2.A.127 The Enterobacterial Cardiolipin Transporter (CLT) Family
During infection, Gram-negative bacteria remodel their OM to promote survival and replication within host tissues. Salmonella rely on the PhoPQ two-component regulators to coordinate OM remodeling in response to environmental cues. In a screen for mediators of PhoPQ-regulated OM remodeling in Salmonella enterica Typhimurium, Dalebroux et al. 2015 identified PbgA (formerly YejM with a DUF3413 transmembrane domain (N-terminus) and a periplasmic alkaline phosphatase (ALP-like) domain (C-terminus)). The latter binds cardiolipin glycerophospholipids (CL) near the inner membrane and promotes their PhoPQ-regulated trafficking to the OM. Purified-PbgA oligomers are tetrameric, and the periplasmic domain contains a globular region that binds to the OM in a PhoPQ-dependent manner. Thus, PbgA forms a complex that may bridge the envelope for regulated cardiolipin delivery. PbgA globular region-deleted mutant bacteria are severely attenuated for pathogenesis, suggesting that increased cardiolipin trafficking to the OM is necessary for Salmonella to survive within host tissues that activate PhoPQ.
The outer membrane of Gram-negative bacteria is asymmetric with the outer leaflet being composed of lipopolysaccharide (LPS) and the inner leaflet is formed by glycerophospholipid (GPL). Cardiolipin (CL) plays an important role in OM biogenesis and pathogenesis, and the inner membrane (IM) protein PbgA, containing five N-terminal TMSs and a globular CL-binding domain in the periplasm. Two crystal structures of the C-terminal soluble periplasmic globular domains of PbgA from S. typhimurium and E. coli have been solved, which revealed that the globular domains of PbgA resemble the structures of the arylsulfatase protein family and contains a novel core hydrophobic pocket that may be responsible for binding and transporting CLs (Dong et al. 2016).
The CLT family is annotated in Pfam/CDD as the YejM, ALP and As1P membrane anchored periplasmic protein YejM alkaline phosphatase/cell wall sulfatase family. Sulfatases catalyze the hydrolysis of sulfate esters from wide range of substrates including steroids, carbohydrates and proteins. Sulfate esters may be formed from various alcohols and amines. The biological roles of sulfatase include the cycling of sulfur in the environment, the degradation of sulfated glycosaminoglycans and glycolipids (in the lysosomes of eukaryotes), and in remodeling sulfated glycosaminoglycans in the extracellular space. Sulfatases are essential for human metabolism. At least eight human monogenic diseases are caused by the deficiency of individual sulfatases.
Strain LH530, a mutant of Escherichia coli K-12, was reported by others to show increased outer membrane permeability, temperature-sensitive growth, and reduced synthesis of lipid A. The mutant gene is suppressed by high-copy-number plasmids carrying the wild-type acpT gene, which encodes a protein that catalyzes the attachment of 4'-phosphopantetheine. De Lay and Cronan 2008 mapped the mutation to yejM. Deletion of the entire yejM gene was lethal. Suppression by AcpT overexpression was specific to AcpT snf did not require that AcpT be enzymatically active.
The generalized transport reaction catalyzed by PbgA is:
CL (inner membrane) → CL (outer membrane).
PbgA (YejM) of 586 aas and 5 N-terminal TMSs with a C-terminal alkaline phosphatase-like domain (Dalebroux et al. 2015). The globular domains of PbgA resemble the structures of the arylsulfatase protein family and contains a novel core hydrophobic pocket that may be responsible for binding and transporting cardiolipin (Dong et al. 2016). PhoPQ is activated within the intracellular phagosome environment of the host animal, where it promotes remodeling of the outer membrane and resistance to innate immune antimicrobial peptides. Maintenance of the PhoPQ-regulated outer membrane barrier requires PbgA, an inner membrane protein with a transmembrane domain essential for growth, and a periplasmic domain required for PhoPQ-activated increases in outer membrane cardiolipin. Fan et al. 2020 reported the crystal structure of cardiolipin-bound PbgA, adopting a transmembrane fold that features a cardiolipin binding site in close proximity to a long and deep cleft spanning the lipid bilayer. The end of the cleft extends into the periplasmic domain of the protein, which is structurally coupled to the transmembrane domain via a functionally critical C-terminal helix. In conjunction with a conserved putative catalytic dyad situated at the middle of the cleft, structural and mutational analyses suggest that PbgA is a multifunction membrane protein that mediates cardiolipin transport, a function essential for growth, and perhaps catalysis of an unknown enzymatic reaction (Fan et al. 2020).
PbgA of Salmonella enterica
Phosphatase PAP2 family protein of 263 aas and 5 TMSs in a 1 + 2 + 2 TMS arrangement.
PAP2 family protein of Tepidimonas taiwanensis
LTA synthase family protein of 641 aas and 5 N-terminal TMSs in a 1 + 2 + 2 TMS arrangement. This protein is homologous to TC# 2.A.22.214.171.124 throughout most of its length. The 5 TMS transmembrane region is similar to several other members of this family as well as the second half of the 11 TMS transmembrane region plus the C-terminal hydropilic domain of TC# 2.A.126.96.36.199.
LTA synthase of Haemophilus influenzae
Phosphatase PAP2 family protein of 307 aas and 6 TMSs, the first 3 TMSs are distant from each other while the last 3 TMSs are close to each other.
PAP2 family protein of Nibricoccus aquaticus
Phosphatase PAP2 family protein of 252 aas and 6 TMSs in a 1 + 2 + 3 TMS arrangement.
PAP2 family protein of Candidatus Cloacimonetes bacterium
Phosphatase PAP2 family protein of 467 aas and 6 TMSs in a 3 + 3 TMS arrangement.
PAP2 family protein of Candidatus Aminicenantes bacterium (sediment metagenome)
PAP2 (acid phosphatase) superfamily protein of 434 aas and 6 or 7 TMSs.
PAP2 family protein of Candidatus Magnetoovum chiemensis
Phosphatase PAP2 family protein of 226 aas and 6 TMSs
PAP2 family protein of Solemya velum gill symbiont
Phosphatase PAP2 family protein of 511 aas and 12 TMSs in a 3 + 3 + 3 + 3 TMS arrangement, where the first and third 3 TMS repeats are separated by larger loops than the second and fourth TMS repeats.
PAP2 family protein of Alphaproteobacteria bacterium
Two domain protein with the N-terminal 6 TMS domain corresponding to those of TC families 2.A.127 and 9.B.105, and the second domain correspoonding to those of TC families 4.D.1 and 4.D.2. These two domains are also found adjacent to each other in the same order in another TC entry with TC# 9.B.27.2.12. The protein is annotated in UniProt as an acidPPc domain-containing protein.
Two domain protein of Fulvimarina sp.
PbgA protein of 586 aas and 5 N-terminal TMSs. The 3-d structure of the C-terminal alkaline phosphatase-like domain has been solved (Dong et al. 2016).
PbgA of E. coli
PbgA of 642 aas and 5 TMSs.
PbgA of Pseudomonas aeruginosa
PbgA homologue of 662 aas and 5 N-terminal TMSs.
PbgA of Myxococcus xanthus
Uncharacterized protein of 619 aas and 5 N-terminal TMSs.
UP of Hymenobacter swuensis
Uncharacterized protein of 896 aas and 11 or 12 TMSs.
UP of Hydrogenophaga palleronii
Putative phosphoethanolamine transferase EptBof 518 aas and 3 N-terminal TMSs.
PE transferase of Thiotrichales bacterium
Uncharacterized protein of 624 aas and 5 N-terminal TMSs.
UP of Rubrobacter radiotolerans
Phosphatase PAP2 family protein of 231 aas and 5 TMSs. This protein is homologous to the first half of TC# 2.A.127.1.6 which is not demonstrably homologous to other members of this family.
PAP2 family protein of Rhodoferax antarcticus