9.B.159 The Inclusion Membrane Protein (IncA) Family

The IncA family includes several proteins that are secreted via Type III Secretion Systems (TC# 3.A.6) in chlamydial species.  No member of this family has been identified with certainty outside of the Chlamydiae.  The proteins enterred into TCDB as IncA proteins usually have a 2 or 4 TMS topology but are in general not demonstrably homologous using TC BLAST.  They are very divergent in sequence, but they all belong to the IncA family of Pfam.  They may be related to mitochodrial Ca²⁺ uniporters and prokaryotic Mg²⁺ channels (TC# 1.A.77), and consequently, these IncA homologues may function as divalent cation channels (Lee et al. 2014). The N-terminal regions appear to be more similar than the remaining parts of the protein.  9.B.159.2.3 retrieves members of 7 sub-families including its own.

Inc proteins may assemble in the membrane of the inclusion (which separates the bacteria from the host cell cytoplasm) to form specific multi-molecular complexes (Gauliard et al. 2015).  IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). The functional core of IncA retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions.This core region of 2 TMS proteins is composed almost entirely of α-helices and assembles into stable homodimers in solution (Ronzone et al. 2014).

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Cingolani et al. 2019 presented the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identified an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during infection. Solithromycin (Sol) impedes homotypic fusion of Chlamydia-containing vacuoles and reduces secretion of the type III secretion (T3S) effector IncA (Gao et al. 2018).

VAMP3 and VAMP4 are eukaryotic SNARE proteins that mediate membrane fusion and are recruited to the inclusion to facilitate inclusion expansion (Bui et al. 2020). VAMPs 3 and 4 are probably recruited by Incs. In chlamydia-infected cells, five Incs temporally and transiently interact with VAMP3. Loss of incA or ct813 expression altered VAMP3 localization to the inclusion. Thus, these transient interactions likely contribute to the chlamydial developmental cycle (Bui et al. 2020).