The Structure of the Haemophilus influenzae HMW1 Pro-piece Reveals a Structural Domain Essential for Bacterial Two-partner Secretion

Abstract

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and In Gram-negative bacteria, the two-partner secretion (TPS) 2 pathway serves as a common secretion mechanism for large protein virulence factors and is essential for the virulence of many human pathogens, including Haemophilus influenzae, Bordetella pertussis, Serratia marcescens, and Proteus mirabilis, among others. The TPS pathway consists of a secreted exoprotein (referred to as a TpsA protein) and a cognate outer membrane translocator (referred to as a TpsB protein) (1-3). TpsA proteins are synthesized as preproteins 100 -500 kDa in size that are processed in the course of secretion across the bacterial inner and outer membranes, yielding functional TpsA proteins (3, 4). Despite limited overall sequence conservation among the TpsA members, functional studies have established that TpsA proteins contain common features, including an atypical N-terminal signal peptide and an adjacent region of about 250 residues that forms the so-called secretion domain (1-4). Although our knowledge of TpsB proteins remains relatively limited, recent studies have established that TpsB proteins have a modular structure with a C-terminal pore-forming domain (5, 6).The H. influenzae HMW1 and HMW2 proteins are high molecular weight, non-pilus adhesins that were originally identified as major targets of the human serum antibody response during acute otitis media (7). These proteins are present in ϳ80% of nontypeable H. influenzae strains and mediate adherence to a variety of epithelial cell types (4, 8). HMW1 and HMW2 are encoded by separate chromosomal loci, with each locus consisting of three genes, designated hmwA, hmwB, and hmwC (8 -10). The hmwA genes encode the surface-exposed adhesins (HMW1 and HMW2), and the hmwB and hmwC genes encode accessory proteins required for proper processing and secretion of the adhesins (5,(11)(12)(13)(14). In H. influenzae strain 12, the HMW1 and HMW2 proteins are synthesized as preproproteins and undergo two discrete cleavage events during the process of maturation and surface localization, the first releasing the N-terminal signal peptide corresponding to residues 1-68 and the second releasing the pro-piece corresponding to residues 69 -441 (see Fig. 1). In H. influenzae strain 12, mature HMW1 corresponds to residues 442-1536 in the HMW1 preproprotein, and mature HMW2 corresponds to residues 442-1477 in the HMW2 preproprotein. In both HMW1 and HMW2, adhesive activity resides in a ϳ360-amino acid region at the N terminus of the mature species, and anchoring to the bacterial surface is mediated by a 20-amino acid region at the very C te...

Translocation path of a substrate protein through its Omp85 transporter

“…3a and Supplementary Table 1 for the list of positions tested). In addition to a few b-helix positions, we particularly targeted segments that form extra-b-helix elements conserved in the TpsA family [4][5][6] . Most substitutions do not affect the secretion of Fha30 alone, enabling us to use the corresponding chimaeras for crosslinking, while those that abolish secretion or cause intracellular proteolytic degradation of the chimaera were discarded (Supplementary Table 1).…”

“…TPS systems are composed of two proteins, collectively called 'TpsA' for the secretory proteins and 'TpsB' for their transporters 2 . The secreted TspA proteins share a conserved, 250-residue-long N-terminal 'TPS' domain essential for secretion and a b-helix fold [3][4][5][6][7] . TpsB transporters are integral outer membrane proteins of the Omp85 superfamily, which is found in all branches of the phylogenetic tree 8 .…”

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast

“…Structural Homology Search-A structural homology search using the program DALI revealed that the best score (Z value of 7.4) was produced by Haemophilus influenzae HMW1 Pro-piece, a structural domain essential for bacterial two-partner secretion (Protein Data Bank code 2ODL) (37). This protein shares 9% sequence identity to LeIBP and can be superimposed with an r.m.s.…”

Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression

“…The Cterminal domain of BamA forms a transmembrane 16-stranded β-barrel core, while the Nterminal region of BamA forms five soluble periplasmic polypeptide-transport-associated domains (POTRA) (Jansen et al, 2015b, Kim et al, 2007, Gatzeva-Topalova et al, 2010, Gatzeva-Topalova et al, 2008. These POTRA domains contribute to BamA stability and directly interact with BamBD within the periplasm, nascent polypeptides and the periplasmic chaperone SurA (Bennion et al, 2010, Kim et al, 2007, Workman et al, 2012, Dong et al, 2012a. The lipoproteins BamC and BamE interact directly with BamD (Kim et al, 2011a, Malinverni et al, 2006, Wu et al, 2005, Stenberg et al, 2005.…”

“…BamA is the major protein that catalyses folding of OMPs and can act alone or in complex with either BamB or with BamC, BamD and BamE (Malinverni et al, 2006, Kim et al, 2007, Hagan et al, 2010, Plummer and Fleming, 2015. The Cterminal domain of BamA forms a transmembrane 16-stranded β-barrel core, while the Nterminal region of BamA forms five soluble periplasmic polypeptide-transport-associated domains (POTRA) (Jansen et al, 2015b, Kim et al, 2007, Gatzeva-Topalova et al, 2010, Gatzeva-Topalova et al, 2008. These POTRA domains contribute to BamA stability and directly interact with BamBD within the periplasm, nascent polypeptides and the periplasmic chaperone SurA (Bennion et al, 2010, Kim et al, 2007, Workman et al, 2012, Dong et al, 2012a.…”