Arne Olsén

Expression of two csg operons is required for production of fibronectin‐ and Congo red‐binding curli polymers in Escherichia coli K‐12

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin‐ and Congo red‐binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non‐polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase‐specific sigma factor σs in wild‐type cells, but not in mutants lacking the nucleoid associated protein H‐NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways.

The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long‐range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C‐terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long‐range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.

EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG

Streptococcus pyogenes is an important human pathogen that selectively interacts with proteins involved in the humoral defense system, such as immunoglobulins and complement factors. In this report we show that S.pyogenes has the ability to hydrolyze the chitobiose core of the asparagine‐linked glycan on immuno globulin G (IgG) when bacteria are grown in the presence of human plasma. This activity is associated with the secretion of a novel 108 kDa protein denoted EndoS. EndoS has endoglycosidase activity on purified soluble IgG as well as IgG bound to the bacterial surface. EndoS is required for the activity on IgG, as an isogenic EndoS mutant could not hydrolyze the glycan on IgG. In addition, we show that the secreted streptococcal cysteine proteinase SpeB cleaves IgG in the hinge region in a papain‐like manner. This is the first example of an endoglycosidase produced by a bacterial pathogen that selectively hydrolyzes human IgG, and reveals a novel mechanism which may contribute to S.pyogenes pathogenesis.

Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins

ABSTRACT Streptococcus pyogenes secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG.

σS‐dependent growth‐phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by σ70 in the absence of the nucleoid‐associated protein H‐NS

The stationary‐phase‐specific sigma factor σS (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin‐binding curli organelles upon reaching stationary phase. We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA. The transcriptional start site of csgBA was determined and an AT‐rich upstream activating sequence (UAS) required for transcriptional activation was identified. The pcsgBA promoter is not specific for σS since the same promoter sequence can be used by Eσ70in vivo in a strain lacking nucleoid‐associated protein H‐NS and σS. Transcription remained growth‐phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon σS for transcription, can be transcribed by Eσ70in vivo in the absence of H‐NS by utilizing the phdeAB promoter. Two other genes known to be under the control of σS for expression, bolA and katE, remained transcriptionally silent in the absence of H‐NS. It is suggested that a subset of E. coli promoters can be recognized by both EσS and Eσ70in vivo but H‐NS interacting with these sequences prevents formation of succesful transcription‐initiation complexes with Eσ70.

Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA)

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue‐type plasminogen activator (t‐PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t‐PA with high affinity (1 × 108 to 2 × 108 M‐1). The binding of plasminogen and t‐PA to curli‐expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli‐expressing E. coli was readily converted to plasmin by t‐PA; both plasmin and t‐PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.

Assembly of human contact phase proteins and release of bradykinin at the surface of curli-expressing Escherichia coli.

Previous work has demonstrated that most strains of the human pathogen Streptococcus pyogenes bind kininogens through M protein, a fibrous surface protein and virulence determinant. Here we find that strains of several other pathogenic bacterial species, both Gram‐positive and Gram‐negative, isolated from patients with sepsis, also bind kininogens, especially H‐kininogen (HK). The most pronounced interaction was seen between HK and Escherichia coli. Among clinical isolates of E. coli, the majority of the entero‐haemorrhagic, enterotoxigenic, and sepsis strains, but none of the enteroinvasive and enteropathogenic strains, bound HK. Binding of HK to E. coli correlated with the expression of curli, another fibrous bacterial surface protein, and the binding of HK to purified curli was specific, saturable, and of high affinity; Ka = 9 107M‐1. Other contact phase proteins such as factor XI, factor XII, and prekallikrein bound to curliated E. coli, but not to an isogenic curli‐deficient mutant strain, suggesting that contact phase activation may occur at the surface of curliated bacteria. Kininogens are also precursor molecules of the vasoactive kinins. When incubated with human plasma, curli‐expressing bacteria absorbed HK. Addition of purified plasma kallikrein to the HK‐loaded bacteria resulted in a rapid and efficient release of bradykinin from surface‐bound HK. The assembly of contact phase factors at the surface of pathogenic bacteria and the release of the potent proinflammatory and vasoactive peptide bradykinin, should have a major impact on the host‐microbe relationship and may contribute to bacterial pathogencity and virulence.

EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis.

ABSTRACT The human pathogen Streptococcus pyogenes primarily infects the upper respiratory tract and skin, but occasionally it disseminates and causes severe invasive disease with high mortality. This study revealed that the activity of extracellular EndoS, which hydrolyzes the functionally important N-linked oligosaccharides on opsonizing immunoglobulin G (IgG), contributes to increased survival of S. pyogenes in human blood ex vivo. The inability to kill the bacteria is due to reduced binding of IgG to Fc receptors and impaired classical pathway-mediated activation of complement. In addition, the activity of extracellular SpeB, which cleaves IgG into Fc and Fab fragments, also increases bacterial survival. This suggests that S. pyogenes expresses two enzymes, EndoS and SpeB, which modulate IgG by different mechanisms in order to evade the adaptive immune system.

Generation of a mature streptococcal cysteine proteinase is dependent on cell wall‐anchored M1 protein

In the present study, we have generated a mutant strain of Streptococcus pyogenes, MC25, which lacks M protein on its surface, and we demonstrate that this strain is unable to generate a mature 28 kDa cysteine proteinase. Furthermore, we show that S. pyogenes bacteria of M1 serotype are dependent on cell wall‐anchored M protein to cleave the secreted zymogen into a mature cysteine proteinase. We also show that MC25 secretes a 40 kDa zymogen, having a conformation different from that secreted by wild‐type bacteria. We provide data showing that the cleavage site is not blocked but, presumably, the active site is. This suggests that M protein, when anchored to the cell wall, is involved in the unfolding of the zymogen and generation of a mature cysteine proteinase that can be activated under reducing conditions. Our data add new aspects to the interaction between two important virulence factors of S. pyogenes, the streptococcal cysteine proteinase and M protein.

Biological properties of a Streptococcus pyogenes mutant generated by Tn916 insertion in mga.

The mga regulon of Streptococcus pyogenes contains genes which contribute to the pathogenicity and virulence of this significant human pathogen. Transposon insertional inactivation of the regulatory mga gene in a S. pyogenes strain of the clinically important M1 serotype, blocked the expression of four genes located downstream of mga. These genes encode the M1 protein, the IgG-binding protein H, protein SIC which is an extracellular inhibitor of complement, and the C5a peptidase which interferes with granulocyte migration. The wild-type strain is resistant to phagocytosis and adheres to human skin tissue sections; properties that were lost in the transposon mutant. Moreover, the mutant was less virulent to mice but more cytolytic to human lymphocytes, the latter due to an increased activity of streptolysin S, whereas the production of streptolysin O, another toxin of S. pyogenes, was not affected. The mga mutation was complemented in trans with an intact mga gene which restored the phenotype of the wild-type strain.