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.

Adhesin related genes as potential markers for the enteroaggregative Escherichia coli category.

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea in children and adults worldwide. This pathotype is phenotypically characterized by the aggregative-adherence (AA) pattern in HEp-2 cells and genetically associated to the presence of the aatA gene. EAEC pathogenesis relies in different virulence factors. At least, three types of adhesins have been specifically associated with EAEC strains: the five variants of the aggregative adherence fimbriae (AAF), the aggregative forming pilus (AFP) and more recently, a fibrilar adhesin named CS22. Our study aimed to evaluate the presence of AAF, AFP and CS22-related genes among 110 EAEC strains collected from feces of children with diarrhea. The presence of aggR (EAEC virulence regulator) and genes related to AAFs (aggA, aafA, agg3A, agg4A, agg5A and agg3/4C), AFP (afpA1 and afpR) and CS22 (cseA) was detected by PCR, and the adherence patterns were evaluated on HeLa cells. aggR-positive strains comprised 83.6% of the collection; among them, 80.4% carried at least one AAF-related gene and presented the AA pattern. aggA was the most frequent AAF-related gene (28.4% of aggR+ strains). cseA was detected among aggR+ (16.3%) and aggR- strains (22.2%); non-adherent strains or strains presenting AA pattern were observed in both groups. afpR and afpA1 were exclusively detected among aggR- strains (77.8%), most of which (71.4%) also presented AA pattern. Our results indicate that AAF- and AFP-related genes may contribute to identify EAEC strains, while the presence of cseA and its importance as an EAEC virulence factor and genotypic marker needs to be further evaluated.

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.