer Mc Cafferty ◽  
Jan Pané-Farré ◽  
Conor O’Byrne ◽  
Aoife Boyd
Keyword(s):  
Stress Response ◽  
Type Species ◽  
Vibrio Vulnificus ◽  
Growth Stress ◽  
Rifampicin Resistance ◽  
Secondary Effects ◽  
Rich Medium ◽  
Content Type ◽  
Link Type ◽  
Resistant Strains

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus , even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml−1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Sign in / Sign up

Export Citation Format

Share Document