Novel IgG-degrading enzymes of the IgdE protease family link substrate specificity to host tropism of <i>Streptococcus</i> species

Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use

Streptococcal immunoglobulin degrading enzymes of the IdeS and IgdE family

Bacteria of the genus Streptococcus are common asymptomatic colonisers of humans and animals. As opportunistic pathogens they can however, depending on their host’s immune status and other circumstances, cause mild to very severe infections. Streptococci are highly intertwined with specific host species, but can also cause zoonosis or anthroponosis in more uncommon hosts. Prolonged and reoccurring infections require immune evasion strategies to circumvent detection and eradication by the host’s immune defence. A substantial part of the immune defence against bacterial pathogens is mediated by immunoglobulins. This thesis is based on work to identify and characterise immunoglobulin degrading enzymes secreted by different Streptococcus species as a means to sabotage and evade antibody-mediated immune responses. Stoichiometric and kinetic analysis of the IgG degrading enzyme IdeS from the important human pathogen S. pyogenes revealed that IdeS cleaves IgG, opposed to previous publications, as a monomer following classical Michaelis-Menten kinetics. The IdeS homologue of S. suis, IdeSsuis, did however not cleave IgG, but was highly specific fo rporcine IgM. S. suis was found to possess yet another protease, IgdE, capable of cleaving porcine IgG. Both of these proteases were shown to promote increased bacterial survival in porcine blood during certain conditions. IgdE is the founding member of a novel cysteine protease family (C113). Novel streptococcal members of this protease family were shown to specifically degrade certain IgG subtypes of the respective Streptococcus species’ main host. The observed substrate specificity of IgdE family proteases reflects the host tropism of these Streptococcus species, thereby giving insights into host-pathogen co-evolution. The abundance of immunoglobulin degrading enzymes among Streptococcus species indicates the importance of evasion from the antibody mediated immune responses for streptococci. These novel identified immunoglobulin degrading enzymes of the IdeS and IgdE protease families are potential valid vaccine targets and could also be of biotechnological use

Correction: Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

[This corrects the article DOI: 10.1371/journal.pone.0164809.]

Rabbit IgA Hinges That Resist IgA1 Protease Action Provide Options for Improved IgA-Based Therapeutic Agents

Immunoglobulin A provides a major line of defence against pathogens and plays a key role in the maintenance of the commensal microbiota in the intestinal tract. Having been shown to be more effective at tumour cell killing than IgG and strongly active against pathogens present in the mucosae, IgA antibodies have been attracting significant attention in recent years for use as therapeutic antibodies. To improve their therapeutic potential, bioengineered IgA forms with increased serum half-life and neutralizing abilities have been developed but the IgA hinge, which impacts susceptibility to bacterial proteases and ability to bridge between target and effector cells, has not yet been explored. The European rabbit has 15 IgA subclasses with exclusive hinge region motifs and varying lengths, constituting a unique model to evaluate the functional capabilities offered by incorporation of longer IgA hinges into immunoglobulins. Hinge regions from rabbit IgAs, featuring different lengths and sequences, were inserted into human IgA1 heavy chain to substitute the IgA1 hinge. These hinges did not appear to affect antigen binding nor the ability of the engineered chimeric IgA1 to bind and trigger FcαRI, as detected by IgA-mediated cell agglutination and release of superoxide by neutrophils. All rabbit hinge-human IgA1 hybrids were resistant to Clostridrum ramosum IgA protease enzyme digestion, as predicted by the lack of the cleavage site in the rabbit hinges. Some IgA1s featuring long rabbit hinges were cleaved by Neisseria meningitidis IgA1 protease cleavage type 1 or 2 enzymes, despite the lack of the predicted cleavage sites. More interestingly, the hybrid featuring the rabbit IgA15 hinge was not affected by any of the IgA proteases. The IgA15 hinge is longer than that found in human IgA1 and is composed by a unique motif with a stretch of nine consecutive Ser residues. These characteristics allow the preservation of a long hinge, with associated ability to bridge distantly spaced antigens and provide higher avidity binding, while remaining resistant to IgA protease degradation. The data suggest that the rabbit Cα15 hinge represents an interesting alternative hinge sequence for therapeutic human IgA antibodies that remains resistant to proteolytic cleavage

IgdE family proteases cleave IgG in the hinge region.

<p>(A) The cleavage sites within IgG molecules were determined through N-terminal Edman sequencing of the 32 kDa IgG cleavage products generated by IgdE family proteases. The identified aa sequences (<b>bold</b>) were found in the hinge regions of the respective IgG heavy chains. Homodimer disulfide bond cysteine residues are <u>underlined</u>. 10 aa N- and C-terminal from the identified cleavage site (scissor symbol) of the respective IgG heavy chain are shown. Porcine IgG4a was chosen as a representative for porcine IgG. (B) Sequences of the hinge regions and adjacent parts of the CH1 and CH2 domains of human, porcine and equine IgG subtypes were aligned using T-COFFEE (Version_8.93) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164809#pone.0164809.ref038" target="_blank">38</a>] to illustrate hinge region diversity. Alignment reliability assessed by TCS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164809#pone.0164809.ref039" target="_blank">39</a>] is color coded (blue to red).</p

Used primers for cloning of <i>igdE</i> genes.

<p>Used primers for cloning of <i>igdE</i> genes.</p

Number of identified putative IgdE family protease sequences and searched genomes.

<p>Number of identified putative IgdE family protease sequences and searched genomes.</p

IgdE family proteases are specific for IgG compared to IgM and IgA.

<p>2% porcine plasma was incubated with (+) or without (-) 20 μg/ml purified rIgdE_porcinus (A) or 5% soluble fraction of E. coli cells expressing rIgdE_{pseudoporcinus} (B) respectively for 16h at 37°C. 2% equine serum was incubated with 5% soluble fraction of E. coli cells expressing rIgdE_equi (C) for 16h at 37°C. The reactions were analyzed by anti-porcine or anti-equine IgG, IgM and IgA Western blots under reducing conditions. Only degradation products of IgG (*) could be observed.</p