Polymerizing activity and regulation of group B Streptococcus pilus 2a sortase C1

Group B Streptococcus [GBS; Streptococcus agalactiae] is the leading cause of life-threatening diseases in newborn and is also becoming a common cause of invasive diseases in non-pregnant, elderly and immune-compromised adults. Pili, long filamentous fibers protruding from the bacterial surface, have been discovered in GBS, as important virulence factors and vaccine candidates. Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date and their mechanisms of transpeptidation and regulation need to be further investigated. The available three-dimensional structures of these enzymes reveal a typical sortase fold except for the presence of a unique feature represented by an N-terminal highly flexible loop, known as the “lid”. This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an auto-inhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme. A further characterization of this sortase active mutant showed promiscuity in the substrate recognition, as it is able to polymerize different LPXTG-proteins in vitro.

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

Mechanism for sortase localization and the role of sortase localization in efficient pilus assembly in Enterococcus faecalis.

Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many secreted virulence factors in gram-positive organisms are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that sortase A (SrtA) and sortase C (SrtC) are colocalized with SecA at single foci in the enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci when SrtA was deleted, implying a single site of secretion for these proteins. Furthermore, in the absence of the pilus-polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that this positively charged domain can act as a localization retention signal for the focal compartmentalization of membrane proteins.

Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif

Surface proteins of Staphylococcus aureus are linked to the bacterial cell wall by sortase, an enzyme that cleaves polypeptides at the threonine of the LPXTG motif. Surface proteins can be released from staphylococci by treatment with hydroxylamine, resulting in the formation of threonine hydroxamate. Staphylococcal extracts, as well as purified sortase, catalyze the hydroxylaminolysis of peptides bearing an LPXTG motif, a reaction that can be inhibited with sulfhydryl-modifying reagents. Replacement of the single conserved cysteine at position 184 of sortase with alanine abolishes enzyme activity. Thus, sortase appears to catalyze surface-protein anchoring by means of a transpeptidation reaction that captures cleaved polypeptides as thioester enzyme intermediates.

Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections

Many Gram-positive bacteria covalently tether their surface adhesins to the cell wall peptidoglycan. We find that surface proteins of Staphylococcus aureus are linked to the cell wall by sortase, an enzyme that cleaves polypeptides at a conserved LPXTG motif. S. aureus mutants lacking sortase fail to process and display surface proteins and are defective in the establishment of infections. Thus, the cell wall envelope of Gram-positive bacteria represents a surface organelle responsible for interactions with the host environment during the pathogenesis of bacterial infections.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.

Sortase as a Tool in Biotechnology and Medicine

We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.

We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.

We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells

Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis.

Background. Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. Methods. S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. Results. We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. Conclusions. This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

Étude des interactions entre streptococcus suis sérotype 2 et des cellules endothéliales porcines

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Untersuchungen zum Vorkommen und molekularen Mechanismus der Biofilm-Bildung bei Enterokokken aus verschiedenen klinischen Bereichen

Im Rahmen der vorliegenden Arbeit wurde eine größere Anzahl an E. faecalis Isolaten aus Vaginalabstrichen, erstmals insbesondere von Patientinnen, die an Bakterieller Vaginose litten, untersucht und mit E. faecalis Stämme aus verschiedenen anderen klinischen Bereichen auf das Vorkommen von Virulenzfaktoren verglichen. Weiterhin wurden Korrelationen zwischen bestimmten Faktoren und der Menge an produziertem Biofilm erstellt, um mögliche Zusammenhänge zum Mechanismus der Biofilm-Bildung zu erfassen. Mittels statistischer Analysen konnte hinsichtlich der 150 untersuchten E. faecalis Isolate nachgewiesen werden, dass keine signifikanten Unterschiede der Inzidenzen von Virulenzfaktoren (esp, asa1, gelE, GelE, cylA, β-Hämolyse) zwischen den Stämmen der verschiedenen Herkunftsbereiche bestanden. In Bezug auf das Auftreten von Biofilm-Bildung zeigte sich ein erhöhtes Vorkommen bei Stämmen aus Urin sowie invasiver Herkunft (insgesamt jeweils ca. 70 % mäßige und starke Biofilm-Bildner) im Vergleich zu E. faecalis Isolaten aus Wunden oder Faeces (je ca. 40 %). Statistische Auswertungen bzgl. des Zusammenhangs eines oder einer Kombination von Virulenzfaktoren mit der Menge an gebildetem Biofilm wiesen darauf hin, dass Isolate, die das esp Gen besaßen, in erhöhtem Maße zur Biofilm-Bildung befähigt waren. Dies zeigte einen gewissen Einfluss des Zellwandproteins auf die Fähigkeit zur Biofilm-Bildung bei E. faecalis. Allerdings wurden stets auch Stämme identifiziert, die die Fähigkeit zur Biofilm-Bildung trotz des Fehlens der jeweils untersuchten genetischen Determinante bzw. der Determinanten aufwiesen, so dass auf das Vorhandensein weiterer, unbekannter Einflussfaktoren auf den Mechanismus der Biofilm-Bildung bei E. faecalis geschlossen werden konnte. Unter 78 untersuchten E. faecium Isolaten aus verschiedenen klinischen Bereichen konnte lediglich ein Stamm (1,3 %) als mäßiger Biofilm-Bildner charakterisiert werden, so dass die Fähigkeit bei dieser Spezies in der hier untersuchten Region unter diesen Bedingungen kaum nachgewiesen werden konnte. Einen Schwerpunkt dieser Arbeit bildete die Untersuchung zum Vorkommen von Virulenzfaktoren und Biofilm-Bildung bei E. faecalis Isolaten aus Vaginalabstrichen. Bzgl. des Auftretens von Virulenzfaktoren und Biofilm-Produktion konnte kein Unterschied zwischen Stämmen assoziiert mit Bakterieller Vaginose und Isolaten einer Vergleichsgruppe festgestellt werden. Allerdings zeigte eine Gegenüberstellung mit den untersuchten E. faecalis Stämmen aus anderen klinischen Bereichen, dass die 80 Isolate aus Vaginalabstrichen eine ähnlich hohe Inzidenz bestimmter Virulenzfaktoren wie Stämme aus Faeces, Urin, Wunden oder invasiver Herkunft sowie eine mit den Isolaten aus Urin und invasiver Herkunft vergleichbar hohe Fähigkeit zur Biofilm-Bildung aufwiesen (ca. 75 % mäßige und starke Biofilm-Bildner). Dies deutete auf eine Verbreitung der Biofilm-Bildungsfähigkeit bei E. faecalis Stämmen der Vaginalflora und somit auf eine große Bedeutung der Eigenschaft für Isolate dieser Herkunft hin. Die statistische Auswertung der Korrelationen von Virulenzfaktoren mit der Menge an gebildetem Biofilm lieferte ähnliche Ergebnisse wie die Analysen bzgl. der 150 E. faecalis Isolate aus anderen klinischen Bereichen und untermauerte die Annahme, dass zusätzliche Faktoren zu den hier untersuchten Determinanten bei E. faecalis vorhanden sein müssen, die Einfluss auf den Mechanismus der Biofilm-Bildung nehmen. Deshalb konzentrierte sich ein weiterer Teil der vorliegenden Arbeit auf die Herstellung und Charakterisierung von E. faecalis Biofilm-Spontanmutanten, um bisher noch ungeklärte Mechanismen oder neue Faktoren zu erkennen, die Einfluss auf die Biofilm-Bildung bei E. faecalis nehmen. Die Untersuchung einer Mutante (1.10.16) und ihres Wildtypstamms lieferte erstmals den phänotypischen Nachweis des HMW-Komplexes der drei Bee-Proteine sowie die Identifizierung konservierter Pili-Motive dieser Proteine. Des Weiteren schien die in diesem Cluster ebenfalls codierte Sortase-1 dasjenige Enzym zu sein, das höchstwahrscheinlich die Bindung des Proteins Bee-2 innerhalb dieses HMW-Komplexes katalysiert. Insofern lieferten diese Untersuchungen neue, konkrete Hinweise zur Rolle des bee Genclusters bei der Pili-Biogenese und Biofilm-Bildung von E. faecalis. Darüber hinaus stellen Erkenntnisse aus der Charakterisierung von zwei weiteren hergestellten Biofilm-Spontanmutanten viel versprechende Ausgangspunkte für zukünftige Untersuchungen dar, die ein weitergehendes Verständnis der molekularen Mechanismen der Biofilm-Bildung bei E. faecalis erzielen könnten.

Untersuchungen zur Biofilmbildung bei dem klinischen Isolat Enterococcus faecalis 1.10

In der vorliegenden Arbeit wurde die Biofilmbildung bei einem klinischen Isolat von Enterococcus faecalis untersucht. Der Prozess der Biofilmbildung ist in mehrere Abschnitte unterteilt und beinhaltet zu Beginn eine Anhaftung von Zellen an Oberflächen. Dieser adhäsive Schritt wird unter anderem durch Pili vermittelt. Pili bei Grampositiven Mikroorganismen sind kovalent mit der Zellwand verknüpfte Proteinstrukturen, die eine Anheftung an biotische und abiotische Oberflächen sowie den Zell-Zell-Kontakt vermitteln. Bei den Analysen dieser Doktorarbeit lag ein besonderes Interesse bei eben diesen Pili, die für Enterococcus faecalis die Namen Ebp (endocarditis and biofilm associated pili) und Bee (biofilm enhancer in enterococci) tragen. Codiert werden sie durch die entsprechenden ebp-/bee-Loci, deren Aufbau unter den Grampositiven Mikroorganismen hochkonserviert ist. Die Loci bestehen aus Pilusuntereinheiten-codierenden Genen und colokalisierten Pilus-spezifischen Sortase Genen. Während in der Regel drei verschiedene Pilusuntereinheiten vorliegen, kann die Anzahl der Sortasen zwischen einer und zwei variieren. Bei den Experimenten wurde neben einer Komplementationsstudie zu einer Bee-Pilus Defekt-Mutante (1.10.16) das Hauptaugenmerk auf die Analyse des zweiten Pilus (Ebp) gelegt, um die Pilisituation bei Isolat 1.10 im Detail darzustellen Zusätzlich sollten weitere Oberflächenassoziierte Proteinstrukturen bei Isolat 1.10 detektiert werden, die gegebenenfalls an der Biofilmbildung beteiligt sind. Weitere Versuche zur Charakterisierung des Bee-Pilus wurden im Laufe dieser Arbeit durchgeführt, blieben jedoch bisher erfolglos. Die Biofilm-/Pilus-Defekt-Mutante 1.10.16 zeigte aufgrund einer Punktmutation (Pm) in der Pilus-spezifischen Sortase 1 des bee-Locus eine geschwächte Fähigkeit zur Anheftung an abiotische Oberflächen, sowie das Fehlen der Bee2 Untereinheit im Pilus. Nach Komplementation der Mutante (1.10.16K) mit dem Wildtyp-srt1 Gen, wurde die starke Biofilmbildungsfähigkeit zurück erlangt. Die Experimente zeigten, dass der Pilus-Defekt auf die Pm im srt1 Gen zurückzuführen war und der Bee-Pilus in Stamm 1.10.16K wieder korrekt gebildet wurde. Zu sehen war dies in Rasterelektronenmikroskopischen Aufnahmen und ebenfalls im massenspektrometrischen Nachweis aller 3 Pilusuntereinheiten im Bee-Pilus charakteristischen High-Molecular-Weight Komplex (~ 250 kDa). Durch Sequenzierungen konnte gezeigt werden, dass zwei Gene des ebp-Locus (ebpR und ebpC) bei Isolat 1.10 durch die Insertion von IS-Elementen IS1062 und IS6770 inaktiviert wurden. Der proteinbiochemische Nachweis über Pilusspezifische Antikörper gegen die Untereinheiten des Ebp-Pilus verlief negativ. Zusätzlich konnte gezeigt werden, dass die mRNA der beiden inaktivierten Gene nicht gebildet wurde. Dies führte folglich zum vollständigen Verlust des Ebp-Pilus bei Isolat 1.10. Zusammen mit den Ergebnissen der Komplementation konnte somit der große Einfluss mindestens eines intakten Pilus auf die Biofilmbildung gezeigt werden. Sind beide Pili durch Insertionen bzw. Mutationen inaktiviert, kommt es zu einer deutlichen Abnahme der Biofilmbildungsstärke. Dass trotzdem noch ein Biofilm gebildet wurde, zeigt den multifaktoriellen Zusammenhang bzw. Einfluss im Biofilmbildungsprozess. Über das gezielte Markieren von Oberflächenproteinen intakter Zellen mittels der Oberflächenbiotinylierung, konnten in der SDS-PAGE Unterschiede im Bandenmuster im Vergleich zur unbehandelten Probe erkannt werden. Die massenspektrometrische Identifikation dieser Proteine erfolgte bisher nicht, jedoch sind diese vorläufigen Ergebnisse vielversprechender Natur für die Identifikation und Aufklärung der Oberflächenproteinsituation bei Isolat 1.10.

Study of two novel streptococcus pneumoniae operons: pilus islet 2 and the lantibiotic operon

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element resembling gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (containing the genes pitA, sipA, pitB, srtG1, and srtG2) codes for a novel functional pilus in pneumococcus. Therefore, there are two pilus islets identified so far in this pathogen (PI-1 and PI-2). Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. PI-2 is associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging in both industrialized and developing countries. Interestingly, strains belonging to clonal complex 271 (CC271) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that may play a role in the initial host cell contact to the respiratory tract. In addition, the pilus proteins are potential antigens for inclusion in a new generation of pneumococcal vaccines. Adherence by pili could represent important factor in bacterial community formation, since it has been demonstrated that bacterial community formation plays an important role in pneumococcal otitis media. In vitro quantification of bacterial community formation by S. pneumoniae was performed in order to investigate the possible role of pneumococcal pili to form communities. By using different growth media we were not able to see clear association between pili and community formation. But our findings revealed that strains belonging to MLST clonal complex CC15 efficiently form bacterial communities in vitro in a glucose dependent manner. We compared the genome of forty-four pneumococcal isolates discovering four open reading frames specifically associated with CC15. These four genes are annotated as members of an operon responsible for the biosynthesis of a putative lanctibiotic peptide, described to be involved in bacterial community formation. Our experiments show that the lanctibiotic operon deletion affects glucose mediated community formation in CC 15 strain INV200. Moreover, since glucose consumption during bacterial growth produce an acidic environment, we tested bacterial community formation at different pH and we showed that the lanctibiotic operon deletion affected pH mediated community formation in CC 15 strain INV200. In conclusion, these data demonstrate that the putative lanctibiotic operon is associated with pneumococcal CC 15 strains in vitro bacterial community formation.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.