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Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes.

ABSTRACT: Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448?pepO mutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCE Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone and as being essential for virulence.

SUBMITTER: Brouwer S

PROVIDER: S-EPMC5869480 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes.

Brouwer Stephan S Cork Amanda J AJ Ong Cheryl-Lynn Y CY Barnett Timothy C TC West Nicholas P NP McIver Kevin S KS Walker Mark J MJ

Journal of bacteriology 20180326 8

Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenoty ...[more]

PMID: 29378883

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Project description:Streptococcus pyogenes causes a variety of diseases ranging from mild diseases to severe invasive infections which result in significant morbidity and mortality. This study focuses on the antibiotic resistance of S. pyogenes and their interaction with cysteine protease. Around 36 beta-hemolytic isolates were collected from the clinical lab, of which seven isolates (19.4%) were identified as Streptococcus pyogenes. One of the seven isolates was collected from a urinary tract infection, which was identified by antibody agglutination and MALTI-TOF-MS, and it is designated as S. pyogenes NBMKU12. Around 8.3 to 66.6 % of the isolates were found to be resistant to one or more antimicrobial agents, especially, penicillin-G resistance was exhibited by 29.1% of the isolates. In the NBMKU12 isolate, the beta lactem (TEM) gene was detected among the 13 antibiotic genes for which it was tested. Furthermore, when analysis for presence of 13 virulence genes were carried out in NBMKU12 isolate, only speJ and speB were detected. The speB (streptococcal pyrogenic exotoxin B) encoding cysteine protease gene was cloned. This was followed by performing DNA sequencing to understand the putative cysteine protease interaction with antibiotics, inhibitors, and substrate. The speB gene consists of 1197 nucleotides and encodes a protein with multiple domains, including a signal peptide (aa 1-22), an inhibitor region (aa 27-156), and a catalytic cysteine domain (aa 160-367). The signal peptide cleavage site is predicted between Ala22 and Asn23. The putative 398 amino acid residues were found to have a theoretical pI of 8.76 and a molecular mass of 43,204.36 Da. The tested culture supernatants of NBMKU12 isolate exhibited the proteolytic activity against casein, papaya and pineapple used as substrates. The proteolytic activity suggests the expression of speB gene. Molecular docking analysis of cysteine protease showed that erythromycin (bond length 2.41 Å), followed by chloramphenicol (2.51 Å), exhibited a strong interaction; while penicillin-G (3.24 Å) exhibited a weak interaction, and this factor could be considered as a cause for penicillin-G resistance. The present study contributes to a better understanding of speB gene encoding cysteine protease, antibiotic resistance, and their interaction in the isolate, S. pyogenes NBMKU12. The antibiotics and cysteine protease interaction study confirms the resistance or sensitivity of S. pyogenes. Hence, it could be hypothesized that the isolate NBMKU12 is resistant to most of the tested antibiotics, and this resistance might be a cause for mutation.

Dataset Information

Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes.

Publications

Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes.

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