Wall teichoic acid protects Staphylococcus aureus from inhibition by Congo red and other dyes

Abstract

Objectives

Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection.

Methods

We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo.

Results

We observed that inhibition of wall teichoic acid expression resulted in an ∼1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus.

Conclusions

Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.

Introduction

Staphylococcus aureus is a leading pathogen of community- and hospital-acquired infection of the skin, soft tissues and other sites.^1,2 Since multidrug-resistant S. aureus, including hospital-acquired methicillin-resistant S. aureus (MRSA), are increasing, new targets for antibiotics are needed.

The Gram-positive S. aureus cell wall includes two negatively charged polymers: lipoteichoic acid (LTA) and wall teichoic acid (WTA), which form a highly hydrated polyanionic matrix that is interwoven through the peptidoglycan. While not all Gram-positive bacteria have teichoic acid polymers identical to those of S. aureus, alternate polymers generally have functional similarity and anionic character, indicating that anionic polymers are central to the normal function of the Gram-positive cell wall.³ WTAs are phosphate-rich, carbohydrate-based polymers, which are initially synthesized on a lipid carrier inserted into the inner leaf of the cytoplasmic membrane, before being transported to the cell surface where they are covalently linked to peptidoglycan.⁴ WTAs affect the cation binding, tensile strength, rigidity and porosity of the Gram-positive cell wall.⁴ They are essential for normal S. aureus adherence to epithelial and endothelial cells, and virulence,^5–10 although S. aureus can grow in vitro without WTA.^6,7

WTA biosynthesis is mediated by enzymes encoded by the tar (teichoic acid ribitol) operon.^3,4 The first steps in the biosynthesis of S. aureus WTA are the addition of two activated sugars to the bactoprenol carrier, which is mediated by enzymes encoded by tarO and tarA. This is followed by the addition of two glycerol 3-phosphate units by the tarB and tarF gene products,¹¹ and, lastly, addition of the polyribitol-phosphate repeat (mediated by the enzyme encoded by tarL and tarK). The nascent chains are then exported by an ABC transporter, consisting of the tarGH gene products, to the external surface of the membrane where the polymer is attached to peptidoglycan.^3,12 The WTA polymer is composed of 11–40 polyribitol-phosphate repeating units.⁹ Since WTA is not essential for in vitro growth, tarO or tarA mutants are viable. However, most of the genes downstream of tarA in the S. aureus WTA pathway cannot be deleted unless tarO (or tarA) is deleted first,^12,13 making these downstream genes conditionally essential. This mixed gene dispensability pattern implies that blocking late-acting WTA biosynthetic enzymes after flux into the pathway has been initiated is deleterious to the bacterium.

In contrast to the WTA polymer, LTA is typically a polymer of poly(1-3)-glycerolphosphate consisting of 18–50 glycerolphosphate repeat units,⁹ which is linked to a diglycosyldiacylglycerol membrane anchor.¹⁴ Polyglycerol phosphate is synthesized by LtaS using the membrane phospholipid,¹⁵ phosphatidylglycerol, as a substrate.^16,17 Limiting ltaS expression in a temperature-susceptible S. aureus mutant decreases membrane LTA content, resulting in cell growth arrest with abnormalities in cell division and separation.^15,18

Azo dyes are compounds bearing the functional group R-N = N-R′, in which R and R′ can be either aryl or alkyl groups. They have been used extensively in industry and the biomedical sciences.¹⁹ Congo red contains two sulphonic acid groups conferring a strong negative charge and is of special interest because it binds amyloid in biological specimens.²⁰ It has also been used to distinguish biofilm-forming properties of staphylococci.^21,22

We recently began to probe the WTA biosynthesis of S. aureus as a novel antimicrobial target.^5,6,23,24 Since WTAs constitute about half the dry weight of the staphylococcal cell wall and make an important contribution to its role as a barrier to dissolved substances, we began to search for compounds that are selectively toxic to WTA-deficient S. aureus with a view towards developing new, synergistic therapeutic combinations. Here, we report that inhibition of S. aureus WTA biosynthesis renders S. aureus highly susceptible to Congo red and related azo dyes.

Materials and methods

Strains and growth conditions

The bacterial strains used are listed in Table 1. S. aureus was grown in tryptic soy broth (TSB), and antibiotic resistances were selected with tetracycline (2.5 mg/L) and kanamycin (50 mg/L).

Table 1.

S. aureus strains used in this study

Strain	Genotype and/or phenotype	Reference
RN6390	prophage-cured derivative of NCTC 8325	31
RN4220	a mutant of NCTC 8325-4 that accepts foreign DNA; partial agr defect	48
Newman	clinical isolate, methicillin-susceptible strain	49
RN6390ΔtarO	RN6390ΔtarO::tetL, tetracycline resistant	45
RN4220ΔtarO	RN4220ΔtarO	50
RN4220ΔtarA	RN4220ΔtarA	51
RN4220ΔtarK	RN4220ΔtarK	51
RN4220ΔltaS	RN4200ΔltaS::phleo/pM101, kanamycin resistant	18
RN4220ΔltaS-ltaS	RN4200ΔltaS::phleo/pM101-ltaS, kanamycin resistant	18
NewmanΔtarO	NewmanΔtarO	50

Congo red susceptibility

Congo red susceptibility tests were conducted as previously described,²⁵ with minor modification. Briefly, 10-fold dilutions, starting at 10⁶ cfu in 5 μL, were spotted on brain heart infusion (BHI) agar or Luria-Bertani (LB) agar, supplemented with 0.08% (w/v) Congo red where appropriate. Congo red agar plates were incubated at 37 or 30°C for 24 h. As a precaution to prevent possible inactivation of Congo red by light, the plates were incubated in the dark. Susceptibility to Congo red was determined by comparing colony density between parental and mutant strains on control and Congo red plates. Sudan red 7B (0.08%, w/v), calcofluor white (0.01%, w/v), direct red (0.08%, w/v), mordant black (0.08%, w/v) and acid red 88 (0.08%, w/v) in BHI agar were tested similarly.

MIC determination

In vitro susceptibility tests (MIC determination) were conducted using the CLSI broth microplate assay guidelines.²⁶ Because of temperature sensitivity, to determine the susceptibility of RN4220ΔltaS, bacteria were cultured at 30°C for 20 h.

Transmission electron microscopy (TEM)

Newman or NewmanΔtarO was inoculated to ∼10⁹ cfu/mL in TSB containing 10 mg/L Congo red and cultured at 37°C for 6 h. Cells were collected at 0 or 6 h, fixed in Karnovsky's fixative [2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer (pH 7.4)] and processed for TEM using standard procedures described previously.⁵ For TEM, 60–90 Å sections were obtained, viewed and photographed with a transmission electron microscope (model 410; Philips Electronics NV, Eindhoven, The Netherlands).

Synergy between Congo red and tunicamycin

A standard chequerboard assay, using Congo red and tunicamycin or Congo red and ampicillin, was performed as described previously.²⁷ To assess the kinetics of inhibition, cultures of strain Newman were started in TSB containing either Congo red or tunicamycin. Briefly, an overnight culture of S. aureus strain Newman was diluted to ∼10⁵ cfu/mL in 10 mL of TSB containing 10 mg/L Congo red or 1 mg/L tunicamycin and cultured at 37°C statically. Following 2 h of incubation, tunicamycin (1 mg/L final concentration) was added to the Congo red culture or Congo red (10 mg/L final concentration) was added to the tunicamycin culture. Following 6 h of additional incubation, bacteria were enumerated by plating serial 10-fold dilutions. The experiments were performed three times, independently.

Caenorhabditis elegans infection

C. elegans glp-4(bn2);sek-1(km4) was used for all experiments and infected essentially as described previously,²⁸ with minor modifications. This mutant line was selected for liquid assay experiments because it is unable to produce progeny at 25°C²⁹ and the deleted sek-1 gene encodes a conserved mitogen-activated protein kinase involved in innate immunity,³⁰ expediting the rate of killing. Worms were cultured and maintained on nematode growth medium agar containing lawns of Escherichia coli HB101.²⁸

To assess the susceptibility of C. elegans to infection by S. aureus RN6390 or an isogenic ΔtarO strain, the bacteria were cultured in TSB at 37°C. Overnight cultures were plated onto 10 cm TSB agar plates for 18 h at 37°C and cooled at room temperature for 30 min. Washed young adult worms were infected on lawns of S. aureus strains for 3 h at 25°C. After washing three times with M9, worms were transferred into the wells of 6-well microtitre plates (40 worms per well). Each well contained 2 mL of assay medium (20% BHI : 80% M9, v/v) supplemented with various concentrations of Congo red, tunicamycin or a combination of Congo red and tunicamycin. The plates were incubated at 25°C and examined for viability at 24 h intervals for 9 days using a Nikon SMZ645 dissecting microscope. Biological replicates of each experiment were conducted.

Statistical analysis

Differences in C. elegans survival were tested for significance by Kaplan–Meier and log-rank tests (STATA6; STATA, College Station, TX, USA). P < 0.05 was considered significant.

Results

Antimicrobial activity of Congo red against teichoic acid-deficient mutants

In examining the virulence and other properties of cells blocked in WTA biosynthesis,⁵ we observed that the tarO WTA-deficient mutant was highly susceptible to Congo red (Figure 1a). To confirm the importance of WTA in this phenotype and to determine the relationship between the point on the WTA pathway blocked and Congo red susceptibility, tarA and tarK mutants were also assessed. Although neither tarA nor tarO strains grew on agar containing 0.08% (w/v) Congo red, growth of the tarK strain was not affected (Figure 1b). A tarK deletion produces a substantial amount of long-chain WTA polymer because tarL compensates.¹²

Figure 1.

Growth of S. aureus and mutant strains on BHI/LB agar or BHI/LB agar supplemented with 0.08% (w/v) Congo red (CR) (indicated on the left-hand side of the figure). All plates were inoculated with 10–10⁶ cells of each strain (top designations) and photographed after 24 h of incubation. Genotypes are indicated on the right-hand side of the figure. (a) S. aureus strains RN6390 and Newman at 37°C. (b) RN4220 strains at 37°C. (c) RN4220 strains at 37°C. (d) RN4220 strains at 30°C.

To determine whether loss of LTA could also generate the Congo red-susceptible phenotype, an ltaS mutant of S. aureus was tested.¹⁸ The ltaS mutant is susceptible to heat and low osmolarity.^15,18 Therefore, Congo red was added at 0.08% (w/v) to LB agar (having a higher NaCl concentration than BHI) and the strain was tested for Congo red susceptibility at 30°C. To determine whether the Congo red effect was temperature related, we retested the ltaS mutant at 37°C, where it grows slowly (Figure 1c and d). In either case, Congo red had no effect on the ltaS mutant (Figure 1c and d). In contrast, the tarO mutant was inhibited by Congo red at both 30 and 37°C (Figure 1c and d).

MICs of Congo red were determined by microdilution assay. Consistent with the results of agar plate tests, the parental strain RN4220 and isogenic tarK and ltaS mutants, as well as an ΔltaS strain complemented on a multicopy plasmid, were all resistant to Congo red at concentrations >1024 mg/L (Table 2). In contrast, tarO and tarA mutants lacking WTA exhibited MICs ∼500–2000-fold lower (0.5–2 mg/L) (Table 2). To determine whether this was unique to the RN6390 lineage, which possesses several known mutations,³¹ clinical isolate Newman was tested along with an isogenic tarO mutant. Again, the wild-type exhibited an MIC >1024 mg/L, whereas the tarO mutant was susceptible to 1 mg/L (Table 2). Hence, the sensitizing effects of deleting tarO are general.

Table 2.

In vitro activities of Congo red against tested strains

S. aureus	MIC (mg/L)
RN6390	>1024
RN6390ΔtarO	4
RN4220	>1024
RN4220ΔtarO	2
RN4220ΔtarA	0.5
RN4220ΔtarK	>1024
RN4220ΔltaS	>1024
RN4220ΔltaS-ltaS	>1024
Newman	>1024
NewmanΔtarO	1

To probe the mechanism of killing, Congo red-inhibited cells were examined by TEM (Figure 2a–d). In contrast to the parental strain, the WTA-deficient mutant before exposure to Congo red shows a rough surface with many protrusions, as previously reported (Figure 2a and b).³² Although the morphology of the parental strain is not detectably affected after 6 h of Congo red treatment, the WTA-deficient mutant at the same timepoint shows disruption and lysis, with some cells showing an aberrantly thickened cell wall (Figure 2c and d). Together, these results show that WTA, but not LTA, protects S. aureus from killing by Congo red, even though both teichoic acids are composed of repetitive polyol phosphate subunits. Since Congo red contains two sulphonic acid groups, we also checked MICs of sulfanilamide, which is developed from prontosil, for Newman and the tarO mutant. However, the MIC for both strains was >128 mg/L.

Figure 2.

Changes in cell morphology induced by Congo red. TEM of S. aureus strain Newman and NewmanΔtarO in TSB containing 10 mg/L Congo red following 0 h (a and b) and 6 h (c and d) of incubation in the presence of drug [(a and c) low magnification; (b and d) high magnification]. White arrowheads show lysis of the ΔtarO mutant at 6 h.

To test whether the loss of WTA conferred susceptibility to other dyes of related structure, Sudan red 7B (0.08%, w/v), calcofluor white (0.01%, w/v), direct red (0.08%, w/v), mordant black (0.08%, w/v) and acid red 88 (0.08%, w/v) were tested for their ability to selectively suppress growth of a tarO mutant but not the parental strain. Loss of WTA conferred susceptibility to each of these dyes (Figure 3).

Figure 3.

Growth of wild-type S. aureus strain Newman and NewmanΔtarO on BHI agar or BHI agar supplemented with 0.08% (w/v) Sudan red 7B, 0.01% (w/v) calcofluor white, 0.08% (w/v) direct red, 0.08% (w/v) mordant black or 0.08% (w/v) acid red 88. All plates were inoculated with 10–10⁶ cells of each strain (top designations), incubated at 37°C for 24 h and photographed. Genotypes are indicated on the right-hand side of the figure.

Tunicamycin and Congo red are highly synergistic

Tunicamycin is an inhibitor of a large class of enzymes that couple sugar phosphates to membrane-embedded lipid phosphates and it inhibits WTA production by blocking TarO, a WTA enzyme belonging to this class.^4,33 However, tunicamycin has not been used extensively to treat infection because inhibitors of TarO are not lethal to S. aureus in vitro and tunicamycin concentrations that block bacterial growth in vitro are toxic to eukaryotic cells.³³ One strategy for circumventing problems of toxicity and lack of antibacterial effect is to incorporate such drugs at subtoxic levels in a mixture with a synergistic compound. Therefore, to determine whether wild-type S. aureus can be inhibited by a synergy between the ability of tunicamycin to inhibit WTA biosynthesis at low concentrations and Congo red, a two-dimensional chequerboard dilution series for each compound was performed. As predicted, Congo red exhibited strong synergy with tunicamycin (Figure 4a). Although the MIC of Congo red for strain Newman as a single agent was >1024 mg/L, levels of tunicamycin as low as 5 mg/L reduced the Congo red MIC to 1 mg/L (Figure 4a). However, tunicamycin may also inhibit an early step in cell wall peptidoglycan biosynthesis. To exclude possible effects on this pathway as contributing to the mechanism for Congo red/tunicamycin synergy, we tested Congo red and ampicillin for possible synergistic effect. In contrast to tunicamycin, ampicillin does not exhibit synergy with Congo red (data not shown). This indicates that Congo red susceptibility is a consequence of WTA biosynthesis inhibition, as inferred from the sensitivity of tarO and tarA mutants, and not from impairment in peptidoglycan biosynthesis.

Figure 4.

Synergy between Congo red (CR) and tunicamycin. (a) Chequerboard assay for interaction between tunicamycin and Congo red against wild-type S. aureus strain Newman, following 20 h of incubation at 37°C. The white line delineates the boundary between growth (right-hand side) and no growth (left-hand side). (b and c) Growth of strain Newman in TSB containing 10 mg/L Congo red or 1 mg/L tunicamycin at 37°C. (b) Addition of Congo red (black arrow) to TSB containing tunicamycin. (c) Addition of tunicamycin (black arrow) to TSB containing Congo red. Addition (open squares) and no addition (filled squares). Data represent mean values ± standard errors of the means (n = 3).

To assess the rate of killing and to obtain evidence for the mechanism of synergy, S. aureus strain Newman was grown in TSB in the presence of either 10 mg/L Congo red or 1 mg/L tunicamycin (values well below the individual MICs). Following a 2 h incubation, the alternate compound was added to the culture and the effect on growth was assessed. When Congo red was added at a level of 10 mg/L to the tunicamycin culture, bacterial death was noticeable 3 h later (Figure 4b). However, when tunicamycin was added at 1 mg/L following 2 h of culture in Congo red, killing was only apparent after 4 h of incubation (Figure 4c). This implies that 4 or 5 h of growth in the presence of tunicamycin are required to deplete WTA levels to the point where the cell becomes susceptible to Congo red killing. These results suggest that a mixture of Congo red and tunicamycin, or a co-drug, could have excellent antimicrobial activity against S. aureus.

Efficacy of Congo red or tunicamycin in vivo

As a proof of principle to determine whether the synergistic activity of Congo red and tunicamycin could be exploited therapeutically using concentrations below levels that exhibit toxicity, the well-studied nematode C. elegans infection model was employed.³⁴ Previous reports showed that C. elegans infected with lethal doses of several human pathogens can be cured by treatment with conventional and novel antibiotics.³⁵ Therefore, Congo red or tunicamycin, or combinations thereof, were first tested for toxicity against uninfected C. elegans. Tunicamycin exhibits dose-dependent toxicity, mirroring what has been found in mammals.³⁶ Congo red showed little toxicity against C. elegans, even at comparatively high concentrations (Figure 5a and b). The compound combination also shows toxicity at higher concentrations of Congo red and tunicamycin (Figure 5c and d). In toxicity tests, a combination of <12.5 mg/L Congo red and 3 or 5 mg/L tunicamycin does not show strong toxicity against C. elegans. Next, C. elegans was infected with S. aureus RN6390 or with WTA-deficient RN6390ΔtarO. While Congo red had little effect on C. elegans infected with RN6390 (Figure 6a), nematodes infected with RN6390ΔtarO were rescued by Congo red in a concentration-dependent manner (Figure 6b), illustrating the potential for Congo red to cure infections caused by S. aureus genetically lacking WTA in vivo (Figure 6b). Next, synergistic combinations of Congo red and tunicamycin were tested. Treatment with a synergistic combination produced a significant curative effect in a Congo red concentration-dependent manner (P < 0.001 for treatments compared with control or tunicamycin only) (Figure 6c and d), providing proof of principle for the strategy of blocking WTA biosynthesis while also treating S. aureus with a compound to which it is now rendered highly susceptible.

Figure 5.

Toxicity of tunicamycin (TUN) or/and Congo red (CR) against C. elegans. (a) Toxicity of tunicamycin (3.0–15.0 mg/L) against C. elegans (n = 40 per well). (b) Toxicity of Congo red (3.1–50.0 mg/L) against C. elegans (n = 40 per well). (c and d) Toxicity of combinations of 3 or 5 mg/L tunicamycin with various concentrations of Congo red (3.1–25.0 mg/L) against C. elegans (n = 40 per well). In pairwise-comparison log-rank tests, the difference in survival curves between control and treatments was P < 0.01.

Figure 6.

Survival of nematodes infected with S. aureus. Congo red (CR) treatment for nematodes infected with S. aureus RN6390 (a) or RN6390ΔtarO (b). The effect of combination therapy with Congo red (3.1–25.0 mg/L) and tunicamycin (TUN) [(c) 3 mg/L or (d) 5 mg/L] for nematodes infected with RN6390. In pairwise-comparison log-rank tests, the difference in survival curves between control and combination therapy treatments was P < 0.001.

Discussion

In this study, Congo red was found to be bactericidal against S. aureus in the absence of WTA. It was previously observed that an msrR mutant, a factor that contributes to methicillin resistance and belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins, also rendered S. aureus susceptible to Congo red.²⁵ Since the msrR mutant was found to possess reduced levels of WTA,²⁵ msrR-mediated resistance to Congo red appears to relate to the WTA content. Congo red has been observed to interact with various β-linked fungal glucans³⁷ and to interact strongly with nascent chitin chains, inhibiting cell wall formation in the alga Poterioochromonas stipitata.³⁸ The addition of Congo red to growing fungal cells results in cell wall-related morphological changes, such as incomplete separation of mother and daughter cells in Saccharomyces cerevisiae,^39,40 which are probably due to chitinase inhibition. Congo red also causes swelling or lysis of hyphal tips in the filamentous fungus Aspergillus niger as the result of the cell wall weakening and internal turgor pressure.^41–43 This cell wall-weakening effect activates the cell wall stress response.⁴⁴

In the present study, sulfanilamide, which is developed from prontosil, was not found to be active against the tarO mutant. The mechanism of sulphonamide action is through competitively inhibiting para-aminobenzoic acid (PABA) biosynthesis, which is needed to produce folic acid. It therefore seems unlikely that tunicamycin synergizes with Congo red as the result of enhanced uptake of a PABA biosynthesis inhibitor. Congo red has a number of functional groups with H-bonding and electrostatic properties, and it can effectively interact as a dimer with targets containing multiple contact points. It was previously observed that blocking the expression of WTA by inhibiting TarO sensitizes MRSA strains to β-lactams, even though the β-lactam-resistant transpeptidase, PBP2A, is still expressed.⁴⁵ This suggests a functional connection between ongoing WTA expression and localization of proteins involved in peptidoglycan assembly in S. aureus. The mechanism of synergy is the subject of ongoing study.

In this study, Congo red exhibited strong synergy with tunicamycin in vitro, and the combination of Congo red and tunicamycin could act antimicrobially in vivo. Despite low-level toxicity and known carcinogenicity,⁴⁶ Congo red has been explored as a drug for Huntington's disease and others because it binds amyloid, and it appears to limit some of the symptoms of disease.⁴⁷ Like Congo red, the use of tunicamycin has been limited because of toxicity.³⁶ However, WTA biosynthesis is inhibited by concentrations of tunicamycin much lower than MIC levels, raising the possibility of its use, or use of a less toxic derivative, at low concentrations in synergistic combinations.

Funding

This work was supported by Harvard-wide project AI083214 and by PHHS grants EY017381 and EY08289 to M. S. G., GM078477 to S. W., F3178727 to J. G. S., F32AI084316 to J. C. and by the Uehara Memorial Foundation and the Japanese Eye Bank Society to T. S.

Transparency declarations

None to declare.