Oral Shedding of an Oncogenic Virus Alters the Oral Microbiome in HIV+ Patients

Abstract

Kaposi's Sarcoma (KS) caused by Kaposi's sarcoma-associated herpesvirus (KSHV) continues to be the most common AIDS-associated tumor. Involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. Numerous types of cancer are associated with the alterations of in components of the microbiome. However, little is known about how KSHV coinfection affects the oral microbiome in HIV+ patients, especially in a "pre-cancer" niche. Using 16S rRNA pyrosequencing, we found that oral shedding of KSHV correlated with altered oral microbiome signatures in HIV+ patients, including a reduction in the microbiota diversity, changing the relative composition of specific phyla and species, and regulating microbial functions. Furthermore, we found that Streptococcus sp., one of the most increased species in the oral cavity of HIV+/KSHV+ patients, induced KSHV lytic reactivation in primary oral cells. Together, these data indicate that oral shedding of KSHV may manipulate the oral microbiome to promote viral pathogenesis and tumorigenesis especially in immunocompromised patients.

Keywords: HIV; KSHV; microbiome; oncogenic virus; oral microbiota.

Figure 1

The components of microbiome at the phylum or species level from individual samples.

Figure 2

Diversity analysis of oral microbiome in HIV patients with or without Kaposi’s sarcoma-associated herpesvirus (KSHV) coinfection. (A,B) The Alpha rarefaction (Shannon index) and Alpha diversity (Pielou’s evenness) analysis of three groups of HIV patients with or without KSHV coinfection.

Figure 3

The top phylum and species in three groups of HIV patients with or without KSHV coinfection.

Figure 4

The significantly changed phyla or species in the group of HIV patients with KSHV oral shedding.

Figure 5

Induction of KSHV lytic gene expression by conditioned medium from Streptococcus species. (A,B) Oral fibroblasts periodontal ligament fibroblasts (PDLF) and human gingival fibroblasts (HGF) were infected with purified rKSHV.219 virus (MOI ~ 5) for 2 h. A 24 h later, cells were treated with filtered conditioned medium from Streptococcus salivarius C699 or Streptococcus mutans NCTC 10449 strain culture or fresh medium as a control (diluted as 1:50) for additional 48 h. Viral gene expression was measured and quantified using qRT-PCR. Error bars represent the S.D. for three independent experiments. *p < 0.05; **p < 0.01 (two-tailed Student’s t-test). PDLF, periodontal ligament fibroblasts; HGF, human gingival fibroblasts.

Figure 6

Induction of KSHV lytic reactivation by Streptococcus species. (A,B) Oral fibroblasts PDLF and HGF were infected with purified rKSHV.219 virus and treated with filtered conditioned medium of Streptococcus species as described above, and then images were captured by using fluorescence microscope.

Figure 7

NF-κB signaling pathway is involved in Streptococcus species induced KSHV lytic reactivation. (A) PDLF and HGF were infected and treated as described above, and then protein expression was detected by using Western blot. (B) PDLF cells were infected first, then transfected with a recombinant construct encoding NF-κB p65 for 48 h, and treated with filtered conditioned medium from Streptococcus species culture for additional 48 h. Viral gene expression was quantified using qRT-PCR. Error bars represent the SD for three independent experiments.