Specificity and Role of the Borrelia burgdorferi CtpA Protease in Outer Membrane Protein Processing

Abstract

To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used sitedirected mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.Borrelia spirochetes, the etiological agents of vector-borne Lyme borreliosis and relapsing fever, are diderm bacteria (bacteria with both an inner and outer membrane) with an unusual envelope structure (reviewed in reference 6). As in Gramnegative bacteria such as Escherichia coli, a periplasmic space separates an inner cytoplasmic membrane (IM) from an outer membrane (OM) (26), but flagella remain sequestered in the periplasm, where they provide both motility and determine bacterial shape (34). Similarly, the Borrelia OM also contains integral membrane proteins (23,43,56), including the porins P66 (15, 41, 51) and P13 (39, 45), as well as BesC, a component of an envelope-spanning type I secretion system involved in virulence and antibiotic resistance (11). However, the cell envelope lacks the lipopolysaccharide coat that typifies Gramnegative bacteria (54). Instead, the borrelial surface is covered by abundant glycolipids (4, 5, 22) and surface lipoproteins (9) such as OspC, a virulence factor that is required for the establishment of mammalian infection upon arthropod-borne transmission (20,44,53,55).Our current understanding of posttranslational protein processing and modification in Borrelia burgdorferi is limited.Based on studies in other Gram-negative or diderm model organisms, lipoproteins are likely modified in a three-step process occurring on the periplasmic face of the IM, involving cleavage by the signal II peptidase Lsp (encoded by open reading frame [ORF] BB0469). Surprisingly, the small B. burgdorferi genome contains genes encoding three signal peptidase I paralogues, LepB1 to LepB3 (BB0030, BB0031, and BB0263) with currently undescribed functions; at...

Comprehensive Spatial Analysis of the Borrelia burgdorferi Lipoproteome Reveals a Compartmentalization Bias toward the Bacterial Surface

“…The His tag epitope was used to assess the sensitivity of the tagged lipoprotein to proteinase K, assuming that the localization of the C-terminal His tag mirrors that of the lipoprotein itself. Of note, C-terminal processing of secreted proteins in B. burgdorferi is rather specific and limited to a small set of proteins (103,104), and C-terminal tags are not known to alter lipoprotein localization (57, 105). Lipoproteins that lost the His tag signal upon proteolysis were considered to be surface exposed (S), whereas lipoproteins that showed no loss in signal relative to the controls were considered to localize to the periplasmic (P) face of the OM or inner membrane (IM).…”

The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence

“…If this hypothesis holds true for Gram-positive bacteria then it seems likely that the proteolytic targets of S. aureus CtpA-mediated hydrolysis are located in the bacterial cell wall. In Gram-negative bacteria, many of the phenotypes associated with ctpA 2 mutants, including altered cell morphology and increased sensitivity to heat and osmotic shock, are proposed to be a consequence of decreased cell-wall integrity (Hara et al, 1991;Kumru et al, 2011;Ostberg et al, 2004;Seoane et al, 1992). Studies in E. coli have demonstrated periplasmic protein leakage in a prc mutant, suggesting increased permeability of the outer membrane in this strain (Hara et al, 1991).…”

“…Other known targets of Gram-negative CTPs include the outer-membrane protein P13 in Borrelia burgdorferi (Noppa et al, 2001). Cterminal processing of P13 by CtpA stabilizes the protein and directs it to the outer membrane where it may be important during infection (Kumru et al, 2011). Although CTP mutants in Gram-negative bacteria display a variety of phenotypes, certain commonalities exist.…”

“…Although CTP mutants in Gram-negative bacteria display a variety of phenotypes, certain commonalities exist. Altered cell morphology, differences in osmotic/thermal stress resistance and altered susceptibility to antibiotics are common phenotypes of mutants from different species (and may result from alterations in the cell-wall integrity) (Hara et al, 1991;Kumru et al, 2011;Seoane et al, 1992). In addition, a number of Gram-negative bacteria, including E. coli, Brucella suis, Chlamydia trachomatis, Salmonella typhimurium, Pseudomonas aeruginosa and Burkholderia mallei, demonstrate reduced virulence upon inactivation of CTP (Bandara et al, 2005(Bandara et al, , 2008Bäumler et al, 1994;Lad et al, 2007;Seo & Darwin, 2013;Wang et al, 2012).…”

Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

“…It forms channels in the outer membrane of Borrelia despite its small 13-kDa molecular mass and its ␣-helical secondary structure (24). The occurrence and function of a periplasmic peptide derived from cleavage of its C-terminal end is not completely understood and is unique among porins (25,26). Another very remarkable feature of P13 is the presence of up to eight paralogues in the genome of B. burgdorferi (27).…”