Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro

Abstract

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn-binding motif (1604-1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N-terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C-terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.

Streptococcus pneumoniae’s Virulence and Host Immunity: Aging, Diagnostics, and Prevention

“…These two isotypes differ in hinge regions—IgA1 has an extended hinge region because of an insertion into this region of a set of duplicated amino acids ( 201 ). IgA1 proteases reduce the binding IgA’s effector region of the heavy chain and hinder killing of the bacterium by these antibodies ( 83 , 85 ). Hydrogen peroxide: S. pneumoniae secretes hydrogen peroxide (H 2 O 2 ) which causes damage to host DNA ( 202 ).…”

Mechanism and inhibition of Streptococcus pneumoniae IgA1 protease

“…The number of interactions between the IgA1P NTD and MD are diminished upon this rearrangement, providing for an energetic rationale for why the enzyme "snaps" back after product release. Such findings also led us to hypothesize that the IgA1P NTD may be soluble alone, similar to its G5 domain and CTD that have been previously shown to be independently folded 21,22 . Indeed, we were able to engineer an NTD construct that gives rise to a well-dispersed 15 N-HSQC analogous to the independently folded IgA1P CTD and the G5 domain (Supplementary Fig.…”

“…The high-resolution structure of the IgA1P catalytic region. In order to identify the S. pneumoniae IgA1P catalytic domain, we engineered several constructs based on our previous results of limited proteolysis on the full, mature IgA1P (residues 154-1963, UniProt accession Q59947; NCBI accession WP_000417171 that corresponds to the common D39 and R6 strains) 21,22 . Only constructs that began at or prior to residue 665 were accessible to enzymatic cleavage of the MBP tag by thrombin ( Supplementary Fig.…”

“…Protein expression and purification. All S. pneumoniae IgA1P constructs of the full catalytic region were engineered and purified similar to that previously described for the mature IgA1P (residues 154-1963), with the exception that the MBP and 6xHis tags were swapped from their original positions 21 . The kanamycinresistant plasmid, pJ401 (DNA2.0/Atum), was used as the backbone with the open reading frame coding for an N-terminal MBP, a thrombin cleavage site, IgA1P residues 665-1963 (or variants thereof described in the main text), and a Cterminal 6xHis tag.…”

“…All NMR data were collected on a Varian 900 equipped with a cryoprobe. Samples were produced as previously described using 15 N M9 minimal media for the S. pneumoniae IgA1P G5 domain (residues 312-393) 22 and 15 N, 2 H M9 minimal media for both the CTD (residues 1611-1963) 21 and the NTD (residues 665-1010). Final size exclusion was performed in NMR buffer (50 mM Na 2 HPO 4 , pH 6.5, and 150 mM NaCl).…”