scholarly journals Pyrimidine Biosynthesis Regulates the Small-Colony Variant and Mucoidy inPseudomonas aeruginosathrough Sigma Factor Competition

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Roy Al Ahmar ◽  
Brandon D. Kirby ◽  
Hongwei D. Yu
Keyword(s):  
Cystic Fibrosis ◽  
Sigma Factor ◽  
Capsular Polysaccharide ◽  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Content Type ◽  
Small Colony Variant ◽  
Small Colony ◽  
Lung Infections ◽  
Alginate Biosynthesis

ABSTRACTMucoidy due to alginate overproduction by the Gram-negative bacteriumPseudomonas aeruginosafacilitates chronic lung infections in patients with cystic fibrosis (CF). We previously reported that disruption inde novosynthesis of pyrimidines resulted in conversion to a nonmucoid small-colony variant (SCV) in the mucoidP. aeruginosastrain (PAO581), which has a truncated anti-sigma factor, MucA25, that cannot sequester sigma factor AlgU (AlgT). Here, we showed that supplementation with the nitrogenous bases uracil or cytosine in growth medium complemented the SCV to normal growth, and nonmucoidy to mucoidy, in thesemucA25mutants. This conversion was associated with an increase in intracellular levels of UMP and UTP suggesting that nucleotide restoration occurred via a salvage pathway. In addition, supplemented pyrimidines caused an increase in activity of the alginate biosynthesis promoter (PalgD), but had no effect on PalgU, which controls transcription ofalgU. Cytosolic levels of AlgU were not influenced by uracil supplementation, yet levels of RpoN, a sigma factor that regulates nitrogen metabolism, increased with disruption of pyrimidine synthesis and decreased after supplementation of uracil. This suggested that an elevated level of RpoN in SCV may block alginate biosynthesis. To support this, we observed that overexpressingrpoNresulted in a phenotypic switch to nonmucoidy in PAO581 and in mucoid clinical isolates. Furthermore, transcription of an RpoN-regulated promoter increased in the mutants and decreased after uracil supplementation. These results suggest that the balance of RpoN and AlgU levels may regulate growth from SCV to mucoidy through sigma factor competition for PalgD.IMPORTANCEChronic lung infections withP. aeruginosaare the main cause of morbidity and mortality in patients with cystic fibrosis. This bacterium overproduces a capsular polysaccharide called alginate (also known as mucoidy), which aids in bacterial persistence in the lungs and in resistance to therapeutic regimens and host immune responses. The current study explores a previously unknown link between pyrimidine biosynthesis and mucoidy at the level of transcriptional regulation. Identifying/characterizing this link could provide novel targets for the control of bacterial growth and mucoidy. Inhibiting mucoidy may improve antimicrobial efficacy and facilitate host defenses to clear the noncapsulatedP. aeruginosabacteria, leading to improved prognosis for patients with cystic fibrosis.

scholarly journals Overproduction of the AlgT Sigma Factor Is Lethal to Mucoid Pseudomonas aeruginosa

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Ashley R. Cross ◽  
Vishnu Raghuram ◽  
Zihuan Wang ◽  
Debayan Dey ◽  
Joanna B. Goldberg
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Continuous Production ◽  
Common Mutation ◽  
Wild Type ◽  
Chronic Infections ◽  
Truncated Form ◽  
Content Type ◽  
Lung Infections

ABSTRACT Pseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon, leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing, since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Based on our findings, we speculate that mutations in mucP stabilize the truncated form of MucA22, rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT. IMPORTANCE Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA, resulting in uncontrolled AlgT and continuous production of alginate in addition to the expression of ∼300 additional genes. Here, we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.

Thymidine‐auxotrophic Staphylococcus aureus small‐colony variant bacteremia in a patient with cystic fibrosis

2020 ◽  
Vol 55 (6) ◽  
pp. 1388-1393
Author(s):  
Dilair C. Souza ◽  
Laura L. Cogo ◽  
Jussara K. Palmeiro ◽  
Libera M. Dalla‐Costa ◽  
Ana P. Oliveira Tomaz ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Small Colony Variant ◽  
Small Colony

scholarly journals Inactivation of thyA in Staphylococcus aureus Attenuates Virulence and Has a Strong Impact on Metabolism and Virulence Gene Expression

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Andre Kriegeskorte ◽  
Desiree Block ◽  
Mike Drescher ◽  
Nadine Windmüller ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Thymidylate Synthase ◽  
Molecular Basis ◽  
De Novo ◽  
Strong Impact ◽  
Small Colony Variants ◽  
Content Type ◽  
Long Term Treatment ◽  
Small Colony ◽  
The Impact

ABSTRACTStaphylococcus aureusthymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronicS. aureusinfections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS;thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations inthyAwere leading to inactivity of TS proteins, and TS inactivity led to tremendous impact onS. aureusphysiology and virulence. Whole DNA microarray analysis of the constructed ΔthyAmutant identified severe alterations compared to the wild type. Important virulence regulators (agr,arlRS,sarA) and major virulence determinants (hla,hlb,sspAB, andgeh) were downregulated, while genes important for colonization (fnbA,fnbB,spa,clfB,sdrC, andsdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyAmutant was strongly attenuated in virulence models, including aCaenorhabditis eleganskilling model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed thatthyAactivity has a major role forS. aureusvirulence and physiology.IMPORTANCEThymidine-dependent small-colony variants (TD-SCVs) ofStaphylococcus aureuscarry mutations in the thymidylate synthase (TS) gene (thyA) responsible forde novosynthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistantS. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact onS. aureusvirulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

scholarly journals Attenuated Virulence and Biofilm Formation in Staphylococcus aureus following Sublethal Exposure to Triclosan

2012 ◽  
Vol 56 (6) ◽  
pp. 3092-3100 ◽  
Author(s):  
Joe Latimer ◽  
Sarah Forbes ◽  
Andrew J. McBain
Keyword(s):  
Staphylococcus Aureus ◽  
Growth Rate ◽  
Growth Rates ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Concentration Gradients ◽  
Content Type ◽  
Small Colony Variant ◽  
Small Colony ◽  
Repeated Passage

ABSTRACTSubeffective exposure ofStaphylococcus aureusto the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype.S. aureusSCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain ofS. aureuscreated by repeated passage on sublethal triclosan concentrations.S. aureusATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using aGalleria mellonellapathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P< 0.01), was defective in biofilm formation and DNase activity, and displayed significantly attenuated virulence. Colony size, hemolysis, coagulase activity, and virulence were only partially restored in strain ×10, whereas the planktonic growth rate was fully restored. However, ×10 was at least as defective in biofilm formation and DNase production as P10. These data suggest that although repeated exposure to triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies.

scholarly journals Capsule Expression and Genotypic Differences among Staphylococcus aureus Isolates from Patients with Chronic or Acute Osteomyelitis

2009 ◽  
Vol 77 (5) ◽  
pp. 1968-1975 ◽  
Author(s):  
Santiago M. Lattar ◽  
Lorena P. N. Tuchscherr ◽  
Roberto L. Caccuri ◽  
Daniela Centrón ◽  
Karsten Becker ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Chronic Osteomyelitis ◽  
Phenotypic Expression ◽  
Genotypic Differences ◽  
Acute Osteomyelitis ◽  
Ample Evidence ◽  
Small Colony Variant ◽  
Small Colony ◽  
Relative Prevalence

ABSTRACT There is ample evidence that Staphylococcus aureus capsular polysaccharide (CP) promotes virulence. Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. This study was conducted to determine the relative prevalence of nonencapsulated S. aureus in patients with chronic and acute osteomyelitis. Only 76/118 (64%) S. aureus isolates from patients with osteomyelitis expressed CP, whereas all 50 isolates from blood cultures of patients with infections other than osteoarticular infections expressed CP (P = 0.0001). A significantly higher prevalence of nonencapsulated S. aureus was found in patients with chronic osteomyelitis (53%) than in those with acute osteomyelitis (21%) (P = 0.0046). S. aureus isolates obtained from multiple specimens from five of six patients with chronic osteomyelitis exhibited phenotypic (expression of CP, α-hemolysin, β-hemolysin, slime, and the small-colony variant phenotype) and/or genotypic (pulsed-field gel electrophoresis and spa typing) differences. Nonencapsulated S. aureus was recovered from at least one specimen from each chronic osteomyelitis patient. Fourteen isolates obtained from two patients with acute osteomyelitis were indistinguishable from each other within each group, and all produced CP5. In conclusion, we demonstrated that nonencapsulated S. aureus is more frequently isolated from patients with chronic osteomyelitis than from those with acute osteomyelitis, suggesting that loss of CP expression may be advantageous to S. aureus during chronic infection. Our findings on multiple S. aureus isolates from individual patients allow us to suggest that selection of nonencapsulated S. aureus is likely to have occurred in the patient during long-term bone infection.

scholarly journals Anex vivocystic fibrosis model recapitulates key clinical aspects of chronicStaphylococcus aureusinfection

2019 ◽  
Author(s):  
Esther Sweeney ◽  
Marwa M. Hassan ◽  
Niamh E. Harrington ◽  
Alan R. Smyth ◽  
Matthew N. Hurley ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Ex Vivo ◽  
Lung Model ◽  
Clinical Aspects ◽  
Small Colony Variant ◽  
Tissue Invasion ◽  
Disease Characteristics ◽  
Small Colony ◽  
Clinically Significant

AbstractStaphylococcus aureusis one of the most prevalent organisms isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. Clinical studies are limited in scope by age and health of participants andin vitrostudies are not always able to accurately recapitulate chronic disease characteristics such as the development of small colony variants. Further, animal models also do not fully represent features of clinical disease: in particular, mice are not readily colonized byS. aureusand when infection is established it leads to the formation of abscesses, a phenomenon almost never observed in the human CF lung. Here, we present details of the development of an existingex vivopig lung model of CF infection to investigate the growth ofS. aureus. We show thatS. aureusis able to establish infection and demonstrates clinically significant characteristics including small colony variant phenotype, increased antibiotic tolerance and preferential localisation in mucus. Tissue invasion and the formation of abscesses were not observed, in line with clinical data.

scholarly journals Scnn1b-Transgenic BALB/c Mice as a Model of Pseudomonas aeruginosa Infections of the Cystic Fibrosis Lung

2020 ◽  
Vol 88 (9) ◽  
Author(s):  
Kristen J. Brao ◽  
Brendan P. Wille ◽  
Joshua Lieberman ◽  
Robert K. Ernst ◽  
Mark E. Shirtliff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Immune Cell ◽  
Opportunistic Pathogen ◽  
Sodium Absorption ◽  
Lung Pathology ◽  
Wild Type ◽  
Content Type ◽  
Lung Infections ◽  
Content Modeling

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1β (IL-1β), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.

scholarly journals Response of Pseudomonas aeruginosa to the Innate Immune System-Derived Oxidants Hypochlorous Acid and Hypothiocyanous Acid

2020 ◽  
Vol 203 (2) ◽  
pp. e00300-20
Author(s):  
Katie V. Farrant ◽  
Livia Spiga ◽  
Jane C. Davies ◽  
Huw D. Williams
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Immune System ◽  
Hypochlorous Acid ◽  
Secretion System ◽  
Content Type ◽  
Type 3 Secretion System ◽  
Lung Infections ◽  
Type 3

ABSTRACTPseudomonas aeruginosa is a significant nosocomial pathogen and is associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and protects itself against HOCl and HOSCN and the contribution of such responses to its success as a CF pathogen. To investigate the P. aeruginosa response to these oxidants, we screened 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators of antibiotic resistance, methionine biosynthesis, catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), which are required for protection against HOCl. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress and responds to both oxidants by upregulating the expression of a putative peroxiredoxin, rclX (PA14_07355). Transcriptional analysis revealed that while there was specificity in the response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated), there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type 3 secretion system, sulfur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinates part of the response to both oxidants, including upregulation of pyocyanin biosynthesis genes, and, in the presence of HOSCN, downregulation of chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa causes devastating infections in immunocompromised hosts, including chronic lung infections in cystic fibrosis patients. To combat infection, the host’s immune system produces the antimicrobial oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Little is known about how P. aeruginosa responds to and survives attack from these oxidants. To address this, we carried out two approaches: a mutant screen and transcriptional study. We identified the P. aeruginosa transcriptional regulator, RclR, which responds specifically to HOCl and HOSCN stress and is essential for protection against both oxidants. We uncovered a link between the P. aeruginosa transcriptional response to these oxidants and physiological processes associated with pathogenicity, including antibiotic resistance and the type 3 secretion system.

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