IL-1 в Localizes to the Cytosolic Face of the Inner Membrane and in the Nucleoids of A. actinomycetemcomitans
In our experimental set up, IL-ip could be detected inside viable A. actinomycetemcomitans biofilm cells grown in close contact with the organotypic gingival mucosa (Paino et al. 2012). In most cases, gold-particle-labeled anti-ILip antibodies were shown by immuno-electron microscopy to localize to either the cytosolic side of the inner membrane or within the nucleoids of A. actinomycetemcomitans (Fig. 21.3a). The uptake of IL-ip could also be detected in single cells of bacteria that were located in close vicinity to the epithelium (Fig. 21.3b). Only viable A. actinomycetemcomitans cells could take up IL-ip; cells treated with antibiotics were smaller and showed no signs of IL-ip uptake (Paino et al. 2012).
Inner Membrane Protein ATP Synthase Subunit в Binds IL-1 в
Because IL-ip localized inside the bacterial cells, we became interested in the possible intracellular proteins that might interact with IL-ip. The intracellular proteins were first separated by native-polyacrylamide gel electrophoresis (PAGE), and blotted onto a membrane that was incubated with biotinylated IL-ip and detected with avidin-HRP. A similar native-PAGE gel with intracellular proteins was also silver-stained. The protein bands that interacted with IL-ip were extracted from the silver-stained gel and identified by mass spectrometry.
One of the intracellular proteins that interacted with IL-ip was F1F0 ATP synthase subunit p. F1F0 ATP synthase is a well-conserved multi-subunit protein
Figure 21.3 Viable A. actinomycetemcomitans cells internalize IL-ip both (a) in biofilm and (b) as planktonic cells. Pre-grown A. actinomycetemcomitans biofilms were co-cultured with an organotypic gingival mucosa, and the localization of IL-1 p was investigated using immuno- electron microscopy. IL-1 p was detected both attached to the inner membrane (IM) and in the outer edges of nucleoids (N).
complex that can be found in all living organisms from bacteria, mitochondria, and chloroplasts to the surfaces of several eukaryotic cell types (Hong and Pedersen 2008). We showed that IL-1p bound specifically to the trimeric form of subunit p, which was produced as a recombinant protein (Paino et al. 2011). The binding of IL-ip to the recombinant protein was detected with an electrophoretic mobility shift assay (EMSA) combined with immunodetection using anti-IL-ip antibodies. The catalytic domain of the F1 part of the ATP synthase consists of three p subunits (Nakamoto et al. 2008), suggesting that IL-ip targets the complete catalytic domain of the enzyme. Different antimicrobial peptides with a-helical structures have been shown to inhibit the F1F0 ATP synthase of E. coli by specifically binding to its p subunit (Laughlin and Ahmad 2010). The positively charged a-helix in antimicrobial peptides interacts with the negatively charged DELSEED sequence in the p-subunit of E. coli (Laughlin and Ahmad 2010). In IL-1p, the potential interacting a-helical portion is the extension loop with a QGQDMEQQ amino acid sequence.
F-type ATP synthase has served as a target for antimicrobial drug development against tuberculosis. The novel drug R207910 binds the c subunit, inhibiting ATP synthesis, which eventually eliminates even drug-resistant Mycobacterium tuberculosis infection (Andries et al. 2005). However, in vitro studies show that M. tuberculosis may also develop resistance to R207910 (Petrella et al. 2006). Of the more conventional antimicrobial agents, iodine deactivates the F1 part of ATP synthase (Petrone et al. 1987) by covalently modifying the p subunit.
A. actinomycetemcomitans is known to be especially resistant to host defenses and antimicrobials due to its ability to invade epithelial cells (Meyer et al. 1991) and vascular endothelial cells (Schenkein et al. 2000). Salmonella enterica, an intracellular pathogen, exploits the specific virulence protein MtgC to inhibit its own F1F0 ATP synthase; this improves persistence within macrophages by balancing ATP levels in an environment with an elevated pH (Lee et al. 2013). Whether the binding of IL-1p to the F1F0 ATP synthase subunit p of A. actinomycetemcomitans is antimicrobial or enhances the virulence and robustness of the biofilm or intracellular planktonic cells needs to be confirmed.