Characterization of the type II secretion system of Chromobacterium violaceum

Author(s)

Kelly Cristina Martins Barroso

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-07-20

Examining board members

José Freire da Silva Neto; Rodrigo Tavanelli Hernandes; Germán Gustavo Sgro; Luis Lamberti Pinto da Silva

• Advisor: José Freire da Silva Neto

Abstract

Bacteria use sophisticated nanomachineries to secrete proteins across the cell envelope. The type II secretion system (T2SS) translocates, via the periplasm, proteins that act as toxins and extracellular hydrolytic enzymes. In this work, we characterized the T2SS from Chromobacterium violaceum, a Gram-negative bacterium that can act as an opportunistic pathogen in humans. The C. violaceum T2SS is encoded by 13 genes (operon gspCDEFGHIJKLMN and pilD). The promoter activity of the T2SS gsp operon increased in the presence of bile salts and in the transition from logarithmic to stationary phase. The increased expression at high cell density was not dependent on the CviIR quorum sensing system. Four T2SS mutant strains were obtained, with deletion of the genes gspD (secretin), gspE (ATPase), pilD (prepilin peptidase), and gspC-N (deletion of the entire gsp operon). Growth curves, CFU counting, and live/dead and electron microscopy assays revealed that the T2SS mutant strains had growth imparaired and drastic reduction in viability in stationary phase cultures. The T2SS mutants showed damage of the outer membrane, as demonstrated by growth curves in the presence of the antibiotic polymyxin B and ethidium bromide permeability assays. The double mutant ΔgspDcviI had improved fitness, suggesting that proteins regulated by the quorum sensing system CviIR would accumulate in the periplasm and impair the fitness of T2SS mutants at high cell density. The hydrolytic activity assays in plate, performed in M9 medium supplemented with specific substrates, revealed that all T2SS mutants showed decreased protease, gelatinase, chitinase, and hemolysin activity. The halos of proteolytic activity of the wild-type strain decreased in the presence of the metal chelating agent EDTA, indicating that C. violaceum secretes metalloproteases via T2SS. Quantitative mass spectrometry analysis comparing the exoproteomes of the wild-type and ΔgspD mutant allowed the identification of several hydrolytic enzymes secreted by T2SS from C. violaceum, including proteases, collagenases, chitinases, and lecithinase. To characterize some substrates secreted by T2SS from C. violaceum, we obtained the mutant strains ΔCV_2001 and ΔCV_3255 (collagenases), ΔCV_2571 and ΔCV_0057 (proteases), ΔCV_1440 and ΔCV_3931 (chitinases), and ΔCV_0362 (hemolysin/lecithinase). Growth curves in LB medium showed that these mutant strains do not have growth impairment. Regarding biofilm production, only the ΔCV_2571 mutant had reduced biofilm. In the hydrolytic activity assays in plate, only ΔCV_0362 had a reduction in the lecithinase/lipase halo. Virulence assays in mice with these seven mutants indicated no or weak attenuation of virulence, suggesting that enzymes secreted by T2SS may play a redundant role. Taken together, the results of this work indicate that C. violaceum expresses a functional T2SS that is necessary for the secretion of several extracellular hydrolytic enzymes. (AU)

• FAPESP's process: 18/03351-2 - Role of type II secretion system in physiology and virulence of Chromobacterium violaceum
• Grantee: Kelly Cristina Martins Barroso
• Support Opportunities: Scholarships in Brazil - Doctorate