Outer Membrane Protein P2 Research Articles

Screening of linear B-cell epitopes and its proinflammatory activities of Haemophilus parasuis outer membrane protein P2.

Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs, but virulent strains can cause Glässer's disease, resulting in significant economic losses to the swine industry. OmpP2 is an outer membrane protein of this organism that shows considerable heterogeneity between virulent and non-virulent strains, with classification into genotypes I and II. It also acts as a dominant antigen and is involved in the inflammatory response. In this study, 32 monoclonal antibodies (mAbs) against recombinant OmpP2 (rOmpP2) of different genotypes were tested for reactivity to a panel of OmpP2 peptides. Nine linear B cell epitopes were screened, including five common genotype epitopes (Pt1a, Pt7/Pt7a, Pt9a, Pt17, and Pt19/Pt19a) and two groups of genotype-specific epitopes (Pt5 and Pt5-II, Pt11/Pt11a, and Pt11a-II). In addition, we used positive sera from mice and pigs to screen for five linear B-cell epitopes (Pt4, Pt14, Pt15, Pt21, and Pt22). After porcine alveolar macrophages (PAMs) were stimulated with overlapping OmpP2 peptides, we found that the epitope peptides Pt1 and Pt9, and the loop peptide Pt20 which was adjacent epitopes could all significantly upregulated the mRNA expression levels of IL-1α, IL-1β, IL-6, IL-8, and TNF-α. Additionally, we identified epitope peptides Pt7, Pt11/Pt11a, Pt17, Pt19, and Pt21 and loop peptides Pt13 and Pt18 which adjacent epitopes could also upregulate the mRNA expression levels of most proinflammatory cytokines. This suggested that these peptides may be the virulence-related sites of the OmpP2 protein, with proinflammatory activity. Further study revealed differences in the mRNA expression levels of proinflammatory cytokines, including IL-1β and IL-6, between genotype-specific epitopes, which may be responsible for pathogenic differences between different genotype strains. Here, we profiled a linear B-cell epitope map of the OmpP2 protein and preliminarily analyzed the proinflammatory activities and effects of these epitopes on bacterial virulence, providing a reliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines.

Molecular Characterization and Phylogenetic Analysis of Outer membrane protein P2 (OmpP2) of Glaesserella (Haemophilus) parasuis Isolates in Central State of Peninsular Malaysia

Glaesserella (Haemophilus) parasuis, the etiological agent of Glässer's disease, is an economically significant pathogen commonly associated with serofibrinous polyserositis, arthritis, fibrinous bronchopneumonia and/or meningitis. This study is the first attempt to molecularly characterize and provide a detailed overview of the genetic variants of G. parasuis present in Malaysia, in reference to its serotype, virulence-associated trimeric autotransporters (vtaA) gene and outer membrane protein P2 (OmpP2) gene. The G. parasuis isolates (n = 11) from clinically sick field samples collected from two major pig producing states (Selangor and Perak) were selected for analysis. Upon multiplex PCR, the majority of the isolates (eight out of 11) were identified to be serotype 5 or 12, and interestingly, serotypes 3, 8 and 15 were also detected, which had never been reported in Malaysia prior to this. Generally, virulent vtaA was detected for all isolates, except for one, which displayed a nonvirulent vtaA. A phylogenetic analysis of the OmpP2 gene revealed that the majority of Malaysian isolates were clustered into genotype 1, which could be further divided into Ia and Ib, while only one isolate was clustered into genotype 2.

Molecular characterisation of a meropenem-resistant Haemophilus influenzae

Background: Haemophilus influenzae β-lactam resistance is mediated by production of β-lactamase and mutations in penicillin-binding protein 3 (PBP3, encoded by ftsI). Only one of the 646 patient-unique H. influenzae isolated in our laboratory between 2012–July 2022 was meropenem (amoxicillin, co-amoxiclav and ceftriaxone) resistant. Methods: Identification and susceptibility of this meropenem-resistant H. influenzae (MRHI) was confirmed by disk diffusion and E-test (bioMérieux) (CLSI M100). WGS was performed on GridION (Nanopore) and reference mapped with Geneious Prime 2022.2. PCR and Sanger sequencing was used to confirm results of WGS. Results: The MRHI isolate was β-lactamase negative and non-typeable. ftsI subgroup IIc1 mutations were present, signifying previously described low level PBP3 alterations that should not solely account for carbapenem resistance. Group VII2 acrR (repressor of AcrAB efflux pump) substitutions were observed but without the reported carbapenem resistance associated frameshift mutation. There were also multiple substitutions and insertions in ompP2 (outer membrane protein P2) porin, associated with carbapenem resistance.2 The 100% concordance between Sanger sequencing and Nanopore increases our confidence for application of this technology for future investigations. Conclusion: Meropenem resistance in this isolate is likely multifactorial, with PBP3 alterations and decreased influx as contributing factors.

Development and clinical application of a endonuclease restriction real-time loop-mediated isothermal amplification (ERT-LAMP) assay for rapid detection of Haemophilus influenzae.

Haemophilus influenzae is a main human pathogen that results in a series of diseases in children and adults, such as pneumonia, bacteremia, and meningitis. Although there are many detection methods, they cannot meet the requirements of an early diagnosis. For the prevention and control of H. influenzae infection, quick, sensitive, and particular diagnostics are crucial. Loop-mediated isothermal amplification (LAMP) coupled with restricted endonuclease digestion and real-time fluorescence (H. influenzae-ERT-LAMP) detection was employed to diagnose H. influenzae. H. influenzae-ERT-LAMP combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence identification into a single-pot reaction, allowing for the rapid identification of H. influenzae in 40 min. The outer membrane protein (OMP) P6 gene of H. influenzae was employed to build a sequence of H. influenzae-ERT-LAMP primers. The limit of detection (LoD) of H. influenzae-ERT-LAMP test was 40 fg of genomic DNA per reaction, and the non-H. influenzae templates did not provide positive outcomes. To investigate the applicability of H. influenzae-ERT-LAMP method in clinical sample detection, 30 sputum specimens were obtained from individuals suspected of being infected with H. influenzae. H. influenzae-ERT-LAMP outcomes were in total agreement with LAMP-LFB and PCR. The H. influenzae-ERT-LAMP assay provides rapid, accurate, and sensitive detection making it a promising screening strategy in clinical and basic lab settings.

Glaesserella parasuis induces IL-17 production might through PKC-ERK/MAPK and IκB/NF-κB signaling pathways

In our previous study, the outer membrane protein P2 of Glaesserella parasuis (G. parasuis) induced the production of interleukin-17A (IL-17A) in host cells. However, how the G. parasuis infection produces IL-17A in the host is unknown. In this study, at 48 hpi in piglets, the proportion of Th17 cells in the peripheral blood significantly increased as determined by flow cytometry, but the overall proportion was low. IL-17 mRNA and protein levels significantly increased in the lungs and spleens of infected piglets. Histopathological examination staining, immunohistochemistry and viability counts, revealed that IL-17 expression might be positively correlated with bacterial content and the degree of pathological injury in lung and spleen tissues. Furthermore, when infected with the G. parasuis Nagasaki strain compared with avirulent strain C5, the primary porcine alveolar macrophages (PAMs) significantly produced IL-17 in a time-dose dependent manner. In addition, we demonstrated that PKC, MEK and NF-κB signaling pathways were essential for G. parasuis-induced IL-17 production because the addition of corresponding inhibitors dramatically reduced IL-17 mRNA production. The phosphorylation levels of PKC, ERK1/2 and IκB in G. parasuis-infected primary PAMs also increased significantly as the infection dose increased as assessed by western blotting. Thus, these finding imply that IL-17 induced by G. parasuis infection is dependent on activation of PKC-ERK/MAPK and IκB/NF-κB signaling pathways.

Mucosal immunity of mannose-modified chitosan microspheres loaded with the nontyepable Haemophilus influenzae outer membrane protein P6 in BALB/c mice.

Nontypeable Haemophilus influenzae (NTHi) is a common opportunistic pathogen that colonizes the nasopharynx. NTHi infections result in enormous global morbidity in two clinical settings: otitis media in children and acute exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Thus, there is an urgent need to design and develop effective vaccines to prevent morbidity and reduce antibiotic use. The NTHi outer membrane protein P6, a potential vaccine candidate, is highly conserved and effectively induces protective immunity. Here, to enhance mucosal immune responses, P6-loaded mannose-modified chitosan (MC) microspheres (P6-MCMs) were developed for mucosal delivery. MC (18.75%) was synthesized by the reductive amination reaction method using sodium cyanoborohydride (NaBH3CN), and P6-MCMs with an average size of 590.4±16.2 nm were successfully prepared via the tripolyphosphate (TPP) ionotropic gelation process. After intranasal immunization with P6-MCMs, evaluation of humoral immune responses indicated that P6-MCMs enhance both systemic and mucosal immune responses. Evaluation of cellular immune responses indicated that P6-MCMs enhance cellular immunity and trigger a mixed Th1/Th2-type immune response. Importantly, P6-MCMs also trigger a Th17-type immune response. They are effective in promoting lymphocyte proliferation and differentiation without toxicity in vitro. The results also demonstrate that P6-MCMs can effectively induce MHC class I- and II-restricted cross-presentation, promoting CD4+-mediated Th immune responses and CD8+-mediated cytotoxic T lymphocyte (CTL) immune responses. Evaluation of protective immunity indicated that immunization with P6-MCMs can reduce inflammation in the nasal mucosa and the lung and prevent NTHi infection. In conclusion, MCMs are a promising adjuvant-delivery system for vaccines against NTHi.

Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay.

BackgroundHaemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical.ResultsIn this study, we used a combined method to detect H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65℃, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection.ConclusionsTaken together, LAMP-LFB could be used as an effective diagnostic approach for H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-022-02547-5.

Establishment and Clinical Application of a RPA-LFS Assay for Detection of Capsulated and Non-Capsulated Haemophilus influenzae.

A recombinase polymerase amplification-lateral flow strip assay was established for detection of the outer membrane protein P6 (omp6) and the capsule encoding gene bexA of Haemophilus influenzae and the detection limit, sensitivity, and specificity were determined. Specific primers and probes were designed based on the published nucleotide sequences of omp6 and bexA. The minimum detection limit was determined with standard strains and the practical applicability of the RPA-LFS assay was assessed by detection of 209 clinical samples. The results confirmed that the RPA-LFS assay was both specific and sensitive for the detection of capsulated and non-capsulated H. influenzae with a detection limit of 1 CFU/µL. The detection rate of the 209 clinical samples was 97.1%, while the detection rate of capsulated H. influenzae was 63.2%. The detection results were consistent with the traditional culture method and dual polymerase chain reaction (PCR), confirming the applicability of the RPA-LFS assay.

A Rapid Detection of Haemophilus influenzae Using Multiple Cross Displacement Amplification Linked With Nanoparticle-Based Lateral Flow Biosensor.

Haemophilus influenzae is a major human pathogenic bacterium, resulting in a series of diseases, such as pneumonia, bacteremia, meningitis. However, it is hard to diagnose H. influenzae quickly. In this study, the multiple cross displacement amplification (MCDA) and nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB) were combined to detect H. influenzae, which has been proven to be reliable, rapid, and not complicated. On the basis of H. influenzae outer membrane protein P6 gene, 10 specific primers were designed. The best MCDA condition was 61°C for 1 h. The sensitivity of H. influenzae-MCD-LFB assay showed, in the pure cultures, the minimum concentration of genomic DNA templates was 100 fg. The specificity of H. influenzae-MCD-LFB assay showed only H. influenzae templates were detected, and no cross-reactivity was found in non-H. influenzae isolates and other Haemophilus species. In 56 sputum samples, with MCDA-LFB method and PCR detection, 21 samples were positive, which was in consistent with the traditional culture method. The accuracy of diagnosis of MCDA-LFB, in comparison with the traditional culture method and PCR detection, can reach 100%, indicating that the MCDA-LFB assay gains an advantage over the cultured-based method for target pathogen detection. In conclusion, the MCDA-LFB assay is suitable for the sensitive, rapid, and specific detection of H. influenzae, which might be used as a potential diagnostic tool for H. influenzae in basic and clinical laboratories.

Molecular and functional characterization of HtrA protein in Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (A.pleuropneumoniae) causes serious economic loss for the swine industry. A high-temperature requirements A (HtrA)-like protease and its homologs have been reported to be involved in protein quality control and expression of important immunoprotective antigens in many pathogens. In this study, we showed that HtrA of A.pleuropneumoniae exhibited both chaperone and proteolytic activities. Moreover, Outer membrane protein P5 (OmpP5) in A.pleuropneumoniae and Heat shock protein 90 (Hsp90) in porcine lung tissues were first discovered and identified as specific proteolytic substrates for rHtrA. The maximum cleavage activity occurs at 50 ℃ in a time-dependent manner. In addition, rHtrA mainly induced IgG 2a subtype of IgG and Th1 (IFN-γ, IL-2) response in a mice model, and promoted a significant proliferation of spleen lymphocytes compare with negative control (P < 0.05). The survival rates of 37.5 % were observed against A.pleuropneumoniae strain. Together, these data demonstrate that rHtrA plays a multi-functional role in A.pleuropneumoniae.

Surface-exposed loops L7 and L8 of Haemophilus (Glaesserella) parasuis OmpP2 contribute to the expression of proinflammatory cytokines in porcine alveolar macrophages

Outer membrane protein P2 (OmpP2) of the virulent Haemophilus (Glaesserella) parasuis has been shown to induce the release of proinflammatory cytokines. The OmpP2 protein is composed of eight or nine surface-exposed loops, but it is unclear which of them participates in the OmpP2-induced inflammatory response. In this study, we synthesized linear peptides corresponding to surface-exposed loops L1–L8 of OmpP2 from the virulent H. parasuis SC096 strain to stimulate porcine alveolar macrophages (PAMs) in vitro. We found that both L7 and L8 significantly upregulated the mRNA expression of interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-17, and IL-23 and the chemokines CCL-4 and CCL-5 in a time- and dose-dependent manner. Additionally, we constructed ompP2ΔLoop7 and ompP2ΔLoop8 mutant SC096 strains and extracted their native OmpP2 proteins to stimulate PAMs. These mutant proteins induced significantly less mRNA expression of inflammatory cytokines than SC096 OmpP2. Next, the amino acid sequences of L7 and L8 from 15 serovars of H. parasuis OmpP2 were aligned. These sequences were relatively conserved among the most virulent reference strains, suggesting that L7 and L8 are the most active peptides of the OmpP2 protein. Furthermore, L7 and L8 significantly upregulated the NF-κB and AP-1 activity levels based on luciferase reporter assays in a dose-dependent manner. Therefore, our results demonstrated that both surface-exposed loops L7 and L8 of H. parasuis OmpP2 induced the expression of proinflammatory cytokines possibly by activating the NF-κB and MAPK signalling pathways in cells infected by H. parasuis.

Colloidal gold immunochromatographic strip for rapid detection of Haemophilus influenzae

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.

First Report of Bacterial Canker of Kiwifruit Caused byPseudomonas syringaepv.actinidiaein Fujian Province, China

In recent years, kiwifruit (Actinidia chinensis cv. Hongyang) was introduced into Fujian Province as an economically important fruit. During 2016 and 2017, symptoms of bacterial canker were observed on kiwifruit in an approximately 15-ha orchard with 3-year-old plants in Fuan city, Fujian Province of China. The orchard exhibited 70 to 80% incidence. Symptoms on leaves began as brown angular spots surrounded by chlorotic margins that later became dark brown spots surrounded by yellow halos, and occasionally red-rusty cankers with reddish exudates were observed on the stems. Two representative strains, FJ-21a and FJ-21d, were isolated onto Luria-Bertani medium (LB) from stems showing a typical symptom of red-rusty cankers with reddish exudates, which were randomly selected from two different plants. These two strains were gram negative, aerobic, rod shaped, and nonfluorescent on King’s B medium or LB; positive for carbohydrate utilization, levan production, and hypersensitive on tobacco and tomato; and negative for oxidase, arginine dihydrolase, urease activity, gelatin liquefaction, and nitrate. The two strains were further identified by polymerase chain reaction (PCR) amplified 16S rDNA region (1,500 bp) and outer membrane protein P1 (ompP1) gene (492 bp) using 27F/1492R (Weisburg et al. 1991) and psaF/psaR (Koh and Nou 2002) primer pairs, respectively. BLASTn analysis showed that the 16S rDNA region (NCBI accession nos. MH071159 and MH071160) and ompP1 (MH084697 and MH084698) gene sequences shared 100 and 100% identity to that of Pseudomonas syringae pv. actinidiae (Psa) strains ICMP 18884 (CP011972) and CRAFRU 12.29 (CP019730), and 11955 (JQ934479) and ISF 8.57 (FR863682), respectively. Based on morphological characteristics, physiological and biochemical tests, and molecular identification, these strains were identified as Psa (Cunty et al. 2015; Takikawa et al. 1989). The two Psa strains were characterized as biovar 3 through PCR using the biovar-specific primers Tac-F/Tac-R (Koh et al. 2014). Pathogenicity was tested with two strains on 6-month-old kiwifruit cv. Jinyan (Fruit Research Institute, Fujian Academy of Agricultural Sciences) using two methods: injecting cell suspensions (10⁸ CFU/ml) into stems with a hypodermic syringe and spraying the bacterial suspension on wounded or unwounded leaves until runoff. Five plants and 10 leaves were used per strain and method. Control plants were inoculated with distilled water or liquid LB medium. The inoculated plants were incubated at 26/22°C (day/night) in a greenhouse (12 h light and >90% relative humidity) for 3 to 4 weeks. As a result, all inoculated plants exhibited the symptoms of twig death or black wilting on stems and yellow or dark brown spots on leaves, which were similar as observed initially in the field. Psa strains were reisolated from each inoculated location. Psa has been reported in other provinces of China, but to our knowledge, this is the first report of Psa causing bacterial canker on kiwifruit in Fujian Province. Psa could have a significant impact on the establishment and productivity of kiwifruit in natural conditions. There is urgency to determine the distribution of Psa in Fujian Province. Furthermore, quarantine measures are needed to prevent further spread of the pathogen to other kiwifruit orchards.

Transcriptional Modulation of Penicillin-Binding Protein 1b, Outer Membrane Protein P2 and Efflux Pump (AcrAB-TolC) during Heat Stress Is Correlated to Enhanced Bactericidal Action of Imipenem on Non-typeable Haemophilus influenzae.

Objective: The purpose of the present study was to investigate the penicillin binding proteins (PBPs), drug influx and efflux modulations during heat stress and their effects on the bactericidal action of imipenem on non-typeable Haemophilus influenzae (NTHi).Methods: The two NTHi clinical isolates (GE47 and GE88, imipenem MICs by E-test > 32 μg/mL) examined in this study were collected at Geneva University Hospitals. The imipenem killing activity was assessed after incubation of the NTHi strains at either 37 or 42°C for 3 h with increasing concentrations of imipenem. The detection of PBPs was carried out by Bocillin-FL. Global transcriptional changes were monitored by RNA-seq after pre-incubation of bacterial cells at either 37 or 42°C, and the expression levels of relevant target genes were confirmed by qRT-PCR.Results: Quantitation of NTHi viable cells after incubation with 0.25 μg/mL of imipenem for 3 h revealed more than a twofold decrease in GE47 and GE88 viable cells at 42°C as compared to 37°C. Transcriptome analysis showed that under heat stress conditions, there were 141 differentially expressed genes with a | log2(fold change)| > 1, including 67 up-regulated and 74 down-regulated genes. The expression levels of ponB (encoding PBP1b) and acrR (regulator of AcrAB-TolC efflux pump) were significantly increased at 42°C. In contrast, the transcript levels of ompP2 (encoding the outer membrane protein P2) and acrB gene (encoding AcrB) were significantly lower under heat stress condition.Conclusion: This study shows that the transcriptional modulation of ponB, ompP2, acrR, and acrB in the heat stress response is correlated to enhanced antimicrobial effects of imipenem on non-typeable H. influenzae.

Genetic variants and phylogenetic analysis of Haemophilus parasuis (HPS) OMPP2 detected in Sichuan, China from 2013 to 2015.

To investigate the genetic variation in Haemophilus parasuis (HPS) in Sichuan, China, 11 isolates were analyzed based on the outer membrane protein P2 (OMPP2) sequence. Sequence analysis showed that the 11 isolates shared 93.0 to 100% nucleotide homology with 15 reference strains, and the consistency between the 26 strains was 89.0%. The isolates of HPS-1, 2, 4, 5, 6, 7, 8, 10 and 11 had a 69-base deletion from 770 base pairs (bp) to 850 bp, which was infrequent in China. The phylogenetic tree showed that HPS-3 and HPS-8 had closer relationships with European and Japanese strains, but shared 98.7% nucleotide homology with the SW114 Japanese strain.

Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation

ObjectiveTo investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). MethodsFifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration–response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. ResultsAll 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL. ConclusionsThe present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18–20/Fc), were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18–20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18–20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such infections, which are often polymicrobial.

IDENTIFICATION OF FUNCTIONALLY AND STRUCTURALLY IMPORTANT RESIDUES OF OUTER MEMBRANE PROTEIN P1 IN HAEMOPHILUSINFLUENZAE

Haemophilusinfluenzae (formerly called Pfeiffer's bacillus or Bacillus influenzae) is a Gram-negative, coccobacillary, facultatively anaerobic pathogenic bacterium belonging to the Pasteurellaceae family. The presence of the capsule in encapsulated type b (Hib), a serotype causing conditions such as epiglottitis, is known to be a major factor in virulence. Their capsule allows them to resist phagocytosis and complement-mediated lysis in the nonimmune host. The unencapsulated strains are almost always less invasive. Haemophilusinfluenzae infections cause some severe and fatal, especially in children such as meningitis and epiglottitis. The property of an antigen to bind specifically complementary antibodies is known as the antigen’s antigenicity; likewise, the ability of an antigen to induce an immune response is called its immunogenicity. Attempts should be made to discover peptides that could mimic protein epitopes and possess the same immunogenicity as the whole protein. Subsequently, theoretical methods for epitope prediction have been developed leading to synthesis of such peptides that are important for development of immunodiagnostic tests and vaccines. The present study was designed to in silico resolving the major obstacles in the control or in prevention of the diseases caused by Haemophilusinfluenzae . We exploitedbioinformatic tools to better understanding and characterizing the Outer Membrane Protein P1 structure in Haemophilusinfluenzae and select appropriate regions as effectiveB cell epitops.

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages.

Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae.

Outer membrane protein P1 is the CEACAM-binding adhesin of Haemophilus influenzae.

Haemophilus influenzae is a Gram-negative pathogen colonizing the upper respiratory tract mucosa. H. influenzae is one of several human-restricted bacteria, which bind to carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on the epithelium leading to bacterial uptake by the eukaryotic cells. Adhesion to CEACAMs is thought to be mediated by the H. influenzae outer membrane protein (OMP) P5. However, CEACAMs still bound to H. influenzae lacking OMP P5 expression, and soluble CEACAM receptor ectodomains failed to bind to OMP P5, when heterologously expressed in Escherichia coli. Screening of a panel of H. influenzae OMP mutants revealed that lack of OMP P1 completely abrogated CEACAM binding and supressed CEACAM-mediated engulfment of H. influenzae by epithelial cells. Moreover, ectopic expression of OMP P1 in E. coli was sufficient to induce CEACAM binding and to promote attachment to and internalization into CEACAM-expressing cells. Interestingly, OMP P1 selectively recognizes human CEACAMs, but not homologs from other mammals and this binding preference is preserved upon expression in E. coli. Together, our data identify OMP P1 as the bona fide CEACAM-binding invasin of H. influenzae. This is the first report providing evidence for an involvement of the major OMP P1 of H. influenzae in pathogenesis.