A pyocin-like T6SS effector mediates bacterial competition in Yersinia pseudotuberculosis.

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa, respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis. Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis. We further identified YPK_0952 as a DNase dependent on Mg2+, Ni2+, Mn2+, and Co2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis.IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis. YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.

Functional versatility of Zur in metal homeostasis, motility, biofilm formation, and stress resistance in Yersinia pseudotuberculosis.

Zur (zinc uptake regulator) is a significant member of the Fur (ferric uptake regulator) superfamily, which is widely distributed in bacteria. Zur plays crucial roles in zinc homeostasis and influences cell development and environmental adaptation in various species. Yersinia pseudotuberculosis is a Gram-negative enteric that pathogen usually serves as a model organism in pathogenicity studies. The regulatory effects of Zur on the zinc transporter ZnuABC and the protein secretion system T6SS have been documented in Y. pseudotuberculosis. In this study, a comparative transcriptomics analysis between a ∆zur mutant and the wild-type (WT) strain of Y. pseudotuberculosis was conducted using RNA-seq. This analysis revealed global regulation by Zur across multiple functional categories, including membrane transport, cell motility, and molecular and energy metabolism. Additionally, Zur mediates the homeostasis not only of zinc but also ferric and magnesium in vivo. There was a notable decrease in 35 flagellar biosynthesis and assembly-related genes, leading to reduced swimming motility in the ∆zur mutant strain. Furthermore, Zur upregulated multiple simple sugar and oligopeptide transport system genes by directly binding to their promoters. The absence of Zur inhibited biofilm formation as well as reduced resistance to chloramphenicol and acidic stress. This study illustrates the comprehensive regulatory functions of Zur, emphasizing its importance in stress resistance and pathogenicity in Y. pseudotuberculosis. IMPORTANCE: Bacteria encounter diverse stresses in the environment and possess essential regulators to modulate the expression of genes in responding to the stresses for better fitness and survival. Zur (zinc uptake regulator) plays a vital role in zinc homeostasis. Studies of Zur from multiple species reviewed that it influences cell development, stress resistance, and virulence of bacteria. Y. pseudotuberculosis is an enteric pathogen that serves a model organism in the study of pathogenicity, virulence factors, and mechanism of environmental adaptation. In this study, transcriptomics analysis of Zur's regulons was conducted in Y. pseudotuberculosis. The functions of Zur as a global regulator in metal homeostasis, motility, nutrient acquisition, glycan metabolism, and nucleotide metabolism, in turn, increasing the biofilm formation, stress resistance, and virulence were reviewed. The importance of Zur in environmental adaptation and pathogenicity of Y. pseudotuberculosis was emphasized.

A TNF-IL-1 circuit controls Yersinia within intestinal pyogranulomas.

Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. We previously reported that Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces the recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas (PG) that control Yersinia infection. Inflammatory monocytes are essential for the control and clearance of Yersinia within intestinal PG, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives the production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptors on non-hematopoietic cells to enable PG-mediated control of intestinal Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative inflammatory circuit that restricts intestinal Yersinia infection.

Yersinia pseudotuberculosis in an alpaca.

A 6-year-old female huacaya alpaca was referred to the clinic for evaluation with a 1-month history of rapid weight loss, inappetence, lethargy, and severe leukocytosis refractory to medical management. Physical examination revealed a body condition score of 1 out of 5 and a large, firm structure palpable in the right caudoventral abdomen. Abdominal ultrasonographic examination revealed 3 masses with hyperechoic, swirling centers. The largest mass measured 15 cm in diameter with a 2-centimeter capsule, and extended from right of midline into the left inguinal region. Transrectal ultrasonography identified a small uterus and clear delineation between the abdominal masses. Complete blood (cell) count findings were consistent with marked systemic inflammation. Based on initial examination and laboratory findings, exploratory laparotomy was elected. Multiple mesenteric masses strongly adhered to the jejunum were observed within the abdomen. Due to the inoperable conditions and the poor long-term prognosis, the alpaca was euthanized under general anesthesia. Bacterial culture of fluid aspirated from the largest mass revealed Yersinia pseudotuberculosis. Key clinical message: Clinical progression and attempted treatment of Yersinia pseudotuberculosis in camelids have not been previously described and the bacterium should be considered as a differential diagnosis for abscessation and persistent leukocytosis. Yersinia pseudotuberculosis is also considered a zoonotic agent and proper precautions should be taken when handling cases of abdominal abscessation.

Yersinia pseudotuberculosis chez un alpaga. Une alpaga huacaya femelle de 6 ans a été référée à la clinique pour évaluation avec des antécédents d'un mois de perte de poids rapide, d'inappétence, de léthargie et de leucocytose sévère réfractaire à la prise en charge médicale. L'examen physique a révélé un score d'état corporel de 1 sur 5 et une structure large et ferme palpable au niveau de l'abdomen caudoventral droit. L'examen échographique abdominal a révélé 3 masses à centres hyperéchogènes et tourbillonnants. La plus grande masse mesurait 15 cm de diamètre avec une capsule de 2 centimètres et s'étendait de la droite de la ligne médiane jusqu'à la région inguinale gauche. L'échographie transrectale a identifié un petit utérus et une délimitation claire entre les masses abdominales. Les résultats de la numération globulaire (cellulaire) sanguine complète étaient compatibles avec une inflammation systémique marquée. Sur la base de l'examen initial et des résultats de laboratoire, une laparotomie exploratoire a été choisie. De multiples masses mésentériques fortement adhérées au jéjunum ont été observées dans l'abdomen. En raison des conditions inopérables et du mauvais pronostic à long terme, l'alpaga a été euthanasié sous anesthésie générale. La culture bactérienne du liquide aspiré de la plus grande masse a révélé Y. pseudotuberculosis.Message clinique clé :La progression clinique et les tentatives de traitement de Y. pseudotuberculosis chez les camélidés n'ont pas été décrites auparavant et la bactérie doit être considérée comme un diagnostic différentiel d'abcès et de leucocytose persistante. Yersinia pseudotuberculosis est également considérée comme un agent zoonotique et des précautions appropriées doivent être prises lors de la manipulation des cas d'abcès abdominal.(Traduit par Dr Serge Messier).

Seroepidemiological survey of pathogenic Yersinia in domestic pigs in Chiba Prefecture, Japan.

This study aimed to investigate the prevalence of antibodies against pathogenic Yersinia such as Y. enterocolitica and Y. pseudotuberculosis in domestic pigs. A total of 650 serum samples from pigs in nine regions of the Chiba Prefecture in Japan, were tested using plasmid-encoded Yersinia outer membrane protein (Yops) antigen ELISA. The cutoff value was calculated using 20 pathogenic Yersinia-free pig serum samples. According to the cutoff value, 246 (37.8%) pigs from seven regions were considered seropositive for pathogenic Yersinia during the study period. These results indicate that pathogenic Yersinia is widespread in pigs in Chiba, which may become the source of human yersiniosis in this region.

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism.

Association of Yersinia Infection With Kawasaki Disease: A Prospective Multicenter Cohort Study.

BACKGROUND: Yersinia infection is known to present with Kawasaki disease (KD)-like symptoms although differentiating the 2 has been a challenge. The present study aimed to describe the clinical characteristics and prevalence of Yersinia infection presenting with KD-like symptoms. METHODS: The present, prospective, multicenter study enrolled patients who received a diagnosis of KD between January 2021 and January 2022 at 2 hospitals in Tokyo. Stool samples were collected within 3 days of the start of KD treatment, and cultures were performed for Yersinia . Clinical history and symptoms suggestive of Yersinia infection were also evaluated. RESULTS: During the study period, 141 KD patients were screened and 117 patients with evaluable stool samples were registered. Only 1 patient was positive for Yersinia pseudotuberculosis , which was detected from both stool and blood cultures. The patient was refractory to KD treatment but improved after initiation of appropriate antibiotic therapy. CONCLUSIONS: Routine screening for Yersinia is not appropriate for patients with KD and should be limited to certain patients in high-risk areas and those who are refractory to the standard KD treatment.

Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants.

Translocon pores formed in the eukaryotic cell membrane by a type III secretion system facilitate the translocation of immune-modulatory effector proteins into the host cell interior. The YopB and YopD proteins produced and secreted by pathogenic Yersinia spp. harboring a virulence plasmid-encoded type III secretion system perform this pore-forming translocator function. We had previously characterized in vitro T3SS function and in vivo pathogenicity of a number of strains encoding sited-directed point mutations in yopD. This resulted in the classification of mutants into three different classes based upon the severity of the phenotypic defects. To investigate the molecular and functional basis for these defects, we explored the effectiveness of RAW 264.7 cell line to respond to infection by representative YopD mutants of all three classes. Signature cytokine profiles could separate the different YopD mutants into distinct categories. The activation and suppression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to alter anti-phagocytosis and programmed cell death pathways. These analyses demonstrated that sub-optimal translocon pores impact the extent and magnitude of host cell responsiveness, and this limits the capacity of pathogenic Yersinia spp. to fortify against attack by both early and late arms of the host innate immune response.

Yersinia deploys type III-secreted effectors to evade caspase-4 inflammasome activation in human cells.

IMPORTANCE: Yersinia are responsible for significant disease burden in humans, ranging from recurrent disease outbreaks (yersiniosis) to pandemics (Yersinia pestis plague). Together with rising antibiotic resistance rates, there is a critical need to better understand Yersinia pathogenesis and host immune mechanisms, as this information will aid in developing improved immunomodulatory therapeutics. Inflammasome responses in human cells are less studied relative to murine models of infection, though recent studies have uncovered key differences in inflammasome responses between mice and humans. Here, we dissect human intestinal epithelial cell and macrophage inflammasome responses to Yersinia pseudotuberculosis. Our findings provide insight into species- and cell type-specific differences in inflammasome responses to Yersinia.

Development of detection methods by multiplex real-time PCR for highly pathogenic Yersinia enterocolitica, low pathogenic Yersinia enterocolitica, and Yersinia pseudotuberculosis based on SYBR Green and TaqMan probes.

This study aimed to develop multiplex real-time PCR methods using SYBR Green and TaqMan probes for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis. Specific primer and probe combinations for differentiating three pathogenic Yersinia groups were designed from three chromosomally encoded genes (ail, fyuA, and inv). Twenty-six stains of pathogenic Yersinia species including 6 strains of low pathogenic Y. enterocolitica serotypes, 7 strains of highly pathogenic Y. enterocolitica serotypes, and 13 strains of pathogenic Y. pseudotuberculosis were used for specificity testing. Specific patterns of real-time amplification signals distinguished three pathogenic Yersinia groups. A detection limit of approximately 101 colony forming units (CFU) /reaction of genomic DNA was determined based on plate counts. Furthermore, the multiplex real-time PCR methods also detected Y. enterocolitica O:8 from the DNA extracted from spiked rabbit blood samples and potentially infected wild rodent fecal samples. These results demonstrated that the multiplex real-time PCR methods developed in this study are useful for rapid detection and differentiation of three pathogenic Yersinia groups. Therefore, these methods provide a new monitoring and detection capability to understand the epidemiology of pathogenic Yersinia and to diagnose three pathogenic Yersinia groups.

The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis.

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

An ultrasensitive aptamer-based fluorescent on/off system for trace amount evaluation of Yersinia enterocolitica in food samples.

An innovative aptamer labeled with 5-FAM has been developed with a high affinity for Yersinia enterocolitica (Y. enterocolitica) using graphene oxide (GO) as a quenching platform. The selectivity of the prepared system was evaluated in the presence of common coexisted bacteria like Yersinia pseudotuberculosis, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium. Some experimental factors like pH and stability were investigated. The results showed that in the absence of Y. enterocolitica, aptamer labeled with 5-FAM was bonded with GO, causing fluorescence to be relatively weak. After the addition of Y. enterocolitica, the aptamer is released from the GO surface and binds to the target bacteria, and significantly increases the fluorescence intensity with an excitation wavelength of 410 nm and an emission wavelength of 530 nm. After optimizing all conditions, the system exhibited a wide linear response for Y. enterocolitica in the concentration range 10 to 1.0 × 109 CFU•mL-1 and the limit of detection (LOD) was 3 CFU•mL-1. This system demonstrated that GO-designed aptamers can be successful in detecting Y. enterocolitica in whole-cell forms, making them potentially useful for screening and rapid detection.

Development and validation of a bacterial gastrointestinal multiplex RT-PCR assay for use on a fully automated molecular system.

PCR-based enteric multiplex panels represent a rapid and reliable alternative to conventional "classical" phenotypic stool diagnostics. The aim of this study was to establish a laboratory-developed non-commercial multiplex Real-Time-PCR panel for the detection of the most important bacterial stool pathogens, Salmonella spp., Shigella spp., Yersinia enterocolitica/ pseudotuberculosis and Campylobacter jejuni/coli. on the "open" cobas omni Utility Channel (UC) of the cobas 6800 system (Roche). The aim was to replace the laborious phenotypical stool diagnostics with a high throughput Real-Time PCR method. The respective primers and probes were designed to cover conserved genomic regions of the pathogens and validated using Ultramer oligonucleotides, positive stool material and reference strains. To further validate the multiplex PCR-assay, the following parameters were evaluated: analytical-sensitivity and -specificity, cross-reactivity, linearity and inter- and intra-assay variance as well as limit of detection (LOD). In addition a retrospective analysis of culture positive and negative samples from daily routine was performed using 745 native stool samples. The Gastro assay was linear over a 5-log-unit and within the expected dynamic range with amplification efficiencies ranging from 94.6% to 120%. In addition, all targets showed excellent coefficients of repeatability (≤ 1.11%), intermediate precision (≤ 1.02%) and total variance (≤ 1.39%). In terms of analytical sensitivity the assay demonstrated detection limits ranging from 7.83 copies per reaction to 14.4 copies per reaction. The assay showed excellent agreement with culture methods (>95%) and a 100% sensitivity and specificity after resolution of discrepant results. The multiplex-PCR assay provides a comprehensive, rapid and sensitive alternative to conventional methods for the detection of the major bacterial stool pathogens in diagnostic laboratories.

Determination of Growth Rate and Virulence Plasmid Copy Number During Yersinia pseudotuberculosis Infection Using Droplet Digital PCR.

Pathogenic bacteria have evolved the ability to evade their host defenses and cause diseases. Virulence factors encompass a wide range of adaptations that allow pathogens to survive and proliferate in the hostile host environment during successful infection. In human pathogenic Yersinia species, the potent type III secretion system (T3SS) and other essential virulence factors are encoded on a virulence plasmid. Here, we investigated the bacterial growth rate and plasmid copy number following a Yersinia infection using droplet digital PCR (ddPCR). ddPCR is an exceptionally sensitive, highly precise, and cost-efficient method. It enables precise quantification even from very small amounts of target DNA. This method also enables analysis of complex samples with large amounts of interfering DNA, such as infected tissues or microbiome studies.

Effect of Non-Specific Porins from the Outer Membrane of Yersinia pseudotuberculosis on Mice Brain Cortex Tissues.

It was found that a single-dose immunization of mice with Yersinia pseudotuberculosis porins OmpF and OmpC causes development of pathological changes in the deep layers of cerebral cortex characterized by dystrophic changes in the cells against the background of the increasing titer of specific antibodies. At the same time, the increased level of caspase-3 expression is observed in the neurons, which indicates induction of proapoptotic signaling pathways. The obtained results indicate potential ability of nonspecific pore-forming proteins of the outer membrane of Gram-negative bacteria to initiate development of degenerative changes in brain cells.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Inflammatory monocytes promote granuloma control of Yersinia infection.

Granulomas are organized immune cell aggregates formed in response to chronic infection or antigen persistence. The bacterial pathogen Yersinia pseudotuberculosis (Yp) blocks innate inflammatory signalling and immune defence, inducing neutrophil-rich pyogranulomas (PGs) within lymphoid tissues. Here we uncover that Yp also triggers PG formation within the murine intestinal mucosa. Mice lacking circulating monocytes fail to form defined PGs, have defects in neutrophil activation and succumb to Yp infection. Yersinia lacking virulence factors that target actin polymerization to block phagocytosis and reactive oxygen burst do not induce PGs, indicating that intestinal PGs form in response to Yp disruption of cytoskeletal dynamics. Notably, mutation of the virulence factor YopH restores PG formation and control of Yp in mice lacking circulating monocytes, demonstrating that monocytes override YopH-dependent blockade of innate immune defence. This work reveals an unappreciated site of Yersinia intestinal invasion and defines host and pathogen drivers of intestinal granuloma formation.

Computational Analysis of the Ligand-Binding Sites of the Molecular Chaperone OppA from Yersinia pseudotuberculosis.

The function of chaperones is to correct or degrade misfolded proteins inside the cell. Classic molecular chaperones such as GroEL and DnaK have not been found in the periplasm of Yersinia pseudotuberculosis. Some periplasmic substrate-binding proteins could be bifunctional, such as OppA. Using bioinformatic tools, we try to elucidate the nature of the interactions between OppA and ligands from four proteins with different oligomeric states. Using the crystal structure of the proteins Mal12 alpha-glucosidase from Saccharomyces cerevisiae S288C, LDH rabbit muscle lactate dehydrogenase, EcoRI endonuclease from Escherichia coli and THG Geotrichum candidum lipase, a hundred models were obtained in total, including five different ligands from each enzyme with five conformations of each ligand. The best values for Mal12 stem from ligands 4 and 5, with conformation 5 for both; for LDH, ligands 1 and 4, with conformations 2 and 4, respectively; for EcoRI, ligands 3 and 5, with conformation 1 for both; and for THG, ligands 2 and 3, with conformation 1 for both. The interactions were analyzed with LigProt, and the length of the hydrogen bridges has an average of 2.8 to 3.0 Å. The interaction within the OppA pocket is energetically favored due to the formation of hydrogen bonds both of OppA and of the selected enzymes. The Asp 419 residue is important in these junctions.