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. 2020 Mar 28;221(8):1331-1341.
doi: 10.1093/infdis/jiz249.

Kaposi Sarcoma-Associated Herpesvirus and Staphylococcus aureus Coinfection in Oral Cavities of HIV-Positive Patients: A Unique Niche for Oncogenic Virus Lytic Reactivation

Lu Dai  1 , Jing Qiao  2 , Jun Yin  2 , Alana Goldstein  3 , Hui-Yi Lin  4 , Steven R Post  1 , Zhiqiang Qin  1
Affiliations

Kaposi Sarcoma-Associated Herpesvirus and Staphylococcus aureus Coinfection in Oral Cavities of HIV-Positive Patients: A Unique Niche for Oncogenic Virus Lytic Reactivation

Lu Dai et al. J Infect Dis. .

Abstract

Collectively, viruses are the principal cause of cancers arising in patients with immune dysfunction, including human immunodeficiency virus (HIV)-positive patients. Kaposi sarcoma (KS) etiologically linked to Kaposi sarcoma-associated herpesvirus (KSHV) continues to be the most common AIDS-associated tumor. The involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. HIV infection incurs an increased risk among individuals with periodontal diseases and oral carriage of a variety of pathogenic bacteria. However, whether interactions involving periodontal bacteria and oncogenic viruses in the local environment facilitate replication or maintenance of these viruses in the oral cavity of HIV-positive patients remain largely unknown. We previously showed that pathogen-associated molecular patterns (PAMPs) from specific periodontal bacteria promoted KSHV entry into oral cells and subsequent establishment of latency. In the current study, we demonstrate that Staphylococcus aureus, one of common pathogens causing infection in HIV-positive patients, and its PAMPs can effectively induce KSHV lytic reactivation from infected oral cells, through the Toll-like receptor reactive oxygen species and cyclin D1-Dicer-viral microRNA axis. This investigation provides further clinical evidence about the relevance of coinfection due to these 2 pathogens in the oral cavities of a cohort HIV-positive patients and reveals novel mechanisms through which these coinfecting pathogens potentially promote virus-associated cancer development in the unique niche of immunocompromised patients.

Keywords: Staphylococcus aureus; HIV; KSHV; Kaposi sarcoma; microRNA.

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Figures

Figure 1.
Figure 1.
Induction of Kaposi sarcoma–associated herpesvirus (KSHV) lytic reactivation by Staphylococcus aureus–conditioned medium and/or S. aureus–derived lipoteichoic acid (LTA). A, Oral fibroblasts were infected by purified KSHV (multiplicity of infection [MOI], approximately 10) for 2 hours. Twenty-four hours later, cells were treated with filtered conditioned medium from overnight S. aureus strain 8325-4 culture or fresh medium control (diluted as 1:50) for an additional 48 hours. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis was used to quantify the expression of representative viral latency (Lana) and lytic genes (Rta, vGpcr, K8.1, and Orf57). B and D, Conditioned medium from periodontal ligament fibroblasts (PDLFs) in panels A or C were collected, ultracentrifuged, and resuspended to infect fresh PDLFs. Lana transcripts (reflecting the level of infectious particles released) were measured by qRT-PCR. C, Cells were infected as described in panel A and then incubated with S. aureus–derived LTA (5.0 µg/mL) for 48 hours, followed by qRT-PCR analysis. Error bars represent the SD for 3 independent experiments. HGF, human gingival fibroblast. *P < .05 and **P < .01, by a 2-tailed Student t test.
Figure 2.
Figure 2.
Toll-like receptor 2 (TLR2)–mediated reactive oxygen species (ROS) production and signaling is required for induction of Kaposi sarcoma–associated herpesvirus (KSHV) lytic reactivation by Staphylococcus aureus–conditioned medium. A, Oral fibroblasts were infected by purified KSHV (multiplicity of infection [MOI], approximately 10) for 2 hours. Twenty-four hours later, cells were treated with filtered conditioned medium from S. aureus 8325-4 culture or fresh medium as a control (diluted as 1:50) for an additional 48 hours. Intracellular levels of ROS were quantified using the ROS-specific dye CM-H2DCFDA and flow cytometry. B, Cells were infected as described in panel A and then were or were not treated with the antioxidant N-acetylcysteine (NAC; 1 mM) for an additional 24 hours. Cells were then incubated with S. aureus–conditioned medium or control for 48 hours, followed by quantitative reverse transcription–polymerase chain reaction analysis. C, Cells were infected as described in panel A. Twenty-four hours later, cells were transfected with either negative control small interfering RNA (n-siRNA), TLR2 siRNA, or TLR4 siRNA for 48 hours and then treated with filtered conditioned medium from S. aureus 8325-4 culture (diluted 1:50) for an additional 48 hours. Intracellular levels of ROS were quantified as described above. D, Human gingival fibroblasts (HGFs) were treated as described in panel C, protein expression was detected by immunoblots, and NADPH oxidase activities were measured using a chemiluminescence-based assay as described in “Methods.” Error bars represent the SD for 3 independent experiments. MFI, median fluorescence intensity; PDLF, periodontal ligament fibroblast. **P < .01, by a 2-tailed Student t test.
Figure 3.
Figure 3.
Higher levels of salivary bacterial pathogen–associated molecular patterns (PAMPs), reactive oxygen species (ROS)/reactive nitrogen species (RNS), and host antioxidant factors are present in patients positive for both human immunodeficiency virus (HIV) and Kaposi sarcoma–associated herpesvirus (KSHV). A and B, The salivary total lipoteichoic acid (tLTA; A) and ROS/RNS (B) levels in HIV-positive patients were measured using an enzyme-linked immunosorbent assay as described in “Methods.” C and D, Levels of the host antioxidant factor uric acid (C) and peroxidase activity (D) were measured using the commercial kits as described in “Methods.” Error bars represent the SD for 3 independent experiments. RFU, relative fluorescence units; −, negative; +, positive. *P < .05 and **P < .01, by a 2-tailed Student t test.
Figure 4.
Figure 4.
Cyclin D1–mediated downregulation of Kaposi sarcoma–associated herpesvirus (KSHV) microRNA (miR) expression from infected oral cells by Staphylococcus aureus–conditioned medium. A, Human gingival fibroblasts (HGFs) were infected by purified KSHV (multiplicity of infection [MOI], approximately 10) for 2 hours. Twenty-four hours later, cells were incubated with S. aureus 8325-4–conditioned medium (1:50 dilution) or control medium (Ctrl) for 48 hours, followed by quantitative reverse transcription–polymerase chain reaction for KSHV miRNA quantification. Protein expression was measured by immunoblots. B–D, Cells were infected with or without purified KSHV (MOI, approximately10) for 2 hours. Twenty-four hours later, cells were incubated with S. aureus 8325-4–conditioned medium (1:50 dilution) or Ctrl for 48 hours. Protein expression, cell viability, and cell cycle distribution were measured using immunoblots, WST-1 assays, and flow cytometry, respectively. Error bars represent the SD for 3 independent experiments. *P < .05 and **P < .01, by a 2-tailed Student t test.
Figure 5.
Figure 5.
Internalization of Staphylococcus aureus and coinfection with Kaposi sarcoma–associated herpesvirus (KSHV) in human primary cells. A, Human umbilical vein endothelial cells (HUVECs) and oral fibroblasts were grown as monolayers in 24-well tissue culture plates and then infected by S. aureus strain MN8 (containing S. aureus major virulence genes SarA or Agr labeled with the YFP reporter in their promoters) at a multiplicity of infection (MOI) of approximately 100 for 1 hour. The cells were washed and treated with lysostaphin (10 µg/mL) for 20 minutes to lyse extracellular bacteria prior to lifting with trypsin. The intracellular YFP signal was detected by flow cytometry, and uninfected cells served as the negative control. B, Cells were first infected by purified KSHV (MOI, approximately 10) for 2 hours. A total of 24 hours later, cells were infected with S. aureus strain MN8 (Agr-YFP). After incubation for an additional 72 hours, immunofluorescence was performed for detection of LANA (representing latently infected KSHV; red) and YFP (representing internalized S. aureus; green), and nuclei were identified by DAPI staining (blue). C, Human gingival fibroblasts (HGFs) were first infected by purified KSHV (MOI, approximately 10) for 2 hours. Twenty-four hours later, cells were infected with S. aureus strain MN8. After additional incubation for 48 hours, cell viability was measured using the WST-1 assay. D, HUVECs with or without KSHV infection were infected with S. aureus strain MN8. The numbers of internalized bacteria at different time points after infection were determined by measuring colony counts on a tryptic soy broth plate. Error bars represent the SD for 2 independent experiments. CFU, colony-forming units; PDLF, periodontal ligament fibroblast. **P < .01, by a 2-tailed Student t test.
Figure 6.
Figure 6.
Clinical prevalence of Staphylococcus aureus and Kaposi sarcoma–associated herpesvirus (KSHV) shedding within saliva samples from cohort human immunodeficiency virus (HIV)–positive patients. A, Total DNA was extracted from saliva samples of cohort HIV-positive patients, using the QIAamp DNA mini-kit (Qiagen). Then, polymerase chain reaction analysis was performed using specific primers designed for the 16S ribosomal RNA (rRNA) of S. aureus or the KSHV-encoded major latency gene Lana. Amplicons were subsequently identified by ethidium bromide–loaded agarose gel electrophoresis; representative bands are shown. Asterisks represent double-positive patients. The KSHV seroprevalence was determined using quantitative enzyme-linked immunosorbent assays (ELISAs) as described in “Methods.” B, Results for samples from 53 HIV-positive patients are shown. C, A hypothetical model of the mechanisms through which S. aureus coinfection induces KSHV lytic reactivation and replication from latently infected oral cells. Ago, Argonaute; LTA, lipoteichoic acid; ROS, reactive oxygen species; −, negative; +, positive.
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References

    1. Cesarman E, Damania B, Krown SE, Martin J, Bower M, Whitby D. Kaposi sarcoma. Nat Rev Dis Primers 2019; 5:9. - PMC - PubMed
    1. Vanni T, Sprinz E, Machado MW, Santana Rde C, Fonseca BA, Schwartsmann G. Systemic treatment of AIDS-related Kaposi sarcoma: current status and perspectives. Cancer Treat Rev 2006; 32:445–55. - PubMed
    1. Engels EA, Biggar RJ, Hall HI, et al. . Cancer risk in people infected with human immunodeficiency virus in the United States. Int J Cancer 2008; 123:187–94. - PubMed
    1. Bonnet F, Lewden C, May T, et al. . Malignancy-related causes of death in human immunodeficiency virus-infected patients in the era of highly active antiretroviral therapy. Cancer 2004; 101:317–24. - PubMed
    1. Maurer T, Ponte M, Leslie K. HIV-associated Kaposi’s sarcoma with a high CD4 count and a low viral load. N Engl J Med 2007; 357:1352–3. - PubMed

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