Gram-positive bacteria are mainly sensed by TLR2 which is expressed on cell surface. Oldenburg et al found that mice lacking TLR2 are still responsive to Staphylococcus aureus or Streptococcus pneumoniae, which indicate that other TLRs play a role in the detection of these bacteria (1). They ruled out the possibility of other classes of pattern recognition receptors such as C-type lectins, RIG-I-like helicases, or nucleotide binding domain-and leucine-rich repeat-containing proteins in the detection of heat inactivated S. aureus or S. pneumoniae by using different knockout macrophages (1). They found that the production of cytokine was dependent on MyD88, which suggest that TLRs are mainly responsible for detection of these bacteria and there is no role of RIG-I-like helicases or nucleotide binding domain-and leucine-rich repeat-containing proteins (1). TLRs are involved in the recognition of viral infection and Tlr13^-/- cells are highly susceptible to vesicular stomatitis virus infection (6).

Oldenburg et al focused on endosomal TLRs including TLR3, -7, -8, -9, -11, and -13 whether these have some role in cell activation. Pretreatment of bafilomycin in Tlr2^-/- macrophages abrogated recognition of Gram-positive bacteria. Bacterial inoculums treated with ribonuclease A were unable to produce TNF-α in Tlr2^-/- and Tlr23479^-/- macrophages. From these results they concluded that an endosomal RNA sensor is involved in detection of heat inactivated S. aureus (1). They generated two types of bone marrow-derived dendritic cells (DCs) called as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Tlr23479^-/- CD8^high cDCs express TLR11, -12, -13 and signal regulatory protein α (Sirp)^high cDCs express TLR13 but lack TLR11, -12 while Tlr23479^-/- pDCs express TLR12 and lack TLR11, -13. Using these cDCs and pDCs they showed that TLR13 acts as bacterial single-stranded RNA sensor. For identification of relevant RNA, they treated heat inactivated S. aureus with calf intestinal phosphatase which affect the integrity of 16S and 23S rRNA or double stranded RNA-specific RNase III or VI (1). They further extended their work and degraded large rRNAs, namely 16S and 23S rRNA and isolated high molecular weight rRNA. Low molecular weight portions from total RNA were unable to stimulate the cells but high molecular weight portions of bacterial RNA activated Tlr23479^-/- cells. From these results they concluded that portion of large bacterial rRNA activates the cDCs in MyD88-dependant manner (1).

For characterization of a TLR responsible for detection of 23S rRNA, TLR13 was focused as they ruled out the involvement of TLR8. They identified the conserved bacterial 23S rRNA sequence "CGGAAAGACC" as a ligand for TLR13 (1). To verify the role of TLR13 in detection of bacterial RNA, TLR13 expression was targeted with small interfering RNA (1). Transfection of DCs with small interfering RNA against TLR13 resulted in significantly less production of IL-12 p40 mRNA upon treatment with bacterial RNA (5). To further explore the role of TLR13 in recognition of bacterial RNA, chinese hamster ovary cells were cotransfected with an NF-κB luciferase reporter and vectors expressing TLR13, -7, or -8, or empty vector, and then treated with bacterial RNA. They found that TLR13-transfected cells significantly induced NF-κB activation. Chinese hamster ovary cells expressing TLR13 induced NF-κB luciferase reporter activity in response to both live and heat killed Streptococcus pyogenes but failed to do so in the presence of ribonuclease A in the heat killed bacterial inoculums. This shows that TLR13 contributes in recognition of group A streptococci (5).

Oldenburg et al showed that bacteria can avoid TLR13 detection by modification in 23S rRNA (1). The erythromycin-resistance methylase family of methyltransferases confers resistance to the macrolide, lincosamide and streptogramin antibiotics by the methylation of 23S ribosomal RNA which leads to abolition of their ability to bind to the ribosome and exhibit their antibiotic activity (1, 7). They found that modification in 23S rRNA via methylation leads to antibiotic resistance and also eradicates immunostimulatory activity in clinical isolates of S. aureus (1).

They show that murine TLR13 is required for detection of bacterial conserved 23S ribosomal RNA sequence and bacteria can evade recognition via TLR13 by specific mechanisms of antibiotic resistance. Hence, they suggested that in humans there may be a related RNA-sensing pattern recognition receptor that can recognize the bacteria which escape TLR13 detection.