Versatile substrates and probes for IgA1 protease activity.

Bacterial meningitis is a severe infectious disease with high mortality. Gram-positive and Gram-negative bacteria that cause meningitis secrete immunoglobulin A1 (IgA1) proteases to assist in mucosal colonization, invasion, and immune evasion. IgA1 proteases have unique selectivity, with few reported substrates other than IgA1 from human tissue. Here we describe the design, characterization, and application of peptide substrates for diverse IgA1 proteases from Neisseria, Haemophilus, and Streptococcus bacteria. IgA1 proteases from diverse strains showed unexpected selectivity profiles among peptide substrates derived from autoproteolytic sites. A fluorescence probe derived from one of these peptides was used to quantitate IgA1 protease activity in buffer and in human cerebrospinal fluid; it was able to detect recombinant Haemophilus influenzae type 1 IgA1 protease at less than 1 µg mL(-1) . We also used the probe to establish the first high-throughput screen for IgA1 protease inhibitors. This work provides tools that will help investigate the roles of IgA1 proteases in bacterial colonization, immune evasion, and infection.

Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans.

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).

Active-site gating regulates substrate selectivity in a chymotrypsin-like serine protease the structure of haemophilus influenzae immunoglobulin A1 protease.

We report here the first structure of a member of the immunoglobulin A protease (IgAP) family at 1.75-A resolution. This protease is a founding member of the type V (autotransporter) secretion system and is considered a virulence determinant among the bacteria expressing the enzyme. The structure of the enzyme fits that of a classic autotransporter in which several unique domains necessary for protein function are appended to a central, 100-A-long beta-helical domain. The N-terminal domain of the IgAP is found to possess a chymotrypsin-like fold. However, this catalytic domain contains a unique loop D that extends over the active site acting as a lid, gating substrate access. The data presented provide a structural basis for the known ability of IgAPs to cleave only the proline/serine/threonine-rich hinge peptide unique to IgA1 (isotype 1) in the context of the intact fold of the immunoglobulin. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique extended loop D in this IgAP sterically occludes the active-site binding cleft in the absence of immunoglobulin binding is presented. Only in the context of binding of the IgA1-Fc domain in a valley formed between the N-terminal protease domain and another domain appended to the beta-helix spine (domain 2) is the lid stabilized in an open conformation. The stabilization of this open conformation through Fc association subsequently allows access of the hinge peptide to the active site, resulting in recognition and cleavage of the substrate.

Milk components inhibit Acanthamoeba-induced cytopathic effect.

PURPOSE: Acanthamoebae provoke a vision-threatening corneal infection known as Acanthamoeba keratitis (AK). It is thought that Acanthamoeba-specific IgA antibodies present in mucosal secretions such as human tears, milk, and saliva provide protection against infection by inhibiting the adhesion of parasites to host cells. The goal of the present study was to determine whether human mucosal secretions have the potential to provide protection against the Acanthamoeba-induced cytopathic effect (CPE) by an additional mechanism that is independent of IgA. METHODS: Breast milk was used as a model of human mucosal secretions. In vitro CPE assays were used to examine the CPE inhibitory effect of IgA-depleted milk and various milk fractions obtained by gel filtration. The activity of amebic proteinases was examined by zymography. RESULTS: IgA-depleted milk inhibited the Acanthamoeba-induced CPE in a concentration-dependent manner. Milk proteins were separated into four major fractions (F1-F4) by gel filtration. Of these four fractions, CPE inhibitory activity was detected largely in fraction F3. In contrast, fractions F1, F2, and F4 lacked CPE inhibitory activity. Moreover, fraction F3, but not F1, F2, or F4, inhibited amebic proteinases. CONCLUSIONS: These data, in conjunction with published findings showing that amebic proteinases are responsible for the induction of Acanthamoeba CPE, led us to propose that human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA and that involves the inhibition of cytotoxic proteinases of amebae.

Microbial IgA protease removes IgA immune complexes from mouse glomeruli in vivo: potential therapy for IgA nephropathy.

The hallmark of IgA nephropathy (IgAN), the most common form of glomerulonephritis, is the presence of mesangial deposits containing IgA, specifically the IgA1 subclass, as the most prominent component. The deposited IgA is considered to be part of an immune complex. The family of enzymes known as bacterial IgA proteases exhibits substrate specificity that is essentially limited to the hinge region of IgA1. Here we demonstrate the ability of systemically administered IgA protease to remove glomerular IgA immune complexes, both the antigen and antibody components, in a passive mouse model of IgAN. Thus, IgA protease may have potential as a therapeutic agent for human IgAN.

Impact of the molecular form of immunoglobulin A on functional activity in defense against Streptococcus pneumoniae.

Antibodies of the immunoglobulin A (IgA) class react with capsular polysaccharides of Streptococcus pneumoniae and support complement-dependent opsonophagocytosis (OPC) of the organism by phagocytes. We characterized the biologic impact of the molecular forms of human monoclonal capsule-specific IgA (monomeric IgA [mIgA], polymeric IgA [pIgA], and secretory IgA [SIgA]) on OPC and susceptibility to cleavage by IgA1 protease. The efficiency of SIgA in support of OPC of S. pneumoniae was comparable to that of pIgA, and both forms exceeded that of mIgA by a fivefold margin. This structure-function relationship was associated with three factors. First, the avidities, or functional affinities, of both pIgA and SIgA for pneumococcal capsules exceeded those of mIgA. Second, both pIgA and SIgA required less complement to achieve similar levels of bacterial OPC than did mIgA, indicating that secretory component does not hinder the effect of complement. Third, both pIgA and SIgA mediated agglutination of the organism, whereas mIgA did not. All three forms of capsule-specific IgA showed comparable susceptibilities to cleavage and functional inhibition by bacterial IgA1 protease, demonstrating that secretory component does not prevent the proteolytic degradation of IgA1 by IgA1 protease. IgA1 cleavage results in formation of identical Fab fragments for each of the molecular forms, thereby abolishing the contribution of multivalence of pIgA and SIgA. In summary, the polymeric forms of IgA (both pIgA and SIgA) provide a substantial advantage in binding, agglutination, and OPC of the organism.

Proteolytic processing of the Cryptosporidium glycoprotein gp40/15 by human furin and by a parasite-derived furin-like protease activity.

The apicomplexan parasite Cryptosporidium causes diarrheal disease worldwide. Proteolytic processing of proteins plays a significant role in host cell invasion by apicomplexan parasites. In previous studies, we described gp40/15, a Cryptosporidium sp. glycoprotein that is proteolytically cleaved to yield two surface glycopeptides (gp40 and gp15), which are implicated in mediating infection of host cells. In the present study, we showed that biosynthetically labeled gp40/15 is processed in Cryptosporidium parvum-infected HCT-8 cells. We identified a putative furin cleavage site RSRR downward arrow in the deduced amino acid sequence of gp40/15 from C. parvum and from all Cryptosporidium hominis subtypes except subtype 1e. Both human furin and a protease activity present in a C. parvum lysate cleaved recombinant C. parvum gp40/15 protein into 2 peptides, identified as gp40 and gp15 by size and by immunoreactivity with specific antibodies. C. hominis gp40/15 subtype 1e, in which the RSRR sequence is replaced by ISKR, has an alternative furin cleavage site (KSISKR downward arrow) and was also cleaved by both furin and the C. parvum lysate. Site-directed mutagenesis of the C. parvum RSRR sequence to ASRR resulted in inhibition of cleavage by furin and the C. parvum lysate. Cleavage of recombinant gp40/15 and a synthetic furin substrate by the C. parvum lysate was inhibited by serine protease inhibitors, by the specific furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk), and by calcium chelators, suggesting that the parasite expresses a Ca2+ dependent, furin-like protease activity. The furin inhibitor Dec-RVKR-cmk decreased C. parvum infection of HCT-8 cells, suggesting that a furin-like protease activity may be involved in mediating host-parasite interactions.

Characteristics of persistent diarrhea in a community-based cohort of young US children.

OBJECTIVE: The objective of the study was to define the characteristics and microbiology of persistent diarrhea (PD) in US children. METHODS: Six-month prospective cohort study of a convenience sample of 604 healthy 6- to 36-month-old children recruited by the Slone Center Office-based Research Network. RESULTS: Of 611 diarrhea episodes, 50 (8.2%) lasted < or = 14 days. The incidence of PD was 0.18 episodes per person-year, and the median duration of episodes was 22.0 days (range, 14-64 days). PD episodes were more likely than acute episodes to result in a medical visit (28.0% vs 8.2%; P = 0.0001). The most commonly used treatments were oral rehydration solution (12.0% of episodes) and antibiotics (6.0%). No bacterial or parasitic pathogens were associated with PD; but norovirus, rotavirus and sapovirus were each significantly more prevalent in PD stools compared with baseline stools, with relative risks of 12.4, 6.9 and 6.2, respectively. Fifty-nine per cent of the PD specimens tested were negative for all studied pathogens. CONCLUSIONS: PD occurs with a frequency of approximately 1 case per 5 person-years in US infants and young children. It seems to be a generally benign illness, with only 28% of cases presenting to medical care. Although viral pathogens seem to cause a minority of PD episodes in this population, most are not due to currently known infectious agents.

Distribution of bacterial proteins in biofilms formed by non-typeable Haemophilus influenzae.

The ability to preserve the fragile ultrastructural organization of bacterial biofilms using cryo-preparation methods for electron microscopy has enabled us to probe sections through non-typeable Haemophilus influenzae (NTHi) biofilms and determine the localization of NTHi-specific lipooligosaccharide (LOS) and proteins within these structures. Some of the proteins we examined are currently being considered as candidates for vaccine development, so it is important that their distribution and accessibility within the biofilms formed by NTHi be determined. We have localized LOS to the extracellular matrix (ECM) of the biofilm and the P6 outer membrane protein to the membrane of what appear to be viable bacteria within the biofilm. The Hap and HWM1/HMW2 adhesive proteins were associated with bacteria within the biofilm and were present in the biofilm ECM. The IgA1 protease is a secreted protein that was also associated with NTHi in the biofilm and was in the ECM, but was more concentrated in the top region of the biofilm, suggesting a role in protecting biofilm bacteria from antibody attack.

Diarrhea in American infants and young children in the community setting: incidence, clinical presentation and microbiology.

OBJECTIVE: The characteristics and microbiology of the full spectrum of pediatric diarrhea occurring in the U.S. community setting are not well-understood. METHODS: Six-month prospective cohort study of 604 healthy 6- to 36-month-old children recruited by the Slone Center Office-based Research Network. RESULTS: The incidence of parent-defined diarrhea was 2.2 episodes per person-year. The median duration of diarrhea was 2 days with a median of 6 stools per episode. Outpatient visits and hospitalization were prompted by 9.7 and 0.3% of episodes, respectively. The most common microorganisms identified in healthy baseline stools were atypical enteropathogenic Escherichia coli (12.2%), enteroaggregative Escherichia coli (3.7%), Clostridium difficile (3.5%) and Clostridium perfringens (2.9%), and each of these was no more common in diarrhea stools. In contrast, all of the viruses analyzed were more prevalent in diarrhea specimens than in baseline specimens: enteric adenovirus (5.7% diarrhea versus 1.4% baseline), rotavirus (5.2% versus 1.4%), astrovirus (3.5% versus 1.4%), Sapporo-like virus (3.0% versus 0.8%) and norovirus (1.9% versus 0.8%). A likely pathogen was detected in 20.6% of diarrhea specimens. Vomiting and > or =16 stools in an episode were predictive of isolating a pathogen from the stool, each with a relative risk of approximately 2. CONCLUSIONS: Healthy young children in this study experienced more than 2 cases of diarrhea per person-year, but most were brief and do not require medical attention. Although most diarrhea-associated pathogens were viruses, no likely pathogen was found in almost 80% of cases; possible etiologies for these cases include currently unknown gastrointestinal infections, nongastrointestinal illnesses and dietary/environmental factors.

Cleavage of the human immunoglobulin A1 (IgA1) hinge region by IgA1 proteases requires structures in the Fc region of IgA.

Secretory immunoglobulin A (IgA) protects the mucosal surfaces against inhaled and ingested pathogens. Many pathogenic bacteria produce IgA1 proteases that cleave in the hinge of IgA1, thus separating the Fab region from the Fc region and making IgA ineffective. Here, we show that Haemophilus influenzae type 1 and Neisseria gonorrhoeae type 2 IgA1 proteases cleave the IgA1 hinge in the context of the constant region of IgA1 or IgA2m(1) but not in the context of IgG2. Both C(alpha)2 and C(alpha)3 but not C(alpha)1 are required for the cleavage of the IgA1 hinge by H. influenzae and N. gonorrhoeae proteases. While there was no difference in the cleavage kinetics between wild-type IgA1 and IgA1 containing only the first GalNAc residue of the O-linked glycans, the absence of N-linked glycans in the Fc increased the ability of the N. gonorrhoeae protease to cleave the IgA1 hinge. Taken together, these results suggest that, in addition to the IgA1 hinge, structures in the Fc region of IgA are required for the recognition and cleavage of IgA1 by the H. influenzae and N. gonorrhoeae proteases.