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. 2017 Aug 18;85(9):e00269-17. doi: 10.1128/IAI.00269-17

Suspected Mycoplasma Contamination in the Study “Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus”

Wiwit Tantibhedhyangkul 1,, Naharuthai Inthasin 1, Patimaporn Wongprompitak 1, Pattama Ekpo 1
Editor: Craig R Roy2
PMCID: PMC5563574  PMID: 28821640

LETTER

In a recent publication (1), Gharaibeh et al. demonstrated that human Toll-like receptor 2 (TLR2) recognizes Orientia tsutsugamushi by using TLR2-transfected HEK293 cells. They demonstrated that murine TLR2 is required for inflammatory cytokine production by bone marrow-derived dendritic cells. Additionally, heat-killed O. tsutsugamushi induced higher levels of cytokine production than live organisms. Next, they performed in vivo experiments with wild-type and TLR2-deficient mice but the results were inconclusive. They revealed that TLR2 is dispensable for intradermal infection with O. tsutsugamushi but might be partially required for bacterial clearance after intraperitoneal infection. Despite its protective role in bacterial restriction, TLR2 seems to cause tissue injury, as well as increase disease severity and the mortality rate.

In contrast to that study, our unpublished data have demonstrated that neither TLR2 nor TLR4 recognizes O. tsutsugamushi. However, if we use O. tsutsugamushi with mycoplasma contamination to stimulate TLR2-transfected HEK293 cells, we obtain false-positive results (Fig. 1). Mycoplasma organisms are well known to possess several lipoproteins, including macrophage-activating lipopeptide 2 (2), which contains diacylated lipopeptides (Pam2Cys) as a TLR2 ligand (3). Heat killing of mycoplasma organisms can cause bacterial lysis and the subsequent release of more lipopeptides into the extracellular milieu. This hypothesis may explain why heat-killed organisms induce a stronger inflammatory response in mouse dendritic cells (1). In contrast, our unpublished data and previous studies have demonstrated that live O. tsutsugamushi without mycoplasma contamination induces higher cytokine response levels in human monocytes and macrophages (4, 5). Moreover, it is noteworthy that Gharaibeh et al. collected O. tsutsugamushi Karp from infected L929 cells at 14 days postinfection. However, on the basis of our experience, infected cells usually show cytopathic effects within 6 to 9 days, whereas mycoplasma-contaminated cells exhibit growth retardation of O. tsutsugamushi Karp and then show cytopathic effects at about 2 weeks postinfection. Therefore, our observations suggest that the results presented by Gharaibeh et al. (1) may be confounded by mycoplasma contamination, which can cause false-positive in vitro and in vivo results.

FIG 1.

FIG 1

O. tsutsugamushi (OT) Karp organisms with or without Mycoplasma orale (Myco) contamination were used to infect HEK-Blue cells (InvivoGen, San Diego, CA), which stably coexpress a human TLR (hTLR) gene and an NF-κB-inducible secreted embryonic alkaline phosphatase reporter gene. Alkaline phosphatase levels in supernatants were measured by spectrophotometry, normalized to those of unstimulated cells, and correlated with fold changes in NF-κB activation. Data represent the mean ± the standard error of the mean of triplicate values and are representative of three independent experiments. Heat-killed Listeria monocytogenes (HKLM) is a positive control for TLR2 activation. To calculate multiplicities of infection (MOIs), O. tsutsugamushi was quantified by real-time PCR. LPS, lipopolysaccharide.

Footnotes

For the author reply, see https://doi.org/10.1128/IAI.00326-17.

REFERENCES

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