The Extracellular Proteases Produced by Vibrio Parahaemolyticus
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World Journal of Microbiology and Biotechnology (2018) 34:68 https://doi.org/10.1007/s11274-018-2453-4 REVIEW The extracellular proteases produced by Vibrio parahaemolyticus George Osei‑Adjei1 · Xinxiang Huang1 · Yiquan Zhang1 Received: 20 February 2018 / Accepted: 8 May 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018 Abstract Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syn- drome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium. Keywords Extracellular protease · Metalloprotease · Serine protease · Vibrio parahaemolyticus · Virulence Introduction incubation period of V. parahaemolyticus is usually 12–24 h post-infection with infection usually occurring in the hot The Gram-negative bacterium, Vibrio parahaemolyticus, summer months because the bacteria is found free swim- lives in marine and estuarine environments and it is a major ming in coastal water or attached to fish and shellfish. V. par- pathogen responsible globally for most cases of seafood- ahaemolyticus has become important as a result of the global associated gastroenteritis in humans (Broberg et al. 2011, emergence of pandemic strains (Newton et al. 2012). The 2010; Miyoshi 2013; Wang et al. 2011a, b). Ingestion of pandemic V. parahaemolyticus serotype O3: K6 has been undercooked or raw seafood contaminated with the bacte- associated with outbreaks in Peru (Martinez-Urtaza et al. rium leads to acute gastroenteritis characterized by diarrhea, 2016), United States, Russia, Mozambique, Mexico, and nausea, vomiting, mild fever and abdominal cramps (Brob- Spain (Martinez-Urtaza et al. 2016; Matlawska-Wasowska erg et al. 2011; Ham and Orth 2012; O’Boyle and Boyd et al. 2010). An estimated 40,000 cases of V. parahaemo- 2014). V. parahaemolyticus strains have in recent times lyticus infection in humans are reported yearly in the United been reported to cause acute hepatopancreatic necrosis syn- States only (Scallan et al. 2011). In China, 9041 illnesses drome (AHPNS) in shrimps with resultant huge losses in and 3948 hospitalizations resulting from 322 V. parahaemo- shrimp farms (Lee et al. 2015; Lin et al. 2017; Tran et al. lyticus outbreaks were reported from 2003 to 2008 (Wu 2013; Zhang et al. 2018). The bacterium can also cause et al. 2014). The growing incidence of V. parahaemolyti- skin infection and septicemia when it comes into contact cus infections has been attributed to leading factors such as with open wounds mostly in individuals with compromised global climate warming (Baker-Austin et al. 2013; Martinez- immunity and persons with an underlying medical condition, Urtaza et al. 2016). The pathogenicity of virulent strains of such as cirrhosis (Broberg et al. 2011; Lee et al. 2015). The V. parahaemolyticus occurs as a result of the expression of virulence factors, such as the thermostable direct hemoly- sin (TDH), TDH-related hemolysin (TRH), two type three * Yiquan Zhang secretion systems (T3SS1 and T3SS2), and two type six [email protected] secretion system (T6SS1 and T6SS2), in addition to some 1 School of Medicine, Jiangsu University, Zhenjiang 212013, adhesins (Letchumanan et al. 2014). V. parahaemolyticus Jiangsu, China Vol.:(0123456789)1 3 68 Page 2 of 7 World Journal of Microbiology and Biotechnology (2018) 34:68 also produces extracellular proteases with diverse patho- induction (Miyoshi et al. 2008; Miyoshi 2013; Yu and Lee logical roles, such as collagenolytic, cytotoxic, hemolytic 1999), but its role in pathogenesis was unclear. PrtV activity and edema-forming activity (Lin et al. 2017; Miyoshi et al. was inhibited by EDTA but was restored by the addition of 2012; Miyoshi 2013). In this review, we focus on the metal- metal ions (Yu et al. 2000). loproteases and serine proteases produced by the pathogenic Vibrio parahaemolyticus VppC, a zinc metallopro- bacteria V. parahaemolyticus (Table 1) and their roles in vir- tease containing the HEXXH consensus motif was cloned, ulence. An insight into the virulence mechanisms involving sequenced and its activity examined by gelatin zymography V. parahaemolyticus proteases is relevant for the diagnosis, (Kim et al. 2002). The novel protease showed strong activ- treatment and infection prevention. ity against native collagen. However, the cytotoxic effects in animal models and contribution towards pathogenesis in vivo were not clarified (Kim et al. 2002). A significant Extracellular proteases in V. collagenolytic activity of VppC was detected at the early parahaemolyticus stationary phase in the culture supernatant when V. para- haemolyticus was cultured at 26 °C in gelatin broth contain- Metalloproteases ing with 3% NaCl (Miyoshi et al. 2008). The 90 kDa protein, generated through truncation of 72 N-terminal amino acid In most pathogenic bacteria, metalloproteases produced are residues was determined by native polyacrylamide gel elec- involved in the degradation of the extracellular matrix of trophoresis (Miyoshi et al. 2008). the host cells. Most metalloproteases are collagenases con- VPM protein, an extracellular zinc metalloprotease from taining zinc ions in their catalytic center and are capable of the vpm gene of V. parahaemolyticus, a putative virulence digesting native collagen, gelatin and other proteins (Duarte factor for host infection, was expressed and characterized et al. 2016; Miyoshi 2013). Thus, their implication in the using E. coli as an expression vector (Luan et al. 2007). virulence of this bacteria. Metalloproteases in the zincins The recombinant protein (rVPM) which showed maximum superfamily contain the consensus zinc-binding HEXXH activity at 37 °C and pH 8 was cytotoxic against flounder gill motif (Luan et al. 2007; Miyoshi et al. 2008; Miyoshi 2013). cells and was pathogenic to fish. The rVPM caused death In the MEROPS database, Vibrio collagenases, are classi- to zebra fish 48 h after 1 mg mL−1 was injected intraperito- fied into the subfamily M9A (Rawlings et al. 2016; Zhang neally. The fish revealed hemorrhage in the peritoneal cav- et al. 2015). ity and hemorrhagic and necrotic symptoms at the injec- PrtV, a 62 kDa protein consisting of a 27 amino acid sig- tion site (Luan et al. 2007). Thus, the authors suggested that nal peptide, encoding a collagenase from V. parahaemolyti- VPM may cause tissue damage by directly degrading host cus and capable of degrading native collagen and the syn- tissue and may cause pathogenesis by promoting bacterial thetic substrate 2-furanacrylyol Leu-Gly-Pro-Ala (FALGPA) invasion. was expressed in E. coli (Miyoshi et al. 2008; Yu and Lee 1999) after it was previously cloned and sequenced (Lee Serine proteases et al. 1995). PrtV, hydrolyzed type I–IV collagens and was thermostable up to 40 °C. The enzyme showed no signifi- Serine proteases are enzymes that cleave peptide bonds in cant effect on the growth of CHO, HeLa and Vero, cells (Yu proteins in which serine serve as the nucleophilic amino et al. 2000). The highest activity of the recombinant protein acid at the active site. Serine proteases are classified into was detected in the culture supernatant after hours of IPTG two broad categories, chymotrypsin-like/trypsin-like or Table 1 Characteristics of extracellular proteases produced by V. parahaemolyticus Name Mol mass Substrates Inhibitors Reference (kDa) Metalloprotease PtrV 62 Type I–IV collagens, FALGPA EDTA Lee et a1. (2002), Miyoshi (2013) VppC 90 Type I collagen, gelatin, Casein EDTA Kim et al. (2002), Miyoshi (2008) VPM 90 Type I–IV collagens, gelatin, casein EDTA Luan et al. (2007) Serine protease VPP1 43 Type I collagen, gelatin EDTA, EGTA, chymostain Miyoshi et al. (2012), Miyoshi (2013) VpSP37 37 Trypsin, gelatin Aprotinin, leupeptin Salamone et al. (2015) PrtA 71 Gelatin EDTA Chang and Lee (2017) 1 3 World Journal of Microbiology and Biotechnology (2018) 34:68 Page 3 of 7 68 subtilisin-like based on their structure (Madala et al. 2010). acids and make them available for growth and survival of V. parahaemolyticus produces VPP1 (protease A), an extra- the pathogen, and also serve as toxic factors processing other cellular serine protease (Miyoshi et al. 2008, 2012; Miyoshi protein toxins (Duarte et al. 2016; Shinoda 2011). Microbial 2013). VVP1/protease A serves as a virulence factor with proteases have also been associated with virulence in fish cytotoxic activity against CHO, HeLa and Vero cells and and shrimp (Kahla-Nakbi et al. 2009; Rui et al. 2009; Tran also showed lethality in mice (Miyoshi et al. 2012). The et al. 2013), however,