Plasmid-encoded toxin of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro.

Plasmid-encoded toxin (Pet) is an autotransporter protein of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the enteroaggregative E. coli (EAEC) virulence plasmid, pAA2. Although this virulence factor was initially described in EAEC, an intestinal E. coli pathotype, pet may also be present in other pathotypes, including extraintestinal pathogenic strains (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of E. coli to human serum.

Hemaglutinação como um teste para avaliar a funcionalidade da toxina Tsh (Temperature-Sensitive Hemagglutinin) purificada por gel filtração

A exposição ou secreção de proteínas relacionadas a fatores de virulência de bactérias Gram-negativas é dependente de sistemas de transporte altamente especializados. O sistema de secreção tipo V é o responsável pelo transporte das proteínas autotransportadoras membros da família das Serino-Proteases Autotransportadoras de Enterobacteriaceae (SPATE), as quais diversas funções de virulência vêm sendo atribuídas. SPATEs têm sido descritas em bactérias isoladas de infecções gastrointestinais, trato urinário, meningite e sepse. O objetivo deste trabalho foi contribuir com os estudos de caracterização do papel das toxinas da família das serino-proteases autotransportadoras de Enterobacteriaceae Vat, Tsh e Sat na infecção bacteriana avaliando, particularmente, a funcionalidade da Tsh purificada por gel filtração. Inicialmente a cinética de crescimento de E. coli patogênicas das cepas EC120 vat+ ou EC077, EC046, EC153 e EC143 tsh+ foi realizada nos meios TSB, LB, DMEM e DMEM acrescido de 1% de triptona a fim de determinar o melhor meio e tempo de cultivo que favorece a secreção das proteínas autotransportadoras. Para a obtenção da Tsh, a cepa EC143 foi cultivada em meio LB. O sobrenadante do cultivo concentrado foi submetido à coluna de gel filtração e as frações foram analisadas por SDS-PAGE e Westen Blotting. A quantidade de proteína das frações contendo Tsh foi determinada e a avaliação da funcionalidade da toxina foi realizada pelo ensaio de hemaglutinação de eritrócitos de galinha, clássico para avaliar a atividade de Tsh. Paralelamente, Vat e Sat foram também submetidas à hemaglutinação. Os resultados mostraram que a funcionalidade da Tsh purificada por gel filtração é mantida, que Vat não tem capacidade hemaglutinante e que a determinação da capacidade hemaglutinante de Sat necessita de maiores estudos visto que em presença de PMSF (inibidor de serino protease) o resultado foi mantido. Como o PMSF pode estar danificando os eritrócitos, novos ensaios utilizando, por exemplo, anticorpos anti-Sat para neutralizar o efeito serão realizados.

Secreted Autotransporter Toxin (Sat) Mediates Innate Immune System Evasion.

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

Secreted autotransporter toxin (Sat) mediates innate immune system evasion

Several strategies are used by Escherichia coli to evade the host innate immune system in the blood, such as the cleavage of complement system proteins by secreted proteases. Members of the Serine Proteases Autotransporters of Enterobacteriaceae (SPATE) family have been described as presenting proteolytic effects against complement proteins. Among the SPATE-encoding genes sat (secreted autotransporter toxin) has been detected in high frequencies among strains of E. coli isolated from bacteremia. Sat has been characterized for its cytotoxic action, but the possible immunomodulatory effects of Sat have not been investigated. Therefore, this study aimed to evaluate the proteolytic effects of Sat on complement proteins and the role in pathogenesis of BSI caused by extraintestinal E. coli (ExPEC). E. coli EC071 was selected as a Sat-producing ExPEC strain. Whole-genome sequencing showed that sat sequences of EC071 and uropathogenic E. coli CFT073 present 99% identity. EC071 was shown to be resistant to the bactericidal activity of normal human serum (NHS). Purified native Sat was used in proteolytic assays with proteins of the complement system and, except for C1q, all tested substrates were cleaved by Sat in a dose and time-dependent manner. Moreover, E. coli DH5α survived in NHS pre-incubated with Sat. EC071-derivative strains harboring sat knockout and in trans complementations producing either active or non-active Sat were tested in a murine sepsis model. Lethality was reduced by 50% when mice were inoculated with the sat mutant strain. The complemented strain producing active Sat partially restored the effect caused by the wild-type strain. The results presented in this study show that Sat presents immunomodulatory effects by cleaving several proteins of the three complement system pathways. Therefore, Sat plays an important role in the establishment of bloodstream infections and sepsis.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are largely distributed among Escherichia coli isolated from the bloodstream

Extraintestinal pathogenic Escherichia coli (ExPEC) is the major cause of Gram-negative-related sepsis. Bacterial survival in the bloodstream is mediated by a variety of virulence traits, including those mediating immune system evasion. Serine protease autotransporters of Enterobacteriaceae (SPATE) constitute a superfamily of virulence factors that can cause tissue damage and cleavage of molecules of the complement system, which is a key feature for the establishment of infection in the bloodstream. In this study, we analyzed 278 E. coli strains isolated from human bacteremia from inpatients of both genders, different ages, and clinical conditions. These strains were screened for the presence of SPATE-encoding genes as well as for phylogenetic classification and intrinsic virulence of ExPEC. SPATE-encoding genes were detected in 61.2% of the strains and most of these strains (44.6%) presented distinct SPATE-encoding gene profiles. sat was the most frequent gene among the entire collection, found in 34.2%, followed by vat (28.4%), pic (8.3%), and tsh (4.7%). Although in low frequencies, espC (0.7%), eatA (1.1%), and espI (1.1%) were detected and are being reported for the first time in extraintestinal isolates. The presence of SPATE-encoding genes was positively associated to phylogroup B2 and intrinsic virulent strains. These findings suggest that SPATEs are highly prevalent and involved in diverse steps of the pathogenesis of bacteremia caused by E. coli.

Identificação de cepas Escherichia coli patogênicas com potencial de secreção da toxina autotransportadora de Enterobacteriaceae Vat (Vacuolating autotransporter toxin) / Identification of pathogenic Escherichia coli strains with potential for secretion of Enterobacteriaceae Vat (Vacuolating autotransporter toxin) autotransporter toxin

As serino-proteases autotransportadoras de Enterobacteriaceae (SPATE) constituem uma família de proteases secretadas pelo sistema de secreção do tipo V, cujos genes foram estudados em Escherichia coli intestinal e extraintestinal. Vat é uma SPATE citotóxica de 140 kDa, a qual o gene foi identificado pela primeira vez em APEC (Avian Pathogenic Escherichia coli) isolado de colibacilose em frangos de corte ocasionando lesões em órgãos internos associados a septicemia. Estudos recentes analisam a relação desta citotoxina em cepas isoladas de infecção extraintestinais (ExPEC), principalmente E. coli isoladas de infecção urinária (UPEC) e septicemia. Além disso, o nosso grupo identificou o gene vat em cepas de E. coli enteropatogênica (EPEC). O objetivo deste trabalho foi contribuir para um projeto amplo sobre a expressão e função de Vat em E. coli patogênica humana, não APEC, identificando cepas de Escherichia coli patogênica (UPEC e EPEC) secretoras de Vat. Na confirmação da presença do gene vat em duas cepas de EPEC (BA 1250 e BA 1244) e uma UPEC (DV 33) constatou-se que a cepa BA 1244 não apresentava mais o gene e por isso esta cepa foi excluída do estudo. A presença de outras SPATEs como Pet e Pic que poderiam se presentes, interferir na identificação de Vat, também foram investigadas nas cepas DV 33 e BA 1250. Em nenhuma das duas cepas a expressão de Pet foi demonstrada. No entanto, a cepa DV 33 secretou Pic sendo está também excluída do estudo. As condições de cultivo da cepa BA 1250 para a obtenção do sobrenadante do cultivo, rico em proteínas, para a identificação de Vat foram padronizadas. Uma banda de massa molecular de 111 kDa que corresponde a massa molecular (sem a porção do β barril e peptídeo sinal) de Vat foi identificada. Uma nova corrida eletroforética será realizada a fim de se obter uma banda mais forte, bem delimitada que possibilite o envio para a identificação.

Serine Protease Autotransporters of the Enterobacteriaceae (SPATEs): Out and About and Chopping It Up.

Autotransporters are secreted proteins with multiple functions produced by a variety of Gram-negative bacteria. In Enterobacteriaceae, a subgroup of these autotransporters are the SPATEs (serine protease autotransporters of Enterobacteriaceae). SPATEs play a crucial role in survival and virulence of pathogens such as Escherichia coli and Shigella spp. and contribute to intestinal and extra-intestinal infections. These high molecular weight proteases are transported to the external milieu by the type Va secretion system and function as proteases with diverse substrate specificities and biological functions including adherence and cytotoxicity. Herein, we provide an overview of SPATEs and discuss recent findings on the biological roles of these secreted proteins, including proteolysis of substrates, adherence to cells, modulation of the immune response, and virulence in host models. In closing, we highlight recent insights into the regulation of expression of SPATEs that could be exploited to understand fundamental SPATE biology.

Curcumin Blocks Cytotoxicity of Enteroaggregative and Enteropathogenic Escherichia coli by Blocking Pet and EspC Proteolytic Release From Bacterial Outer Membrane.

Pet and EspC are toxins secreted by enteroaggregative (EAEC) and enteropathogenic (EPEC) diarrheagenic Escherichia coli pathotypes, respectively. Both toxins are members of the Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) family. Pet and EspC are important virulence factors that produce cytotoxic and enterotoxic effects on enterocytes. Here, we evaluated the effect of curcumin, a polyphenolic compound obtained from the rhizomes of Curcuma longa L. (Zingiberaceae) on the secretion and cytotoxic effects of Pet and EspC proteins. We found that curcumin prevents Pet and EspC secretion without affecting bacterial growth or the expression of pet and espC. Our results show that curcumin affects the release of these SPATEs from the translocation domain, thereby affecting the pathogenesis of EAEC and EPEC. Curcumin-treated EAEC and EPEC did not induce significant cell damage like the ability to disrupt the actin cytoskeleton, without affecting their characteristic adherence patterns on epithelial cells. A molecular model of docking predicted that curcumin interacts with the determinant residues Asp1018-Asp1019 and Asp1029-Asp1030 of the translocation domain required for the release of Pet and EspC, respectively. Consequently, curcumin blocks Pet and EspC cytotoxicity on epithelial cells by preventing their release from the outer membrane.

Three new serine-protease autotransporters of Enterobacteriaceae (SPATEs) from extra-intestinal pathogenic Escherichia coli and combined role of SPATEs for cytotoxicity and colonization of the mouse kidney.

Serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted proteins that contribute to virulence and function as proteases, toxins, adhesins, and/or immunomodulators. An extra-intestinal pathogenic E. coli (ExPEC) O1:K1 strain, QT598, isolated from a turkey, was shown to contain vat, tsh, and three uncharacterized SPATE-encoding genes. Uncharacterized SPATEs: Sha (Serine-protease hemagglutinin autotransporter), TagB and TagC (tandem autotransporter genes B and C) were tested for activities including hemagglutination, autoaggregation, and cytotoxicity when expressed in E. coli K-12. Sha and TagB conferred autoaggregation and hemagglutination activities. TagB, TagC, and Sha all exhibited cytopathic effects on a bladder epithelial cell line. In QT598, tagB and tagC are tandemly encoded on a genomic island, and were present in 10% of UTI isolates and 4.7% of avian E. coli. Sha is encoded on a virulence plasmid and was present in 1% of UTI isolates and 20% of avian E. coli. To specifically examine the role of SPATEs for infection, the 5 SPATE genes were deleted from strain QT598 and tested for cytotoxicity. Loss of all five SPATEs abrogated the cytopathic effect on bladder epithelial cells, although derivatives producing any of the 5 SPATEs retained cytopathic activity. In mouse infections, sha gene-expression was up-regulated a mean of sixfold in the bladder compared to growth in vitro. Loss of either tagBC or sha did not reduce urinary tract colonization. Deletion of all 5 SPATEs, however, significantly reduced competitive colonization of the kidney supporting a cumulative role of SPATEs for QT598 in the mouse UTI model.

First investigation of the presence of SPATE genes in Shigella species isolated from children with diarrhea infection in Ahvaz, southwest Iran.

Background: SPATE (serine protease autotransporters of enterobacteriaceae) genes are considered as a group of the main virulence factors of Shigella species This study aimed to investigate for the first time the distribution of SPATE genes among Shigella spp. isolated from children with diarrhea infection in Ahvaz, Iran. Methodology: In this study, a total of 74 Shigella isolates were collected between August 2016 and June 2017 from feces of children with diarrhea and identified by biochemical and molecular methods for Shigella species. The frequency distribution of the SPATE genes, including pic, pet, sat, sigA and sepA, was evaluated using PCR. The genetic relationship of all isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR. Results: The most common species of Shigella was S. flexneri, followed by S. sonnei and S. boydii. In total, 95.94% of Shigella isolates had at least one of the SPATE genes. The presence of pic, pet, sat, sigA and sepA genes was confirmed among 35.13%, 27%, 47.29%, 58.1% and 39.18% of Shigella isolates, respectively. Of these SPATE genes, the sat and sigA genes were recognized as the most common autotransporters among S. flexneri and S. sonnei isolates, respectively. Also, either S. flexneri or S. sonnei isolates belonging to a same clone type had similar SPATE genes profile. Conclusion: Our results revealed that the high distribution of SPATE genes among Shigella isolates in our region. Hence, this study highlights a need for epidemiological programs to monitor the distribution of SPATE genes locally for prevention from further dissemination of the Shigella isolates harboring them.

Distribution of serine protease autotransporters of Enterobacteriaceae in typical and atypical enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is an agent of acute and persistent diarrhea worldwide, categorized in typical or atypical subgroups. Some EAEC virulence factors are members of the serine protease autotransporters of Enterobacteriaceae (SPATE). The presence of SPATE-encoding genes of different E. coli pathotypes was searched in a large collection of EAEC strains, and a possible association between SPATEs and E. coli phylogroups was investigated. Among 108 typical and 85 atypical EAEC, pic was the most prevalent gene, detected in 47.1% of the strains, followed by sat (24.3%), espI (21.2%), pet (19.2%), sepA (13.5%), sigA (4.1%), eatA (4.1%), vat (1.0%), espP and tsh, detected in one strain (0.5%) each; while epeA and espC were not detected. Phylogenetic analysis demonstrated that 39.9% of the strains belonged to group A, 23.3% to B1, 10.9% to B2, 7.8% to D, 8.8% to E and 1.5% to F. The majority of the SPATE genes were distributed in typical and atypical strains without association with any phylogroup. In addition, pic and pet were strongly associated with typical EAEC and sepA was detected in close association with atypical EAEC. Our data indicate that SPATEs may represent important virulence traits in both subgroups of EAEC.

Bacterial Control of Pores Induced by the Type III Secretion System: Mind the Gap.

Type III secretion systems (T3SSs) are specialized secretion apparatus involved in the virulence of many Gram-negative pathogens, enabling the injection of bacterial type III effectors into host cells. The T3SS-dependent injection of effectors requires the insertion into host cell membranes of a pore-forming "translocon," whose effects on cell responses remain ill-defined. As opposed to pore-forming toxins that damage host cell plasma membranes and induce cell survival mechanisms, T3SS-dependent pore formation is transient, being regulated by cell membrane repair mechanisms or bacterial effectors. Here, we review host cell responses to pore formation induced by T3SSs associated with the loss of plasma membrane integrity and regulation of innate immunity. We will particularly focus on recent advances in mechanisms controlling pore formation and the activity of the T3SS linked to type III effectors or bacterial proteases. The implications of the regulation of the T3SS translocon activity during the infectious process will be discussed.

The serine protease Pic as a virulence factor of atypical enteropathogenic Escherichia coli.

Autotransporter proteins (AT) are associated with bacterial virulence attributes. Originally identified in enteroaggregative Escherichia coli (EAEC), Shigella flexneri 2a and uropathogenic E. coli, the serine protease Pic is one of these AT. We have previously detected one atypical enteropathogenic E. coli strain (BA589) carrying the pic gene. In the present study, we characterized the biological activities of Pic produced by BA589 both in vitro and in vivo. Contrarily to other Pic-producers bacteria, pic in BA589 is located on a high molecular weight plasmid. PicBA589 was able to agglutinate rabbit erythrocytes, cleave mucin and degrade complement system molecules. BA589 was able to colonize mice intestines, and an intense mucus production was observed. The BA589Δpic mutant lost the capacity to colonize as well as the above-mentioned in vitro activities. Thus, Pic represents an additional virulence factor in aEPEC strain BA589, associated with adherence, colonization and evasion from the innate immune system.

X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC).

Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a ß-pleaded sheet at the sequence region of residues 181-190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

Autotransporters and virulenceof enteroaggregative E. coli

EAEC, enteroaggregative E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli; EIEC,enteroinvasive E. coli; STEC, shiga-toxin producing E. coli; DAEC, diffusely adhering E. coli; AA, agregative adherence; OMP, outer membrane protein; AAF, aggregative adherence fimbriae; ECP, E. coli common pilus; T1SS, type I secretion system; EAST-1,enteroaggregative E. coli heat stable enterotoxin 1; ORF, open reading frame; Pet, plasmid-encoded toxin; Pic, protein involved in colonization; ShET1, Shigella enterotoxin 1; HlyE, hemolysin E; OM, outer membrane; T5SS, type V secretion system; TPS, twopartner secretion; SPATE, serine protease autotransporter of the Enterobacteriaceae; ER, endoplasmic reticulum; Shmu, Shigellamucinase; cAMP, cyclic adhenosin monophosphate; cGMP, cyclic guanosine monophosphate; STa, heat-stable toxin a; GC-C, guanylyl cyclase C; IL, interleukin; IEC, intestinal epithelial cells; MAPK, mitogen-activating protein kinase; TLR5, toll-like receptor 5; TNFá, tumour necrosis factor á; GRO, growth-related gene product; ICAM-1, intercellular adhesion molecule 1;GM-CSF, granulocyte-macrophage colony-stimulating factor.

Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function.

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.