Identification and Characterization of BicistronicspeBandprsAGene Expression in the Group A Streptococcus

Abstract

Severe, invasive group A streptococcal infections have reemerged worldwide, and extracellular toxins, including streptococcal pyrogenic exotoxin B (SpeB), have been implicated in pathogenesis. The genetic regulation of SpeB is not fully understood, and the mechanisms involved in the processing of the protoxin to its enzymatically active form have not been definitively established. The present work demonstrated that the genes encoding SpeB (speB) and a peptidyl-prolyl isomerase (prsA) constitute an operon with transcription initiated from two promoters upstream of speB. Further, the speB-prsA operon was transcribed as a bicistronic mRNA. This finding is in contrast to the generally accepted notion that speB is transcribed only as a monocistronic gene. In addition, prsA has its own promoter, and transcription from this promoter starts in early log phase, prior to the transcription of speB. Genomic disruption of prsA decreased the production of enzymatically active SpeB but not the level of the pro-SpeB zymogen. Taken together, these results demonstrate that prsA is required for production of fully mature, enzymatically active SpeB.Group A streptococcus (GAS) causes many diseases in humans, ranging in severity from milder infections such as pharyngitis, simple cellulitis, erysipelas, and scarlet fever to life-threatening necrotizing fasciitis, septicemia, and toxic shock syndrome. One of the many potentially important virulent factors produced by this organism is streptococcal pyrogenic exotoxin B (SpeB). As a potent cysteine proteinase, SpeB cleaves multiple streptococcal virulence factors, including M protein (3), as well as many host factors controlling inflammation (18,20).The gene for SpeB (speB) is chromosomally located on every GAS strain studied and consists of a 1,196-base pair (bp) open reading frame yielding a 371-amino-acid polypeptide with a predicted molecular weight of 40,000 (16). SpeB is secreted strictly in the late log/early stationary phase of growth as a proteinase precursor that must be proteolytically cleaved to the mature active form having a calculated molecular mass of approximately 28 kDa. SpeB is also found on the surfaces of the bacteria and possesses glycoprotein and laminin binding activities (19). While all strains of GAS are endowed with the gene for SpeB, not all strains produce the toxin in vitro, and even among strains that do, the quantity produced varies greatly from strain to strain (6,15,16,22,31). Other environmental factors, such as acidic pH, concentration of NaCl, the availability of nutrients, the presence of kanamycin, etc., also affect speB expression (7, 9, 39).Current knowledge regarding SpeB's transcriptional regulation and maturation is derived from many labs around the world. At the transcriptional level, rgg (also known as ropB) positively regulates SpeB expression and production (5), as does the global regulator mga (35). In addition, inactivation of both oligopeptide and dipeptide transport systems diminished speB mRNA levels (33, 34). At the posttranscriptiona...

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

“…The genomic coordinates are shown and the putative promoters and terminators are indicated. prsA (encoding the foldase protein PrsA) is involved in the maturation of SpeB protease [28]. SPy_2038 was described to encode an inhibitor of SpeB protease activity [58].…”

“…In an effort to provide coherence for future studies – the annotation of the speB 5′ UTR has been inconsistent in previous works [23,26–28] –, we first determined speB transcript boundaries. We demonstrated that RNase Y and so far unidentified RNase(s) process the 5′ UTR of speB mRNA.…”

“…It has recently been shown that a partial deletion of the speB 5′ UTR results in a global accumulation of mRNAs at the stationary phase of growth [27]. speB harbours a long 5′ UTR where two transcriptional start sites (TSSs), named P1 and P2, were originally predicted 697 nt and 137 nt upstream of the speB start codon, respectively [28]. Despite the important regulatory role of the intergenic region in speB expression, the annotation of this region is not consistent in the literature [21,23,26–29].…”

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

“…Previous studies have demonstrated that Nuc requires PPIase activity for optimal protein folding (3,4). Based on this observation and the fact that PPIase-mediated folding is required for subsequent activity of secreted virulence factors in a number of bacterial pathogens (8)(9)(10)(11)(27)(28)(29)(30), we hypothesized that a staphylococcal PPIase may be required for the activity of Nuc. To begin to investigate this, we examined the genome of the community-associated methicillin-resistant S. aureus (MRSA) isolate USA300 for genes that encode PPIase enzymes.…”

“…It is thought to assist in the refolding of proteins as they are exported from the bacterial cell. PrsA has been well studied in two bacterial pathogens, Listeria monocytogenes and Streptococcus pyogenes, where alteration of prsA levels (either increased or decreased) leads to defects in protein secretion and/or virulence of the organism (7)(8)(9)(10). The cyclophilins are by far the least-studied and least well understood group of PPIases in bacteria.…”