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Standardization of Pneumococcal Biofilm Release to PncO Expression, a Predictive Measurement of Virulence.

ABSTRACT: A critical component in clinical trials for vaccines against pneumococcal disease is the establishment of robust preclinical models and clinical correlates of protection, which, in the case of the causative bacterial agent Streptococcus pneumoniae, include standard sepsis/pneumonia mouse models and opsonophagocytic activity (OPA), respectively. Despite broad usage, these gold-standard measures are ill equipped to evaluate nontraditional antigens that target virulence factors beyond capsular polysaccharides and/or proteins not associated with colonization or routine growth. These assays are further complicated by observed inconsistencies in the expression of target protein antigens and in the quantity of usable bacteria provided from respective growth processes. In an effort to overcome these issues, we performed an extensive optimization study of the critical steps in a bacterial biofilm dispersion model (termed "the biofilm model") to identify conditions that yield the greatest quantity of released pneumococci displaying a consistent virulence phenotype. Using this knowledge, we developed a secondary immune absorbance assay to provide immediate insight into the phenotypic state of bacteria conditioned using the biofilm model. Specifically, positive correlations between the expression of PncO (a key virulence-associated protein antigen) and immune absorbance (R 2 = 0.96), capsule shedding, and OPA assay titers were translated into a predictive readout of virulence in sepsis and pneumonia challenge models. These results present a methodology for generating consistent lots of virulent bacteria to standardize inputs in preclinical and clinical models for testing vaccines against biofilm-associated bacteria.

SUBMITTER: Hill A

PROVIDER: S-EPMC6105903 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Standardization of Pneumococcal Biofilm Release to PncO Expression, a Predictive Measurement of Virulence.

Hill A A Beitelshees M M Pfeifer B A BA Jones C H CH

Infection and immunity 20180822 9

A critical component in clinical trials for vaccines against pneumococcal disease is the establishment of robust preclinical models and clinical correlates of protection, which, in the case of the causative bacterial agent <i>Streptococcus pneumoniae</i>, include standard sepsis/pneumonia mouse models and opsonophagocytic activity (OPA), respectively. Despite broad usage, these gold-standard measures are ill equipped to evaluate nontraditional antigens that target virulence factors beyond capsul ...[more]

PMID: 29986896

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Project description:Aquaporins, integral membrane proteins widely distributed in organisms, facilitate the transport of water, glycerol, and other small uncharged solutes across cellular membranes and play important physiological roles in eukaryotes. However, characterizations and physiological functions of the prokaryotic aquaporins remain largely unknown. Here, we report that Streptococcus pneumoniae (pneumococcus) AqpC (Pn-AqpC), representing a new aquaporin subfamily possessing a distinct substrate-selective channel, functions as an oxygen porin by facilitating oxygen movement across the cell membrane and contributes significantly to pneumococcal virulence. The use of a phosphorescent oxygen probe showed that Pn-AqpC facilitates oxygen permeation into pneumococcal and Pn-AqpC-expressing yeast cells. Reconstituting Pn-AqpC into liposomes prepared with pneumococcal and Escherichia coli cellular membranes further verified that Pn-AqpC transports O2 but not water or glycerol. Alanine substitution showed that Pro232 in the substrate channel is key for Pn-AqpC in O2 transport. The deletion of Pn-aqpC significantly reduced H2O2 production and resistance to H2O2 and NO of pneumococci, whereas low-H2O2 treatment helped the ΔPn-aqpC mutant resist higher levels of H2O2 and even NO, indicating that Pn-AqpC-facilitated O2 permeation contributes to pneumococcal resistance to H2O2 and NO. Remarkably, the lack of Pn-aqpC alleviated cell autolysis, thus reducing pneumolysin (Ply) release and decreasing the hemolysis of pneumococci. Accordingly, the ΔPn-aqpC mutant markedly reduced survival in macrophages, decreased damage to macrophages, and significantly reduced lethality in mice. Therefore, the oxygen porin Pn-AqpC, through modulating H2O2 production and pneumolysin release, the two major pneumococcal virulence factors, controls the virulence of pneumococcus. Pn-AqpC orthologs are widely distributed in various pneumococcal serotypes, highlighting that the oxygen porin is important for pneumococcal pathogenicity. IMPORTANCE Pneumococcus is the leading cause of community-acquired pneumonia, bacteremia, and meningitis. This work reports that a novel aquaporin subfamily represented by pneumococcal Pn-AqpC functions as an oxygen porin facilitating O2 influx into cells. Importantly, by mediating O2 influx, Pn-AqpC controls the production and release of H2O2 and Ply, the two major pneumococcal virulence factors. Moreover, by enhancing endogenous H2O2 production, Pn-AqpC significantly increases pneumococcal resistance to H2O2 and even NO, the major bactericidal chemical produced by macrophages. Consequently, the deletion of Pn-aqpC markedly decreased pneumococcal survival in macrophages and reduced damage to macrophages. Accordingly, the ΔPn-aqpC mutant displays significantly attenuated virulence in a murine pneumonia model. Given that Pn-AqpC orthologs are widely distributed in all pneumococcal serotypes, this new subfamily of aquaporins is identified as novel virulence-related proteins.

Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is a deleterious human pathogen responsible for severe morbidity and mortality worldwide. The pathogen has attained high priority in the World Health Organization (WHO) - Multidrug-resistant (MDR) pathogens list. Emerging MDR strains of S. aureus are clinically challenging due to failure in conventional antibiotic therapy. Biofilm formation is one of the underlying mechanisms behind the antibiotic resistance. Hence, attenuating biofilm formation has become an alternative strategy to control persistent infections. The current study is probably the first that focuses on the antibiofilm and antivirulence potential of myrtenol against MRSA and its clinical isolates. Myrtenol exhibited a concentration-dependent biofilm inhibition without causing any harmful effect on cell growth and viability. Further, microscopic analysis validated the biofilm inhibitory efficacy of myrtenol against MRSA. In addition, myrtenol inhibited the synthesis of major virulence factors including slime, lipase, α-hemolysin, staphyloxanthin and autolysin. Inhibition of staphyloxanthin in turn sensitized the MRSA cells to healthy human blood and hydrogen peroxide (H2O2). Notably, myrtenol treated cells were deficient in extracellular DNA (eDNA) mediated autoaggregation as eDNA releasing autolysis was impaired by myrtenol. Biofilm disruptive activity on preformed biofilms was observed at concentrations higher than minimum biofilm inhibitory concentration (MBIC) of myrtenol. Also, the non-cytotoxic effect of myrtenol on human peripheral blood mononuclear cell (PBMC) was evidenced by trypan blue and Alamar blue assays. Transcriptional analysis unveiled the down-regulation of global regulator sarA and sarA mediated virulence genes upon myrtenol treatment, which is well correlated with results of phenotypic assays. Thus, the results of the present study revealed the sarA mediated antibiofilm and antivirulence potential of myrtenol against MRSA.

Project description:The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.

Project description:BackgroundIn recent years, the idea of a highly immunogenic protein-based vaccine to combat Streptococcus pneumoniae and its severe invasive infectious diseases has gained considerable interest. However, the target proteins to be included in a vaccine formulation have to accomplish several genetic and immunological characteristics, (such as conservation, distribution, immunogenicity and protective effect), in order to ensure its suitability and effectiveness. This study aimed to get comprehensive insights into the genomic organization, population distribution and genetic conservation of all pneumococcal surface-exposed proteins, genetic regulators and other virulence factors, whose important function and role in pathogenesis has been demonstrated or hypothesized.ResultsAfter retrieving the complete set of DNA and protein sequences reported in the databases GenBank, KEGG, VFDB, P2CS and Uniprot for pneumococcal strains whose genomes have been fully sequenced and annotated, a comprehensive bioinformatic analysis and systematic comparison has been performed for each virulence factor, stand-alone regulator and two-component regulatory system (TCS) encoded in the pan-genome of S. pneumoniae. A total of 25 S. pneumoniae strains, representing different pneumococcal phylogenetic lineages and serotypes, were considered. A set of 92 different genes and proteins were identified, classified and studied to construct a pan-genomic variability map (variome) for S. pneumoniae. Both, pneumococcal virulence factors and regulatory genes, were well-distributed in the pneumococcal genome and exhibited a conserved feature of genome organization, where replication and transcription are co-oriented. The analysis of the population distribution for each gene and protein showed that 49 of them are part of the core genome in pneumococci, while 43 belong to the accessory-genome. Estimating the genetic variability revealed that pneumolysin, enolase and Usp45 (SP_2216 in S. p. TIGR4) are the pneumococcal virulence factors with the highest conservation, while TCS08, TCS05, and TCS02 represent the most conserved pneumococcal genetic regulators.ConclusionsThe results identified well-distributed and highly conserved pneumococcal virulence factors as well as regulators, representing promising candidates for a new generation of serotype-independent protein-based vaccine(s) to combat pneumococcal infections.

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Standardization of Pneumococcal Biofilm Release to PncO Expression, a Predictive Measurement of Virulence.

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Standardization of Pneumococcal Biofilm Release to PncO Expression, a Predictive Measurement of Virulence.

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