Molecular Characterization of Protease Activity in Serratia sp. Strain SCBI and Its Importance in Cytotoxicity and Virulence

Abstract

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex.

Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria

“…This may compromise the ability of infected bees to limit expansion of the bacterium in the hemolymph. In the lepidopteran insect pathogen Serratia sp SCBI, (representing the closest genomic match to Ss1 as shown in Fig 5), a hemolysin was detected in the bacterial culture [48] and cytotoxicity was connected with protease expression [49]. Similar factors in Ss1 may deplete hemocyte populations in hemolymph of infected bees.…”

RNA modification enzymes encoded by the gid operon: Implications in biology and virulence of bacteria

“…A recent study by Petersen et al showed inactivation of gidA influences virulence and protease activity in a newly discovered Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI) [69]. In their study, a gidA mutant was attenuated in proteolytic activity, cytotoxicity to Buffalo green monkey kidney (BGMK) cells, hemolytic activity, swarming motility, and overall virulence by a decreased inability to kill third-instar Manduca sexta larva.…”

“…In their study, a gidA mutant was attenuated in proteolytic activity, cytotoxicity to Buffalo green monkey kidney (BGMK) cells, hemolytic activity, swarming motility, and overall virulence by a decreased inability to kill third-instar Manduca sexta larva. Furthermore, their transcriptome analysis suggests a role for global regulation by GidA as seen in other Gram-negative bacteria [69]. Several reports show GidA-associated virulence mechanisms in several species of Pseudomonas [62,63,70].…”

Structure of a thermostable serralysin fromSerratiasp. FS14 at 1.1 Å resolution

“…are facultative anaerobic organisms that ubiquitously inhabit soil, water, animals, plants and humans. Serratia species can secrete a variety of exoenzymes including proteases, nucleases, phospholipases and chitinases (Monreal & Reese, 1969;Braun & Schmitz, 1980;Givskov et al, 1988;Chen et al, 1992;Petersen & Tisa, 2014). In particular, serralysin, a zinc-dependent metalloprotease, has been well studied (Miyata et al, 1970;Braunagel & Benedik, 1990;Baumann, 1994;Baumann et al, 1995;Aiyappa & Harris, 1976;Hamada et al, 1996).…”

“…Serralysin has been found to not only play important roles in bacterial nutrition but also in virulence as a cytotoxic factor capable of infecting larvae, mammalian cells and the human immune system (Marty et al, 2002;Kida et al, 2007;Petersen & Tisa, 2014;Shanks et al, 2015;Pineda-Castellanos et al, 2015).…”