Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri

Abstract

ABSTRACT. Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ( HIS CPXAC) recognized the purified recombinant cysteine peptidase HIS CPXAC, confirming the correct production of this protein in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicityinducing medium. Kinetic analysis revealed that HIS CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k cat /K m ) of 47 μM -1 •s -1 . The purified HIS CPXAC displayed maximal catalytic activity at pH 5.5 and 30°C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.

The sulfur/sulfonates transport systems in Xanthomonas citri pv. citri

“…To verify if the putative genes involved with sulfate or organic sources of sulfate uptake and assimilation were expressed during the bacterium growth in two defined conditions, we performed a RT-PCR analysis from samples obtained from rich medium LB modified and virulence inducing medium XAM1, which final sulfate concentration is 7.8 mM [ 10 , 30 ]. Total RNA samples were extracted from the X. citri cells grown in the different culture media and used for cDNA amplification (Fig.…”

“…The X. citri strain used in this study was grown at 28 °C overnight in Luria-Bertani (LB) modified broth (without NaCl) supplemented with ampicillin (100 μg/ml) at 28 °C at 200 rpm. After the growth period, the cultures were diluted 50 times, washed two times in sterile water and incubated in virulence induction medium to mimic the plant environment, XAM1 [ 10 , 30 ], or LB until the mid end-exponential growth phase. Samples normalized by O.D.…”

“…X. citri is one of the relevant species from the genus because it is the causative agent of the citrus canker, a disease that affects citrus plants and causes significant economic losses in Brazil and many other countries in the world. Although studies have demonstrated the importance of specific genes for biofilm formation [ 23 ], infection [ 20 ] and pathogenesis [ 30 ], there is a lack of information regarding the sulfur or sulfate assimilation pathways in this bacterium and the relevance of these compounds for infection and pathogenesis.…”

The periplasmic binding protein NrtT affects xantham gum production and pathogenesis in Xanthomonas citri

“…In vitro growth of the samples was monitored after inoculation of 10 6 CFU of X. citri and Xac::nrtT cultures into 50 mL (1 : 100) of LB broth, XAM1 and XAM1 modified media (XAM1a and XAM1b), where XAM1 is 3.0 m m (NH 4 )SO 4 , 16.5 m m KH 2 PO 4 , 30 m m K 2 HPO 4 , 0.85 m m Na 3 C 6 H 5 O 7 , 100 m m MgSO 4 , 0.18% (m/v) fructose, 0.34% (m/v) sucrose, 0.03% (m/v) casamino acids and 0.1% (m/v) BSA 30; in XAM1a 3.0 m m MOPS and 100 m m MgCl 2 replace (NH 4 )SO 4 and MgSO 4 , respectively; and in XAM1b 3.0 m m taurine replaces (NH 4 )SO 4 . Growth was maintained at 30 °C, 250 r.p.m.…”

Sugarcane cystatin CaneCPI‐1 promotes osteogenic differentiation in human dental pulp cells: a new insight into cysteine proteases inhibitors

“…This recombinant cathepsin inhibitor (Soares-Costa et al 2002) interfered with the human cathepsins B, K, L and V, as well as the cysteine peptides from plant source papain and baupain (Oliva et al 2004), and the growth of the filamentous fungus Trichoderma reesei (Soares-Costa et al 2002). Currently, there are five recombinant cystatins derived from sugarcane named CaneCPI-1, CaneCPI-2, CaneCPI-3, CaneCPI-4 and CaneCPI-5 (Gianotti et al 2006, Oliveira et al 2011, Soares-Costa et al 2012, Santiago et al 2017. Studies have shown that CaneCPI-4 has reduced invasion of tumour cells in vitro (Gianotti et al 2008), inhibited the development of melanoma in vivo and decreased the angiogenesis process in vitro and in vivo (Oliveira et al 2011).…”