Pleiotropic Effects of Inactivating a Carboxyl-Terminal Protease, CtpA, in Borrelia burgdorferi

Abstract

A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

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

“…either in Gram-negative bacteria (where many have been shown to affect virulence) or in non-pathogenic Grampositive species (where one example is known to affect sporulation) (Bandara et al, 2008;Lad et al, 2007;Marasco et al, 1996;Ostberg et al, 2004;Pan et al, 2003). In this work we have, for the first time, to our knowledge, identified and characterized a C-terminal protease from a Grampositive, pathogenic bacterial species.…”

“…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).…”

A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice

“…The Ctp protein is also believed to be involved in protection of the bacterium from thermal and osmotic stresses [20,21] and degradation of certain aberrant cellular proteins [22]. In Borrelia burgdorferi, Ctp is involved in up-or down-regulation of protein expression [14,16,21]. The putative Ctp protein of Brucella suis influences cell morphology, salt-sensitive growth, and in vitro and in vivo persistence [23].…”

“…The endoproteases responsible for cleaving of amino-terminal peptides are called signal peptidases or amino-terminal processing proteases and have been well characterized [13]. A relatively new class of endoproteases with carboxyl-terminal processing activities has been identified from higher plants, algae, and bacteria [14][15][16][17][18]. These carboxyl-terminal proteases (Ctp) are serine proteases that utilize a Ser/Lys catalytic dyad instead of the well-known Ser/His/Asp catalytic triad [19].…”

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

“…Ö stberg and colleagues identified CtpA (BB0359) as a B. burgdorferi homologue of carboxyl-terminal proteases, a family of unusual serine-like proteases (38). Inactivation of ctpA had a pleiotropic effect on the B. burgdorferi proteome, affecting the processing of both lipidated and nonlipidated OM-associated proteins, including the OM porin P13 (37)(38)(39).…”