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. 2014 Jun 27;9(6):e101326.
doi: 10.1371/journal.pone.0101326. eCollection 2014.

Lipoteichoic acid (LTA) and lipopolysaccharides (LPS) from periodontal pathogenic bacteria facilitate oncogenic herpesvirus infection within primary oral cells

Lu Dai  1 , Michael R DeFee  2 , Yueyu Cao  3 , Jiling Wen  4 , Xiaofei Wen  4 , Mairi C Noverr  5 , Zhiqiang Qin  6
Affiliations

Lipoteichoic acid (LTA) and lipopolysaccharides (LPS) from periodontal pathogenic bacteria facilitate oncogenic herpesvirus infection within primary oral cells

Lu Dai et al. PLoS One. .

Abstract

Kaposi's sarcoma (KS) remains the most common tumor arising in patients with HIV/AIDS, and involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. HIV infection incurs an increased risk for periodontal diseases and oral carriage of a variety of bacteria. Whether interactions involving pathogenic bacteria and oncogenic viruses in the local environment facilitate replication or maintenance of these viruses in the oral cavity remains unknown. In the current study, our data indicate that pretreatment of primary human oral fibroblasts with two prototypical pathogen-associated molecular patterns (PAMPs) produced by oral pathogenic bacteria-lipoteichoic acid (LTA) and lipopolysaccharide (LPS), increase KSHV entry and subsequent viral latent gene expression during de novo infection. Further experiments demonstrate that the underlying mechanisms induced by LTA and/or LPS include upregulation of cellular receptor, increasing production of reactive oxygen species (ROS), and activating intracellular signaling pathways such as MAPK and NF-κB, and all of which are closely associated with KSHV entry or gene expression within oral cells. Based on these findings, we hope to provide the framework of developing novel targeted approaches for treatment and prevention of oral KSHV infection and KS development in high-risk HIV-positive patients.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. LTA and LPS from periodontal pathogenic bacteria increase KSHV entry and latent gene expression during de novo infection of primary oral cells.
(A) HGF and PDLF cells were pre-treated with 5 µg/mL of purified LTA from S. aureus or LPS from P. gingivalis for 24 h, then infected with purified KSHV (MOI∼3). IFA to identify LANA expression were performed 24 h p.i. using an anti-LANA monoclonal antibody and a secondary antibody conjugated to Texas Red, along with DAPI for nuclear colocalization (blue). (B–C) HGF and PDLF were pre-treated with LTA or LPS (2.5, 5, 10 µg/mL, respectively) for 24 h, then infected with purified KSHV as (A). qRT-PCR was used to quantify ORF73 (Lana) transcripts at 24 h p.i (B). qPCR was used to quantify intracellular KSHV DNA contents 2 h after incubation of cells with the virions (C). Error bars represent the standard errors of the means for 3 independent experiments. * = p<0.05, ** = p<0.01.
Figure 2
Figure 2. Heparan sulfate is the major cellular receptor responsible for KSHV entry into oral fibroblasts.
(A) HGF and PDLF were first treated with 0.4 mg/mL mannan (the inhibitor of DC-SIGN) or 20 µg/mL xCT Ab, or purified virions were first treated with 0.5 mg/mL heparin (the competitor for heparan sulfate) or 15 µg/mL soluble integrin α3β1 and αvβ3 for 1 h at 4°C, then the cells were infected with purified virions (MOI∼3) for 2 h at 37°C. After that, cells were trypsinized and washed to remove extracellular KSHV virions. Total cellular DNA was prepared and the internalized viral copies were measured by qPCR as described in Methods. Error bars represent the standard errors of the means for 3 independent experiments. * = p<0.05, ** = p<0.01. (B–C) PDLF were incubated with 10 µg/mL LTA or LPS for 24 h, and purified virions (MOI∼3) were incubated with or without 0.5 mg/mL heparin for 1 h at 4°C. Cells were subsequently infected for 2 h at 37°C, then DNA (2 h p.i.) and RNA (24 h p.i.) were isolated for quantification of intracellular viral copies or ORF73 (Lana) transcripts using qPCR (B) or qRT-PCR (C), respectively. **/##/formula image formula image = p<0.01 relative to K (**), LTA+K (##), and LPS+K (formula image formula image).
Figure 3
Figure 3. LTA and LPS from periodontal pathogenic bacteria increase heparan sulfate proteoglycans (HSPG) expression in HGF and PDLF.
(A) HGF and PDLF cells were pre-treated with 10 µg/mL of LTA from S. aureus or LPS from P. gingivalis for 24 h, then proteins expression was detected by immunoblots. (B) HGF were treated as (A), and total HSPG expression was detected by IFA as described in Methods.
Figure 4
Figure 4. LTA and LPS from periodontal pathogenic bacteria induce ROS production from oral cells through increasing NADPH oxidase complex activities.
(A) HGF and PDLF cells were treated with indicated concentrations of LTA from S. aureus or LPS from P. gingivalis for 24 h, then intracellular ROS production was measured as described in Methods. (B–C) Cells were treated as above, then proteins expression was detected by immunoblots (B), and NADPH oxidase activities were measured using a chemiluminescence-based assay as described in Methods (C). Error bars represent the standard errors of the means for 3 independent experiments. * = p<0.05, ** = p<0.01.
Figure 5
Figure 5. The antioxidant NAC reduces viral entry and gene expression during KSHV de novo infection.
(A, C) HGF cells were pre-treated with 10 µg/mL of LTA from S. aureus or LPS from P. gingivalis for 24 h, then treated with or without NAC (10 mM) for 2 h, followed by infected with KSHV for 2 h and internalized viral DNA copies were measured by qPCR. (B, D) HGF were pretreated and infected as above, then treated with or without NAC (1 mM) for additional 24 h and Lana transcripts were measured by qRT-PCR. Error bars represent the standard errors of the means for 3 independent experiments. ** = p<0.01.
Figure 6
Figure 6. LTA and LPS from periodontal pathogenic bacteria increase viral latent gene expression through activating intracellular signaling pathways.
(A) HGF and PDLF cells were pre-treated with indicated concentrations of LTA from S. aureus or LPS from P. gingivalis for 24 h, then infected with purified KSHV (MOI∼3). Proteins expression was detected by immunoblots. (B–C) HGF were pre-treated with 10 µg/mL of LTA from S. aureus or LPS from P. gingivalis for 24 h, then treated with 10 µM of the MEK/MAPK inhibitor U0126 or NF-κB inhibitor Bay11-7082 for 1.5 h, respectively, followed by infection with KSHV. qRT-PCR was used to quantify ORF73 (Lana) transcripts. Error bars represent the standard errors of the means for 3 independent experiments. ** = p<0.01.
Figure 7
Figure 7. Schematic representation of mechanisms for facilitating KSHV entry and latency establishment in oral cells by periodontal bacterial LTA and LPS.
See this image and copyright information in PMC

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