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

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.

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Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

Infection and Immunity ◽

10.1128/iai.01487-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1600-1605 ◽

Cited By ~ 32

Author(s):

Melissa A. Dean ◽

Randall J. Olsen ◽

S. Wesley Long ◽

Adriana E. Rosato ◽

James M. Musser

Keyword(s):

Staphylococcus Aureus ◽

Pathway Analysis ◽

Molecular Mechanisms ◽

Point Mutations ◽

Biosynthesis Pathway ◽

Wild Type ◽

Small Colony Variants ◽

Content Type ◽

Genes Encoding ◽

Small Colony

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of fiveS. aureusSCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

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Pharmacodynamic Evaluation of the Activity of Antibiotics against Hemin- and Menadione-Dependent Small-Colony Variants of Staphylococcus aureus in Models of Extracellular (Broth) and Intracellular (THP-1 Monocytes) Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00285-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3700-3711 ◽

Cited By ~ 23

Author(s):

L. G. Garcia ◽

S. Lemaire ◽

B. C. Kahl ◽

K. Becker ◽

R. A. Proctor ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Concentration Effect ◽

Intracellular Bacteria ◽

Maximal Effect ◽

Small Colony Variants ◽

Content Type ◽

Pharmacodynamic Profile ◽

Lower Amplitude ◽

Small Colony ◽

Antibiotic Failure

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate ofmenDandhemBmutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistantS. aureus(MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for themenDSCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for themenDmutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

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Tomatidine Inhibits Replication of Staphylococcus aureus Small-Colony Variants in Cystic Fibrosis Airway Epithelial Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01468-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1937-1945 ◽

Cited By ~ 29

Author(s):

Gabriel Mitchell ◽

Mariza Gattuso ◽

Gilles Grondin ◽

Éric Marsault ◽

Kamal Bouarab ◽

...

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Electron Transport ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Plant Secondary Metabolite ◽

Airway Epithelial ◽

Small Colony Variants ◽

Content Type ◽

Small Colony

ABSTRACTSmall-colony variants (SCVs) often are associated with chronicStaphylococcus aureusinfections, such as those encountered by cystic fibrosis (CF) patients. We report here that tomatidine, the aglycon form of the plant secondary metabolite tomatine, has a potent growth inhibitory activity against SCVs (MIC of 0.12 μg/ml), whereas the growth of normalS. aureusstrains was not significantly altered by tomatidine (MIC, >16 μg/ml). The specific action of tomatidine was bacteriostatic for SCVs and was clearly associated with their dysfunctional electron transport system, as the presence of the electron transport inhibitor 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) caused normalS. aureusstrains to become susceptible to tomatidine. Inversely, the complementation of SCVs' respiratory deficiency conferred resistance to tomatidine. Tomatidine provoked a general reduction of macromolecular biosynthesis but more specifically affected the incorporation of radiolabeled leucine in proteins of HQNO-treatedS. aureusat a concentration corresponding to the MIC against SCVs. Furthermore, tomatidine inhibited the intracellular replication of a clinical SCV in polarized CF-like epithelial cells. Our results suggest that tomatidine eventually will find some use in combination therapy with other traditional antibiotics to eliminate persistent forms ofS. aureus.

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In Vivo Mutations of Thymidylate Synthase (Encoded by thyA) Are Responsible for Thymidine Dependency in Clinical Small-Colony Variants of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00912-07 ◽

2007 ◽

Vol 190 (3) ◽

pp. 834-842 ◽

Cited By ~ 79

Author(s):

Indranil Chatterjee ◽

Andre Kriegeskorte ◽

Andreas Fischer ◽

Susanne Deiwick ◽

Nadine Theimann ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Thymidylate Synthase ◽

Point Mutations ◽

Underlying Mechanism ◽

Nucleotide Sequences ◽

Transcriptional Analysis ◽

Small Colony Variants ◽

Reverse Transcription Pcr ◽

Small Colony

ABSTRACT Trimethoprim-sulfamethoxazole (SXT)-resistant Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from the airways of cystic fibrosis (CF) patients, often in combination with isogenic normal strains if patients were treated with SXT for extended periods. As SXT inhibits the synthesis of tetrahydrofolic acid, which acts as a cofactor for thymidylate synthase (thyA), the survival of TD-SCVs depends exclusively on the availability of external thymidine. Since the underlying mechanism for thymidine dependency is unknown, we investigated if alterations in the thyA nucleotide sequences were responsible for this phenomenon. Sequence analysis of several clinical TD-SCVs and their isogenic normal strains with reference to previously published S. aureus thyA nucleotide sequences was performed. Three clinical TD-SCVs were complemented by transforming TD-SCVs with the vector pCX19 expressing ThyA from S. aureus 8325-4. Transcriptional analysis of metabolic and virulence genes and regulators (agr, hla, spa, citB, thyA, and nupC) was performed by quantitative reverse transcription-PCR. The previously published sequences of thyA and two normal clinical strains were highly conserved, while thyA of four normal strains and four SCVs had nonsynonymous point mutations. In 8/10 SCVs, deletions occurred, resulting in stop codons which were located in 4/10 SCVs close to or within the active site of the protein (dUMP binding). Complementation of TD-SCVs with thyA almost fully reversed the phenotype, growth characteristics, and transcription patterns. In conclusion, we demonstrated that mutations of the thyA gene were responsible for the phenotype of TD-SCVs. Complementation of TD-SCVs with thyA revealed that a functional ThyA protein is necessary and sufficient to change the SCV phenotype and behavior back to normal.

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Staphylococcus aureus Adapts to Oxidative Stress by Producing H2O2-Resistant Small-Colony Variants via the SOS Response

Infection and Immunity ◽

10.1128/iai.03016-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 1830-1844 ◽

Cited By ~ 60

Author(s):

Kimberley L. Painter ◽

Elizabeth Strange ◽

Julian Parkhill ◽

Kathleen B. Bamford ◽

Darius Armstrong-James ◽

...

Keyword(s):

Oxidative Stress ◽

Staphylococcus Aureus ◽

Time Course ◽

Strand Break ◽

Host Cells ◽

Wild Type ◽

Small Colony Variants ◽

Content Type ◽

Dna Double Strand Break ◽

Small Colony

The development of chronic and recurrentStaphylococcus aureusinfections is associated with the emergence of slow-growing mutants known as small-colony variants (SCVs), which are highly tolerant of antibiotics and can survive inside host cells. However, the host and bacterial factors which underpin SCV emergence during infection are poorly understood. Here, we demonstrate that exposure ofS. aureusto sublethal concentrations of H2O2leads to a specific, dose-dependent increase in the population frequency of gentamicin-resistant SCVs. Time course analyses revealed that H2O2exposure caused bacteriostasis in wild-type cells during which time SCVs appeared spontaneously within theS. aureuspopulation. This occurred via a mutagenic DNA repair pathway that included DNA double-strand break repair proteins RexAB, recombinase A, and polymerase V. In addition to triggering SCV emergence by increasing the mutation rate, H2O2also selected for the SCV phenotype, leading to increased phenotypic stability and further enhancing the size of the SCV subpopulation by reducing the rate of SCV reversion to the wild type. Subsequent analyses revealed that SCVs were significantly more resistant to the toxic effects of H2O2than wild-type bacteria. With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H2O2. Taken together, these data reveal a mechanism by whichS. aureusadapts to oxidative stress via the production of a subpopulation of H2O2-resistant SCVs with enhanced catalase production.

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Impact of Staphylococcus aureus Small Colony Variants on Human Lung Epithelial Cells with Subsequent Influenza Virus Infection

Microorganisms ◽

10.3390/microorganisms8121998 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1998

Author(s):

Janine J. Wilden ◽

Eike R. Hrincius ◽

Silke Niemann ◽

Yvonne Boergeling ◽

Bettina Löffler ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Immune Response ◽

Respiratory Tract ◽

Influenza Viruses ◽

Lung Epithelial Cells ◽

Human Beings ◽

Small Colony Variants ◽

Small Colony ◽

Tract Infections ◽

The Impact

Human beings are exposed to microorganisms every day. Among those, diverse commensals and potential pathogens including Staphylococcus aureus (S. aureus) compose a significant part of the respiratory tract microbiota. Remarkably, bacterial colonization is supposed to affect the outcome of viral respiratory tract infections, including those caused by influenza viruses (IV). Since 30% of the world’s population is already colonized with S. aureus that can develop metabolically inactive dormant phenotypes and seasonal IV circulate every year, super-infections are likely to occur. Although IV and S. aureus super-infections are widely described in the literature, the interactions of these pathogens with each other and the host cell are only scarcely understood. Especially, the effect of quasi-dormant bacterial subpopulations on IV infections is barely investigated. In the present study, we aimed to investigate the impact of S. aureus small colony variants on the cell intrinsic immune response during a subsequent IV infection in vitro. In fact, we observed a significant impact on the regulation of pro-inflammatory factors, contributing to a synergistic effect on cell intrinsic innate immune response and induction of harmful cell death. Interestingly, the cytopathic effect, which was observed in presence of both pathogens, was not due to an increased pathogen load.

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Evolution of Staphylococcus aureus under Vancomycin Selective Pressure: the Role of the Small-Colony Variant Phenotype

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04508-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1347-1351 ◽

Cited By ~ 14

Author(s):

Justin R. Lenhard ◽

Christof von Eiff ◽

Irene S. Hong ◽

Patricia N. Holden ◽

Michael D. Bear ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Therapy ◽

Selective Pressure ◽

Type Model ◽

Methicillin Resistant ◽

Small Colony Variants ◽

Content Type ◽

Small Colony Variant ◽

Small Colony

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) often persist despite antibiotic therapy. Against a 108-CFU/ml methicillin-resistantS. aureus(MRSA) (strain COL) population of which 0%, 1%, 10%, 50%, or 100% was an isogenichemBknockout (Ia48) subpopulation displaying the SCV phenotype, vancomycin achieved maximal reductions of 4.99, 5.39, 4.50, 3.28, and 1.66 log10CFU/ml over 48 h. Vancomycin at ≥16 mg/liter shifted a population from 50% SCV cells at 0 h to 100% SCV cells at 48 h, which was well characterized by a Hill-type model (R2> 0.90).

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Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00144-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 8

Author(s):

Franziska Kleinert ◽

René Kallies ◽

Michael Hort ◽

Annegret Zweynert ◽

Christiane Szekat ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Acquired Resistance ◽

Laboratory Strain ◽

Resistance Mechanisms ◽

Transposition Frequency ◽

Related Sequence ◽

Small Colony Variants ◽

Content Type ◽

Small Colony ◽

Insertion Sites

ABSTRACT Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256. When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00742-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7265-7272 ◽

Cited By ~ 25

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.

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Guanine Limitation Results in CodY-Dependent and -Independent Alteration ofStaphylococcus aureusPhysiology and Gene Expression

Journal of Bacteriology ◽

10.1128/jb.00136-18 ◽

2018 ◽

Vol 200 (14) ◽

Cited By ~ 6

Author(s):

Alyssa N. King ◽

Samiksha A. Borkar ◽

David J. Samuels ◽

Zachary Batz ◽

Logan L. Bulock ◽

...

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

De Novo ◽

Expression Profiles ◽

Sugar Transport ◽

Health Care Facilities ◽

Gene Products ◽

Nucleotide Synthesis ◽

Content Type ◽

The Impact

ABSTRACTInStaphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) onS. aureusphysiology and CodY activity by constructing aguaAnull mutant (ΔguaAstrain).De novobiosynthesis of guanine monophosphate is abolished due to theguaAmutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked outguaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaAmutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaAcells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of theS. aureustranscriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting thatS. aureusexhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureusinfections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence ofS. aureusin response to environmental cues is critical for developing efficient diagnostics and treatments.De novopurine biosynthesis is essential for both fitness and virulence inS. aureussince inhibiting production cripplesS. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affectsS. aureusfitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combatS. aureusinfections.

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