Laboratory Plasticware Induces Expression of a Bacterial Virulence Factor

Abstract

Pollution with microplastic has become a prime environmental concern. The various ways in which human-made polymers and microorganisms interact are little understood, and this is particularly true for microplastic and pathogenic microorganisms. Previous reports demonstrated that expression of central virulence-associated protein A (VapA) of the pathogenic bacterium Rhodococcus equi is shut off at 30°C, whereas it is strongly expressed at 37°C, a temperature which may serve as an intrahost cue. Here, we show that cultivation at 30°C in disposable plastic tubes increases mRNA levels of vapA 70-fold compared to growth in conventional glass tubes. Strong expression of vapA in plastic tubes does not seem to be caused by a compound leaching from plastic but rather by tube surface properties. Expression stimulation during growth in plastic is regulated by the R. equi transcription regulators VirR and VirS, indicating that plastic-induced vapA expression is (co)regulated through the canonical vapA expression pathway. Our observations have important implications for the future analysis and assessment of environmental microplastic contaminations in that they show that, in principle, contact of pathogens with environmental plastic can increase their virulence. IMPORTANCE Millions of tons small plastic pieces (microplastic) find their way into the environment every year. They pose digestive and toxicity problems to various life forms in soil, freshwater, and seawater. Additionally, microplastic offers an opportunity for microorganisms to attach and to become an important part of a "plastisphere community." The significance of our study lies in the documentation of a sharp increase in production of a central virulence factor by a bacterial pathogen when the bacterium is in touch with certain makes of plastic. Although this feature may not reflect an increased health risk in case of this particular soilborne pathogen, our data disclose a new facet of how microplastics can endanger life.

Keywords: Rhodococcus; gene expression; microplastic; pathogen; pollution; virulence.

FIG 1

VapA expression in various plastic tubes at 30°C by different R. equi isolates. (A) An overnight culture of R. equi 33701 at 30°C was diluted into fresh BHI at an OD₆₀₀ of 0.1 and further grown at 30°C or 37°C in glass or plastic tubes for 18 h, as indicated. Proteins from equal numbers of bacteria were analyzed by anti-VapA Western blotting. VapA typically runs as up to three separate proteins with molecular weights between 15 and 18 kDa (11, 49). (B and C) Strain ATCC 33701 was grown in Teflon tubes (B) or in untreated (mock) or siliconized (silic) Sarstedt 13-mL Snap Cap polypropylene tubes (C) at 30°C or 37°C and processed as described above. (D) Cultures of R. equi strains 103, ATCC 33701, and 85F were grown in glass (G) or Sarstedt 13-mL Snap Cap polypropylene tubes and processed as described above. The immunoblots are representative of three independent experiments each. Corn, Corning Inc.; Epp, Eppendorf AG; Falc, Falcon Corning Inc.; Grei, Greiner; PP, polypropylene SC; Sar, Sarstedt AG; SC, Sarstedt Snap Cap; TPP, Techno Plastic Products AG; VWR, VWR International.

FIG 2

VapA expression at 30°C is not induced by supplements commonly used in plastic manufacturing. (A and B) A 30°C overnight culture of R. equi 33701 was diluted into fresh BHI at an OD₆₀₀ of 0.1 and grown for 18 h at 30°C or 37°C in glass tubes supplemented with (A) oleamide or stearamide or (B) erucamide or di-HEMDA at the indicated concentrations (micromolar units). Lanes labeled “0” contain the respective carrier controls. Sample buffer extracts of equal numbers of bacteria were analyzed as for Fig. 1. Top panels show an approximately 6-kDa loading control protein detected by R. equi antiserum. The protein was less strongly expressed in the presence of ethanol. (C) An overnight culture of R. equi 33701 at 30°C was diluted into fresh BHI broth at an OD₆₀₀ of 0.1 and grown for 18 h in Sarstedt Snap Cap tubes that had been pretreated for 2 days at 21°C as indicated or in untreated glass tubes. Bacteria were grown at 30°C for 18 h (as for Fig. 1), and the bacteria were analyzed (Tube) or shaken in glass tubes supplemented with 5% (by volume) of the respective 2 day wash supernatant solution (Solvent). EtOH, ethanol; Isop, isopropanol; MeOH, methanol; DMSO, dimethyl sulfoxide. The immunoblots are representative of three independent experiments.

FIG 3

Sarstedt Snap Cap culture tubes lose the ability to induce VapA expression during heating at 121°C. (A) Sarstedt Snap Cap tubes left for 2 h at ambient temperature (mock) or 121°C dry heat are shown. Note the increased turbidity of the heated tube. (B) Autoclaving. An overnight culture of R. equi 33701 at 30°C was diluted into fresh BHI broth at an OD₆₀₀ of 0.1 and grown for 18 h in glass tubes or Snap Cap tubes that had been untreated (mock) or autoclaved (autocl) at 121°C. “BHI transfer” represents a sample in which the BHI broth from an autoclaved Snap Cap tube was transferred into a glass tube and vapA expression during growth at 30°C was assessed. In all cases, vapA expression was quantified by VapA Western blotting and blot scanning, and the blot signal for the glass/37°C sample was set as 100% in each experiment. Three independent experiments were performed, and the means and standard deviations (SD) are shown. (C) Dry heat. A Western blot representative of three experiments developed with anti-VapA shows samples as generated for panel B but in regular glass tubes or in Snap Cap tubes pretreated (121°C) or not (mock) in a dry heat cabinet for 120 min. Growth temperature was 30°C or 37°C, as indicated. As a loading control, we used rabbit antiserum to R. equi as for Fig. 2A and B.

FIG 4

Induction of VapA expression at 30°C depends on the virR/virS regulatory system. Overnight cultures of R. equi 103 and its isogenic deletion mutants 103/ΔvirR, 103/ΔvirS, and 103/ΔvcgB, grown at 30°C, were diluted into fresh BHI broth at an OD₆₀₀ of 0.1 and grown for 18 h in glass tubes (G) or Sarstedt Snap Cap tubes (PP) at the indicated temperatures. Extracts of equal numbers of bacteria were analyzed for vapA expression by Western blotting. A representative blot from three independent experiments is shown.

FIG 5

Quantitative real-time PCR of genes involved in vapA expression. Total RNA of R. equi 33701 was isolated from cultures grown for 18 h in glass tubes at 30°C or 37°C, as indicated, or in Sarstedt Snap Cap polypropylene (PP) tubes at 30°C and further analyzed by quantitative real-time PCR. Values shown indicate the fold change in vapA mRNA abundance (log₁₀) between bacteria from 30°C/glass cultures and from the respective test sample. alkB denotes the transcripts from an alkane monooxygenase gene. The dotted line marks the “no change” horizon. Data are means and standard deviations from three independent experiments.

FIG 6

Working model of VapA transcriptional regulation modified from reference . Increased temperature and moderately low pH act on the constitutively expressed LysR-type transcription regulator VirR in an unknown way. VirR then stimulates transcription of the icgA operon, including the virS gene. The newly produced VirS protein, an orphan two-component signal transduction response regulator, and then strongly upregulates vapA transcription. We show in this study that plastic-mediated induction of vapA expression at 30°C uses at least part of the same pathway through VirR, although the high-temperature requirement is circumvented. P, promoter.