Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles

Abstract

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

“…A later immunocytochemical analysis demonstrated that LtxA was located on the OMV surface (Berthold et al, 1992). This surface localization of LtxA on the OMV was validated by subsequent reports that trypsin, which is unable to access the vesicle lumen, completely digests OMV‐associated LtxA (Nice et al, 2018), and the demonstration that treatment of the OMVs with DNase, which likewise can only access surface‐associated LtxA, is able to release LtxA from the OMV surface (Ohta et al, 1991). Release of the RTX toxins via association with OMVs seems to be highly conserved among this family of toxins, as this mechanism of toxin release has been reported for B. pertussis CyaA, K. kingae RtxA, V. cholerae cytolysin, and E. coli α‐hemolysin (Balsalobre et al, 2006; Donato et al, 2012; Elluri et al, 2014; Maldonado et al, 2011).…”

“…LtxA and outer membrane protein A (OmpA) were found in OMVs collected in lighter layers (larger OMVs), whereas Cdt was concentrated in OMVs found in heavier layers (smaller OMVs; Rompikuntal et al, 2012). We have similarly found that A. actinomycetemcomitans strain JP2 produces at least two populations of OMVs, one highly abundant population of small OMVs (less than 100 nm in diameter), and a less abundant population of large OMVs (greater than 300 nm in diameter; Nice et al, 2018). Our preliminary evidence indicates that LtxA is concentrated in the “large” OMVs and excluded from the “small” OMVs, leading us to hypothesize that each population of OMV likely serves entirely different purposes.…”

“…JP2 and 652 OMVs, as well as OMVs produced by an isogenic, leukotoxin‐deficient strain of JP2, associated with the cytoplasmic membrane of HL‐60 cells to a similar extent within two min (Demuth et al, 2003). We have recently reported that JP2 OMVs are delivered to host (THP‐1) cells in a cholesterol‐ and LFA‐1‐independent manner (Nice et al, 2018), unlike free LtxA, which requires both cholesterol (Brown et al, 2013; Fong et al, 2006) and LFA‐1 (DiFranco et al, 2012; Dileepan et al, 2007; Kieba et al, 2007). This finding is important as it suggests that OMV packaging may permit or even promote delivery of LtxA to cells that do not express LFA‐1, that is, non‐immune cells.…”

Differential Cell Lysis Among Periodontal Strains of JP2 and Non-JP2 Genotype of Aggregatibacter actinomycetemcomitans Serotype B Is Not Reflected in Dissimilar Expression and Production of Leukotoxin

“…Environmental factors are of great importance and have been reported to influence on both the production and the secretion of the LtxA [18,[22][23][24][25]. Previous studies have reported that the secretion of LtxA occurs into the environment both as a water-soluble protein and as an attached protein to membrane vesicles [26][27][28]. The LtxA attached to the vesicle is expected to be found in the supernatant used in the ELISA and the Western blot; however, this is not the case for the cell lysis assay used by Åberg and coworkers [20].…”

Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA) Requires Death Receptor Fas, in Addition to LFA-1, To Trigger Cell Death in T Lymphocytes

“…LtxA has been shown to kill lymphocytes via a caspase-dependent mechanism (18,20,27). Additionally, previous studies have suggested that LtxA can induce certain cellular events independent of LFA-1 expression (16,28). To determine if LtxA is able to activate caspases in the absence of CD18 or CD11a, Jurkat E6.1 cells and Jurkat CD18 Ϫ/Ϫ clones or Jurkat CD11a Ϫ/Ϫ clones were treated with various concentrations of LtxA for 24 h, and caspase activation was assessed using a fluorescent polycaspase reagent, FAM-VAD-FMK FLICA (where FLICA is fluorescent-labeled inhibitor of caspases).…”