Virulence Factor In Infections Research Articles

Should Antimicrobial Resistance Limit Access to an Organ Transplant?

Burkholderia cenocepacia (B cenocepacia) is a gram-negative bacteria associated with significant morbidity and mortality following lung transplantation. Most US transplant programs consider B cenocepacia colonization to be an absolute contraindication to transplantation. This article argues that, if clinicians have good clinical reasons to expect poor outcomes for patients with B cenocepacia, then offering transplantation anyway is an abrogation of clinicians' fiduciary duties. This article also discusses other fiduciary obligations transplant programs might have to patients with B cenocepacia, such as referring to another transplant center, considering novel treatment options, and investigating how the infection's virulence factors stratify that patient's risk for poor transplant outcomes.

The Role of Selected Virulence Factors in Infections Caused by Enterococcus spp.

Streszczenie Ziarenkowce z rodzaju Enterococcus spp. są składnikiem mikrobioty ludzi i zwierząt oraz powszechnie występują w środowisku naturalnym. Należą do patogenów warunkowo chorobotwórczych, mogących wywoływać zakażenia o różnej lokalizacji. Bakterie te rzadko wywołują zakażenia pozaszpitalne. Mimo, że do niedawna były uważane za drobnoustroje o niskim potencjale chorobotwórczym, stały się jednym z istotnych patogenów szpitalnych. Ich powszechne występowanie i zdolność do przeżycia w środowisku szpitalnym przyczyniają się do notowanej od kilku lat i nadal wzrastającej częstości ich izolacji, również z zakażeń inwazyjnych. Gatunki najczęściej izolowane z przypadków zakażeń to E. faecalis i E. faecium, stwarzające problemy terapeutyczne w związku z ich narastającą wielolekoopornością. Z powodu rosnącego znaczenia klinicznego, mechanizmów naturalnej i nabytej oporności na antybiotyki oraz potencjalnych czynników wirulencji, ziarenkowce z rodzaju Enterococcus stały się przedmiotem wielu badań. Celem pracy jest przedstawienie aktualnej wiedzy dotyczącej najistotniejszych czynników wirulencji mogących występować u bakterii z rodzaju Enterococcus, do których należą – antygen wydzielniczy SagA, białko EfaA, białko powierzchniowe Esp, białko wiążące kolagen Ace, cytolizyna, hialuronidaza, hemaglutynina, lipaza, proteaza serynowa, substancja agregująca, zewnątrzkomórkowe nadtlenki oraz żelatynaza.

Farnesol: An approach on biofilms and nanotechnology.

Farnesol is a natural compound that inhibits the formation of biofilms from different microbial species. The encapsulation of this molecule in nanoparticles is a promising alternative for the development of more effective therapies against biofilms.

Efficiency of Plant Active Compounds as Anti-Adhesion Factors for Clinical Candida albicans Isolates in vitro

The aim of this study is the determination of the anti-adhesion activity of active compounds extracted from plants in the inhibition of adhesion ability of C. albicans that is used as a virulence factor for infection. Adherence to host surfaces is a primary factor in the colonization of human tissues by fungi, which can also adhere to the surfaces of medical devices and form biofilms. Medicinal plants possess therapeutic properties or beneficial pharmacological effects on the body. A total of one hundred samples were collected from female patients with vaginal infection in two hospitals in Baghdad city for three months. The fungi were isolated and identified by microscopic morphology, morphological features on culture media, and Vital Index of Traditional Environmental Knowledge (VITEK 2) System. Extraction and purification of plant active compounds, vasicine from Adhatoda vasica and gallic acid from Punica granatum L. , were performed. The active compounds were used as anti-adhesion factors and the minimum inhibition concentration (MIC) against Candida albicans was determined. We also used the antifungal drug fluconazole for comparison as a control. The results of vaginal discharge characterization of clinical samples showed that 30% of the infections are caused by fungi (100% were Candida albicans), 47% by Gram negative bacteria, and 23% by Gram positive bacteria. The results of MIC assays showed that the plant extract and the antifungal drug do not seem to have fungicidal effects on the isolates of C. albicans tested. The ability of C. albicans isolates for adhesion to the epithelial cells was detected by the calculation of the number of adherent C. albicans cells under light microscope. The activity of vasicin and gallic acid appeared to be approximately equal on the number of C. albicans adherent on epithelial cells. Vasicin concentration activity used in mg/ ml was higher than that used in µg/ ml, with less numbers of C. albicans adhered on epithelial cell. However, treatment with fluconazole showed that more than half of the number of C. albicans were non-adherent on epithelial cells when added to the suspension, in contrast to the control (C. albicans without the extracts or antifungal drug).

Human Enteric α-Defensin 5 Promotes Shigella Infection by Enhancing Bacterial Adhesion and Invasion

Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection invitro in a structure-dependent manner. Shigella, commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium throughinteractions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella-induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen.

Pyomelanin produced by Vibrio cholerae confers resistance to predation by Acanthamoeba castellanii

Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii.

A putative spermidine synthase interacts with flagellar switch protein FliM and regulates motility in Helicobacter pylori.

The flagellar motor is an important virulence factor in infection by many bacterial pathogens. Motor function can be modulated by chemotactic proteins and recently appreciated proteins that are not part of the flagellar or chemotaxis systems. How these latter proteins affect flagellar activity is not fully understood. Here, we identified spermidine synthase SpeE as an interacting partner of switch protein FliM in Helicobacter pylori using pull-down assay and mass spectrometry. To understand how SpeE contributes to flagellar motility, a speE-null mutant was generated and its motility behavior was evaluated. We found that deletion of SpeE did not affect flagellar formation, but induced clockwise rotation bias. We further determined the crystal structure of the FliM-SpeE complex at 2.7 Å resolution. SpeE dimer binds to FliM with micromolar binding affinity, and their interaction is mediated through the β1' and β2' region of FliM middle domain. The FliM-SpeE binding interface partially overlaps with the FliM surface that interacts with FliG and is essential for proper flagellar rotational switching. By a combination of protein sequence conservation analysis and pull-down assays using FliM and SpeE orthologues in E. coli, our data suggest that FliM-SpeE association is unique to Helicobacter species.

Determining the Structure of NRPS‐Independent Siderophore (NIS) Synthetase DesD Using X‐ray Crystallography

A lack of iron leads to secretion of siderophores (small molecule metal chelators) by bacteria. Siderophores have high affinity for iron outcompeting host chelators, and making them virulence factors in infection. Some siderophores (of particularly virulent pathogens) are assembled via an NRPS‐independent siderophore (NIS) pathway. Understanding the structure of NIS siderophore biosynthesis enzymes may lead to the design of specific antibiotics to fight pathogenic bacteria.The Hoffmann lab is using x‐ray crystallography to determine the protein structure of NIS synthetase DesD, from Streptomyces coelicolor. DesD makes the siderophore desferrioxamine B, requiring three iterative bonds and a macrocyclization event. DesD is one of the best biochemically characterized NIS synthetases so far, and we are solving this structure as a model for the rest. The goal of this project is to solve the x‐ray structure of DesD bound to substrate and ATP, as well as apo (empty of substrate) structures, in order to characterize the conformational changes that may occur with binding. DesD crystalizes in 20% PED 8,000, 15% glycerol, and 15% ethylene glycol conditions, and crystals diffract to 2.48 Å with good statistics. We are solving the structure using molecular replacement and are pursuing substrate‐bound crystals. This structural information may allow us to design an inhibitor that will prevent the binding of substrate in future work.

A potential therapeutic peptide-based neutralizer that potently inhibits Shiga toxin 2 in vitro and in vivo.

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which can cause serious clinical complications in humans, such as hemolytic uremic syndrome (HUS). Recently, we screened and identified two peptide-based Stx2 neutralizers, TF-1 and WA-8, which specifically and directly bind to Stx2. Computer simulations suggested that the majority of TF-1 or WA-8 binds tightly at the receptor-binding site 3 of Stx2. The two peptides also effectively inhibited the cytotoxic activity of Stx2 by blocking the binding of Stx2 to target cells. TF-1 exhibits remarkable therapeutic potency in both mice and rat toxicity models. In mice toxicity models, TF-1 provided full protection when mice were injected with 5 LD50 of Stx2. In rat toxicity models, TF-1 reduced fatal tissue damage and completely protected rats from the lethal challenges of Stx2. In these rats, TF-1 significantly decreased the concentration of Stx2 in blood and diminished tissue distribution levels of Stx2. Furthermore, TF-1 effectively protected rats from the pathological effects caused by Stx2, especially in the kidney, thymus, adrenal gland, and lung. Taken together, these results indicate that TF-1 is a promising therapeutic agent against the pathogenicity of Stx2.

Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa.

Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR. Here, it was shown that MucR impairs alginate production in response to nitrate in P. aeruginosa. Subsequent site-specific mutagenesis of MucR revealed that the second MHYT sensor motif (MHYT II, amino acids 121-124) of MucR sensor domain was involved in nitrate sensing. We also showed that both c-di-GMP synthesizing and degrading active sites of MucR were important for alginate production. Although nitrate and deletion of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post-translational level through a localized pool of c-di-GMP. Nitrate increased pel promoter activity in the mucR mutant while in the same mutant the psl promoter activity was independent of nitrate. Nitrate and deletion of mucR did not impact on swarming motility but impaired attachment to solid surfaces. Nitrate and deletion of mucR promoted the formation of biofilms with increased thickness, cell density, and survival. Overall, this study provided insight into the functional role of MucR with respect to nitrate-mediated regulation of alginate biosynthesis.

Characterization of the biofilm forming ability of Staphylococcus pseudintermedius from dogs

BackgroundStaphylococcus pseudintermedius is an opportunistic pathogen of dogs and has emerged as a leading cause of skin, wound and surgical site infections worldwide. Methicillin resistance is common and clinical infections as a result of methicillin-resistant S. pseudintermedius (MRSP) pose a clinical challenge. In other staphylococci, biofilm formation has been shown to be a virulence factor for infection, however, it has received little attention in S. pseudintermedius. The objectives of this study were to evaluate the biofilm forming ability of clinical isolates of S. pseudintermedius obtained from dogs using phenotypic and genotypic techniques.Results96% (136/140) of S. pseudintermedius isolates were classified as strong or moderate biofilm producers, with the majority of isolates being able to produce biofilm. There was no difference in biofilm formation between MRSP and MSSP (p=0.8), amongst isolates from clinical infections compared with isolates obtained from colonized dogs (p=0.08), and between isolates from sequence type (ST) 71 and ST 68 (P=0.09). icaA was detected in 77.9% (109/140) of isolates and icaD was detected in 75.7% (106/140) of isolates. Scanning electron microscopic evaluation of S. pseudintermedius biofilm production revealed aggregates of cocci and irregularly produced extracellular polymeric matrix.ConclusionThe majority of S. pseudintermedius isolates evaluated in this study were able to produce biofilm and this may be an important virulence factor in the rapid emergence of this bacterium in veterinary hospitals worldwide. Further study into the mechanisms of biofilm formation by S. pseudintermedius is warranted.

Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2

CPS are major virulence factors in infections caused by Neisseria meningitidis and form the basis for meningococcal serogroup designation and protective meningococcal vaccines. CPS polymers are anchored in the meningococcal outer membrane through a 1,2-diacylglycerol moiety, but the innate immunostimulatory activity of CPS is largely unexplored. Well-established human and murine macrophage cell lines and HEK/TLR stably transfected cells were stimulated with CPS, purified from an endotoxin-deficient meningococcal serogroup B NMB-lpxA mutant. CPS induced inflammatory responses via TLR2- and TLR4-MD-2. Meningococcal CPS induced a dose-dependent release of cytokines (TNF-α, IL-6, IL-8, and CXCL10) and NO from human and murine macrophages, respectively. CPS induced IL-8 release from HEK cells stably transfected with TLR2/6, TLR2, TLR2/CD14, and TLR4/MD-2/CD14 but not HEK cells alone. mAb to TLR2 but not an isotype control antibody blocked CPS-induced IL-8 release from HEK-TLR2/6-transfected cells. A significant reduction in TNF-α and IL-8 release was seen when THP-1- and HEK-TLR4/MD-2-CD14- but not HEK-TLR2- or HEK-TLR2/6-transfected cells were stimulated with CPS in the presence of Eritoran (E5564), a lipid A antagonist that binds to MD-2, and a similar reduction in NO and TNF-α release was also seen in RAW 264.7 cells in the presence of Eritoran. CD14 and LBP enhanced CPS bioactivity, and NF-κB was, as anticipated, the major signaling pathway. Thus, these data suggest that innate immune recognition of meningococcal CPS by macrophages can occur via TLR2- and TLR4-MD-2 pathways.

The C-Terminal Tail of Yersinia pseudotuberculosis YopM Is Critical for Interacting with RSK1 and for Virulence

Yersinia spp. undermine the immune responses of infected animals by translocating Yops directly into host cells with a type III secretion system. YopM, a leucine-rich repeat protein, is a critical virulence factor in infection. YopM localizes to both the nucleus and the cytoplasm in cultured cells, interacts with mammalian p90 ribosomal S6 kinase 1 (RSK1), and causes a decrease in NK cell populations in spleens. Little is known about the molecular interaction between YopM and RSK1 and its significance in pathogenesis. We performed a systematic deletion analysis of YopM in Yersinia pseudotuberculosis to determine which regions are required for RSK1 interactions, nuclear localization, virulence, and changes in immune cell populations during infection of mice. Full-length YopM associated with RSK1 in at least two protein complexes in infected cells, and deletion of its C-terminal tail abrogated all RSK1 interactions. The C-terminal tail was required for tissue colonization, as yopM mutants that failed to interact with RSK1 were as defective for tissue colonization as was a DeltayopM mutant; however, nuclear localization of YopM was not dependent on its RSK1 interaction. Mutants expressing YopM proteins which do not interact with RSK1 caused more pathology than did the DeltayopM mutant, suggesting that there are other RSK1-independent functions of YopM. Histopathological and flow cytometric analyses of spleens showed that infection with wild-type Y. pseudotuberculosis caused an influx of neutrophils, while mice infected with yopM mutants had increased numbers of macrophages. Decreases in NK cells after Y. pseudotuberculosis infection did not correlate with YopM expression. In conclusion, the C terminus of YopM is essential for RSK1 interactions and for virulence.

Cloning and overexpression of Pochonia chlamydosporia chitinase gene pcchi44, a potential virulence factor in infection against nematodes

The fungus Pochonia chlamydosporia is a biocontrol agent with commercial potential for root-knot and cyst nematodes. In this study, a gene (designated pcchi44) encoding an extracellular endochitinase was isolated for the first time from P. chlamydosporia using degenerate primers and a genome walking technique. The 2385-bp pcchi44 sequence is interrupted by three introns and encodes a 424-amino acid polypeptide. The cDNA sequence of pcchi44 was amplified via reverse transcription polymerase chain reaction and overexpressed in Escherichia coli BL21. The recombinant chitinase PCCHI44 was purified as a protein of ca. 44 kDa with an optimal activity at pH 6.0 and 50 °C. The V max and K m value of PCCHI44 using 4-methylumbelliferyl-β- d- N, N′, N″-triacetylchitotrioside as substrate were 8.37 × 10 −3 μM s −1 and 9.65 μM, respectively. The hydrolytic activity of PCCHI44 could be completely inhibited by acetazolamide. The chitinase PCCHI44 could damage eggs of both the root-knot nematode Meloidogyne incognita and the insect Bombyx mori.

Role of Toll-Like Receptors 2 and 4 in Pulmonary Inflammation and Injury Induced by Pneumolysin in Mice

BackgroundPneumolysin (PLN) is an intracellular toxin of Streptococcus pneumoniae that has been implicated as a major virulence factor in infections caused by this pathogen. Conserved bacterial motifs are recognized by the immune system by pattern recognition receptors among which the family of Toll-like receptors (TLRs) prominently features. The primary objective of the present study was to determine the role of TLR2 and TLR4 in lung inflammation induced by intrapulmonary delivery of PLN.Methodology/ResultsFirst, we confirmed that purified PLN activates cells via TLR4 (not via TLR2) in vitro, using human embryonic kidney cells transfected with either TLR2 or TLR4. Intranasal administration of PLN induced an inflammatory response in the pulmonary compartment of mice in vivo, as reflected by influx of neutrophils, release of proinflammatory cytokines and chemokines, and a rise in total protein concentrations in bronchoalveolar lavage fluid. These PLN-induced responses were dependent in part, not only on TLR4, but also on TLR2, as indicated by studies using TLR deficient mice.ConclusionThese data suggest that although purified PLN is recognized by TLR4 in vitro, PLN elicits lung inflammation in vivo by mechanisms that may involve multiple TLRs.

An Orally Applicable Shiga Toxin Neutralizer Functions in the Intestine To Inhibit the Intracellular Transport of the Toxin

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells.

Lack of protection of mice against Staphylococcus aureus despite a significant immune response to immunization with a DNA vaccine encoding collagen-binding protein

Collagen-binding protein (CNA) is the major Staphylococcus aureus adhesin responsible for high affinity binding to collagen and is assumed to be a major virulence factor in infection and disease. Mutants lacking the cna gene are less virulent than the parent strain in models of septic arthritis, osteomyelitis, keratinitis, and endocarditis. In order to investigate the immunological and protective properties of a CNA-based DNA vaccine, a eukaryotic expression vector pCNA was constructed which expressed the collagen-binding domain of this adhesin in transfected cells. Three groups of 11 Balb/c mice received three injection of either pCNA, the empty expression vector (pCI) or PBS. Those injected with pCNA showed hi titre (64000) antibody and evidence of a cell-mediated immune response (CMI). The anti-CNA antibodies recognized the intact bacteria and prevented binding to collagen in vitro. However, the vaccination did not protect against bacterial challenge using the intra-peritoneal route of infection. Moreover, S. aureus that had been treated with sera from vaccinated mice caused a more severe infection than bacteria treated with sera from non-vaccinated mice. In summary, DNA vaccination against CNA produced a strong antibody and cellular response in mice but failed to protect from i.p. infection by S. aureus.

Detailed characterization of the lipid A fraction from the nonpathogen Acinetobacter radioresistens strain S13

The genus Acinetobacter is composed of ubiquitous, generally nonpathogen environmental bacteria. Interest concerning these microorganisms has increased during the last 30 years, because some strains, belonging to the so-called A. baumannii-A. calcoaceticus complex, have been implicated in some severe pathological states in debilitated and hospitalized patients. The involvement of lipopolysaccharides (LPSs) as virulence factors in infections by Acinetobacter has been proven, and ongoing studies are aimed toward the complete serological characterization of the O-polysaccharides from LPSs isolated in clinical samples. Conversely, no characterization of the lipid A fraction from Acinetobacter strains has been performed. Here, the detailed structure of the lipid A fraction from A. radioresistens S13 is reported for the first time. A. radioresistens strains have never been isolated in cases of infectious disease. Nevertheless, it is known that the lipid A structure, with minor variations, is highly conserved across the genus; thus, structural details acquired from studies of this nonpathogen strain represent a useful basis for further studies of pathogen species.

Autoprocessing of Helicobacter pylori γ-Glutamyltranspeptidase Leads to the Formation of a Threonine-Threonine Catalytic Dyad

Helicobacter pylorigamma-glutamyltranspeptidase (HpGT) is a glutathione-degrading enzyme that has been shown to be a virulence factor in infection. It is expressed as a 60-kDa inactive precursor that must undergo autocatalytic processing to generate a 40-kDa/20-kDa heterodimer with full gamma-glutamyl amide bond hydrolase activity. The new N terminus of the processed enzyme, Thr-380, is the catalytic nucleophile in both the autoprocessing and enzymatic reactions, indicating that HpGT is a member of the N-terminal nucleophile hydrolase superfamily. To further investigate activation as a result of autoprocessing, the structure of HpGT has been determined to a resolution of 1.9 A. The refined model contains two 40-kDa/20-kDa heterodimers in the asymmetric unit and has structural features comparable with other N-terminal nucleophile hydrolases. Autoprocessing of HpGT leads to a large conformational change, with the loop preceding the catalytic Thr-380 moving >35 A, thus relieving steric constraints that likely limit substrate binding. In addition, cleavage of the proenzyme results in the formation of a threonine-threonine dyad comprised of Thr-380 and a second conserved threonine residue, Thr-398. The hydroxyl group of Thr-398 is located equidistant from the alpha-amino group and hydroxyl side chain of Thr-380. Mutation of Thr-398 to an alanine results in an enzyme that is fully capable of autoprocessing but is devoid of enzymatic activity. Substrate docking studies in combination with homology modeling studies of the human homologue reveal additional mechanistic details of enzyme maturation and activation, substrate recognition, and catalysis.

Uncoupling the Enzymatic and Autoprocessing Activities of Helicobacter pylori γ-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.