Epidemiological analysis of serum anti‐Pseudomonas aeruginosa PcrV titers in adults

Of the various virulence mechanisms of the opportunistic pathogen Pseudomonas aeruginosa, the type III secretion system (TTSS) has been characterized as a major factor associated with acute lung injury, bacteremia and mortality. In addition, PcrV, a component protein of the TTSS, has been characterized as a protective antigen against infection with P. aeruginosa. This study comprised an epidemiological analysis of serum anti‐PcrV titers in a cohort of Japanese adults. From April 2012 to March 2013, serum anti‐PcrV titers of 198 volunteer participants undergoing anesthesia for scheduled surgeries were measured. The median, minimum and maximum serum anti‐PcrV titers among the 198 participants were 4.09 nM, 1.01 nM and 113.81 nM, respectively. The maximum peaks in the histogram were within the anti‐PcrV 2.00–4.99 nM titer range; values for 115 participants (58.1%) were within this range. Anti‐PcrV titers were more than approximately three‐fold greater (>12 nM) than the median value in 21 participants (10.6%). Ten‐year interval age increases, history of treatment for traffic trauma, and a history of past surgery each showed statistically significant associations with higher anti‐PcrV titers (i.e., >10 nM) than did the other factors assessed by binomial analysis. This study revealed a considerable variation in anti‐PcrV titers in adult subjects without any obvious histories of infection with P. aeruginosa.     

Synthesis of Catechin‐Rare Earth Complex with Efficient and Broad‐Spectrum Anti‐Biofilm Activity

Biofilm is the crucial reason of clinical infections. Herein, green tea based polyphenol (catechin) and rare earth (RE) metal ions were employed for the preparation of catechin–RE complexes with significant anti‐biofilm properties. The complexes were characterized by FT‐IR, Raman spectroscopy, X‐ray photoelectron spectroscopy (XPS) and dynamic light scattering (DLS), which suggested that catechin coordinated with RE³⁺ through its ortho phenolic hydroxy groups. The prepared catechin‐RE showed significant effects in anti‐biofilm growth against P. aeruginosa (Gram‐negative bacteria), S. sciuri (Gram‐positive bacteria), and A. niger (fungi), which significantly exceeded the utilization of catechin or RE³⁺. Morphological observations indicated that catechin supplied cell affinity to transfer RE³⁺ and helped to damage cell membrane, which act as a carrier to exert cytotoxicity of RE³⁺ to realize anti‐biofilm. Differential gene expression analysis described gene expression changes induced by catechin‐RE, including 56, 272 and 2160 downregulated genes for P. aeruginosa, S. sciuri and A. niger, respectively, which suggested critical changes in cellular metabolism, growth and other processes. These results illustrate the outstanding superiority of catechin‐RE complexes in anti‐infection aspect, i. e., the green tea based rare earth complexes are promising candidates for anti‐biofilm applications to address serious challenges in the prevention of multiple infections.     

A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.     

Effects of Psidium guajava leaf extract on secretion systems of gram‐negative enteropathogenic bacteria

In this study, 672 plant‐tissue extracts were screened for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, an extract from the leaves of Psidium guajava (guava) was found to inhibit secretion of EPEC‐secreted protein B (EspB) from EPEC and EHEC without affecting bacterial growth. Guava extract (GE) also inhibited EPEC and EHEC from adhering to, and injecting EspB into, HEp‐2 cells. GE seemed to block translocation of EspB from the bacterial cells to the culture medium. In addition, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped secreting Yersinia outer proteins and was unable to induce apoptosis of mouse bone marrow‐derived macrophages. S. typhimurium exposed to GE stopped secreting Sip proteins and was unable to invade HEp‐2 cells. GE inhibited secretion of EspC, the type V secretion protein of EPEC, but not secretion of Shiga toxin 2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used to treat and prevent gram‐negative enteropathogenic bacterial infections.

     

A recombinant carboxy‐terminal domain of alpha‐toxin protects mice against Clostridium perfringens

Clostridium perfringens alpha‐toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C‐terminal domain (CP251‐370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S‐transferase (GST) fusion proteins. Antibodies that cross‐reacted with alpha‐toxin were produced after immunization with recombinant proteins including GST‐CP251‐370, GST‐CP281‐370, GST‐CP311‐370, CB1‐372 and GST‐CB251‐372. Anti‐GST‐CP251‐370, anti‐GST‐CP281‐370 and anti‐GST‐CP311‐370 sera neutralized both the PLC and hemolytic activities of alpha‐toxin, whereas anti‐CB1‐372 and anti‐GST‐CB251‐372 weakly neutralized these activities. Immunization with GST‐CP251‐370 and GST‐CP281‐370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST‐CP311‐370 and CB1‐372. GST‐CP251‐370 and GST‐CP281‐370 are promising candidates for vaccines for clostridial‐induced gas gangrene.     

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore‐forming toxin is detrimental for the host

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase‐1, which in turn triggers macrophage pyroptosis and IL‐1β/IL‐18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore‐forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase‐11 activation. Surprisingly, previous studies indicated that a T3SS‐induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS‐negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore‐forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll‐like receptors, and thus enhanced the expression of inflammatory proteins including pro‐IL‐1β and TNF‐α. However, mature‐IL‐1β and IL‐18 were undetectable in wild‐type mice, suggesting that ExlA failed to effectively activate caspase‐1. Nevertheless, caspase‐1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA‐induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome‐dependent process.     

Genetic fusion protein 3×STa‐ovalbumin is an effective coating antigen in ELISA to titrate anti‐STa antibodies

Heat‐stable toxin type I (STa)‐ovalbumin chemical conjugates are currently used as the only coating antigen in ELISA to titrate anti‐STa antibodies for ETEC vaccine candidates. STa‐ovalbumin chemical conjugation requires STa toxin purification, a process that can be carried out by only a couple of laboratories and often with a low yield. Alternative ELISA coating antigens are needed for anti‐STa antibody titration for ETEC vaccine development. In the present study, we genetically fused STa toxin gene (three copies) to a modified chicken ovalbumin gene for genetic fusion 3×STa‐ovalbumin, and examined application of this fusion protein as an alternative coating antigen of anti‐STa antibody titration ELISA. Data showed fusion protein 3×STa‐ovalbumin was effectively expressed and extracted, and anti‐STa antibody titration ELISA using this recombinant protein (25 ng per well) or STa‐ovalbumin chemical conjugates (10 ng/well) showed the same levels of sensitivity and specificity. Furthermore, mice immunized with this fusion protein developed anti‐STa antibodies; induced antibodies showed in vitro neutralization activity against STa toxin. These results indicate that recombinant fusion protein 3×STa‐ovalbumin is an effective ELISA coating antigen for anti‐STa antibody titration, enabling a reliable reagent supply to make standardization of STa antibody titration assay feasible and to accelerate ETEC vaccine development.

     

Molecular cloning and expression of a C‐type lectin‐like protein from orange‐spotted grouper Epinephelus coioides

A C‐type lectin‐like protein (Ec‐CTLP) was cloned from the grouper Epinephelus coioides. The full‐length cDNA of Ec‐CTLP was composed of 905 bp with a 522 bp open reading frame that encodes a 174‐residue protein. The putative amino acid sequence of Ec‐CTLP contains a signal peptide of 19 residues at the N‐terminus and a CLECT domain from Cys43 to Arg169 and a conserved imperfect WND (Trp‐Asn‐Asp) motif. The homologous identity of deduced amino acid sequences is from 32 to 42% with other fishes. The expression of Ec‐CTLP was differently upregulated in E. coioides spleen (germline stem) cells after being challenged at 16 and 4° C. Intracellular localization revealed that Ec‐CTLP was distributed only in the cytoplasm. Recombinant Ec‐CTLP (rEc‐CTLP) was expressed in Escherichia coli BL21 (DE3) and purified for mouse Mus musculus anti‐Ec‐CTLP serum preparation. The rEc‐CTLP fusion protein does not possess haemagglutinating activity, but improves survival from frozen bacteria. The survival of bacteria (including gram‐negative E. coli and gram‐positive Staphylococcus aureus) was positively correlated with the concentration of the rEc‐CTLP. These findings can provide clues to help understand the probable C‐type lectin in marine fish innate immunity.     

Central role of PAFR signalling in ExoU‐induced NF‐κB activation

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU‐driven NF‐κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF‐κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR‐dependent NF‐κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR?/? or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU‐induced NF‐κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF‐κB inhibitor Bay 11–7082, or transfection with IκBα negative‐dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11–7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF‐κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.     

Processing of cell‐surface signalling anti‐sigma factors prior to signal recognition is a conserved autoproteolytic mechanism that produces two functional domains

Cell‐surface signalling (CSS) enables Gram‐negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti‐sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP‐mediated proteolysis of the anti‐sigma factors is key to σ^ECF activation. Using the Pseudomonas aeruginosa FoxR anti‐sigma factor, we show here that RseP is responsible for the generation of an N‐terminal tail that likely contains pro‐sigma activity. Furthermore, it has been reported previously that this anti‐sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti‐sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti‐sigma factors.     

Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N5‐formyltetrahydrofolate and UDP‐Ara4N

ArnA from Escherichia coli is a key enzyme involved in the formation of 4‐amino‐4‐deoxy‐l ‐arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram‐negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N‐ and C‐terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N¹⁰‐formyltetrahydrofolate. Here we describe the structure of the isolated N‐terminal domain of ArnA in complex with its UDP‐sugar substrate and N⁵‐formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.     

Cross‐protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv

Gram‐negative Vibrio species secrete multifunctional autoprocessing repeats‐in‐toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross‐reactivity and cross‐protectivity of mAbs against V. vulnificus RtxA1/MARTX_Vv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTX_Vc, inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins.     

Diagnosis of toxic shock syndrome by two different systems; clinical criteria and monitoring of TSST‐1‐reactive T cells

Two methods of TSS diagnosis were evaluated: comparison of symptoms with clinical criteria and monitoring for evidence of selective activation of Vβ2⁺ T cells by the causative toxin, TSS toxin‐1 (TSST‐1). Ten patients with acute and systemic febrile infections caused by Staphylococcus aureus were monitored for increase in TSST‐1‐reactive Vβ2⁺ T cells during their clinical courses. Nine of the ten patients were diagnosed with TSS based on evidence of selective activation of Vβ2⁺ T cells by TSST‐1; however, clinical symptoms met the clinical criteria for TSS in only six of these nine patients. In the remaining patient, clinical symptoms met the clinical criteria, but selective activation of Vβ2⁺ T cells was not observed. Time taken to reach the diagnosis of TSS could be significantly shortened by utilizing the findings from tracing Vβ2⁺ T cells. In vitro studies showed that TSST‐1‐ reactive T cells from TSS patients were anergic in the early phase of their illness. Examining selective activation of Vβ2⁺ T cells could be a useful tool to supplement clinical criteria for early diagnosis of TSS.     

Metformin prevents the effects of Pseudomonas aeruginosa on airway epithelial tight junctions and restricts hyperglycaemia‐induced bacterial growth

Lung disease and elevation of blood glucose are associated with increased glucose concentration in the airway surface liquid (ASL). Raised ASL glucose is associated with increased susceptibility to infection by respiratory pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. We have previously shown that the anti‐diabetes drug, metformin, reduces glucose‐induced S. aureus growth across in vitro airway epithelial cultures. The aim of this study was to investigate whether metformin has the potential to reduce glucose‐induced P. aeruginosa infections across airway epithelial (Calu‐3) cultures by limiting glucose permeability. We also explored the effect of P. aeruginosa and metformin on airway epithelial barrier function by investigating changes in tight junction protein abundance. Apical P. aeruginosa growth increased with basolateral glucose concentration, reduced transepithelial electrical resistance (TEER) and increased paracellular glucose flux. Metformin pre‐treatment of the epithelium inhibited the glucose‐induced growth of P. aeruginosa, increased TEER and decreased glucose flux. Similar effects on bacterial growth and TEER were observed with the AMP activated protein kinase agonist, 5‐aminoimidazole‐4‐carboxamide ribonucleotide. Interestingly, metformin was able to prevent the P. aeruginosa‐induced reduction in the abundance of tight junction proteins, claudin‐1 and occludin. Our study highlights the potential of metformin to reduce hyperglycaemia‐induced P. aeruginosa growth through airway epithelial tight junction modulation, and that claudin‐1 and occludin could be important targets to regulate glucose permeability across airway epithelia and supress bacterial growth. Further investigation into the mechanisms regulating metformin and P. aeruginosa action on airway epithelial tight junctions could yield new therapeutic targets to prevent/suppress hyperglycaemia‐induced respiratory infections, avoiding the use of antibiotics.     

Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles

Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.     

Emerging Technologies for the Electrochemical Detection of Bacteria

Infections are a huge economic liability to the health care system, although real‐time detection can allow early treatment protocols to avoid some of this cost and patient morbidity and mortality. Pseudomonas aeruginosa (PA) is a drug‐resistant gram‐negative bacterium found ubiquitously in clinical settings, accounting for up to 27% of hospital acquired infections. PA secretes a vast array of molecules, ranging from secondary metabolites to quorum sensing molecules, of which many can be exploited to monitor bacterial presence. In addition to electrochemical immunoassays to sense bacteria via antigen–antibody interactions, PA pertains a distinct redox‐active virulence factor called pyocyanin (PYO), allowing a direct electrochemical detection of the bacteria. There has been a surge of publications relating to the electrochemical tracing of PA via a myriad of novel biosensing techniques, materials, and methodologies. In addition to indirect methods, research approaches where PYO has been sensitively detected using surface modified electrodes are reviewed and compared with conventional PA‐sensing methodologies. This review aims at presenting indirect and direct electrochemical methods currently developed using various surface modified electrodes, materials, and electrochemical configurations on their electrocatalytic effects on sensing of PA and in particular PYO.     

Epigenetic acquisition of inducibility of type III cytotoxicity in P. aeruginosa

Background  

Pseudomonas aeruginosa, an opportunistic pathogen, is often encountered in chronic lung diseases such as cystic fibrosis or chronic obstructive pneumonia, as well as acute settings like mechanical ventilation acquired pneumonia or neutropenic patients. It is a major cause of mortality and morbidity in these diseases. In lungs, P. aeruginosa settles in a biofilm mode of growth with the secretion of exopolysaccharides in which it is encapsulated, enhancing its antibiotic resistance and contributing to the respiratory deficiency of patients. However, bacteria must first multiply to a high density and display a cytotoxic phenotype to avoid the host's defences. A virulence determinant implicated in this step of infection is the type III secretion system (TTSS), allowing toxin injection directly into host cells. At the beginning of the infection, most strains isolated from patients' lungs possess an inducible TTSS allowing toxins injection or secretion upon in vivo or in vitro activation signals. As the infection persists most of the bacteria permanently loose this capacity, although no mutations have been evidenced. We name "non inducible" this phenotype. As suggested by the presence of a positive feedback circuit in the regulatory network controlling TTSS expression, it may be due to an epigenetic switch allowing heritable phenotypic modifications without genotype's mutations.     

Regulatory RNAs and the HptB/RetS signalling pathways fine‐tune Pseudomonas aeruginosa pathogenesis

Bacterial pathogenesis often depends on regulatory networks, two‐component systems and small RNAs (sRNAs). In Pseudomonas aeruginosa, the RetS sensor pathway downregulates expression of two sRNAs, rsmY and rsmZ. Consequently, biofilm and the Type Six Secretion System (T6SS) are repressed, whereas the Type III Secretion System (T3SS) is activated. We show that the HptB signalling pathway controls biofilm and T3SS, and fine‐tunes P. aeruginosa pathogenesis. We demonstrate that RetS and HptB intersect at the GacA response regulator, which directly controls sRNAs production. Importantly, RetS controls both sRNAs, whereas HptB exclusively regulates rsmY expression. We reveal that HptB signalling is a complex regulatory cascade. This cascade involves a response regulator, with an output domain belonging to the phosphatase 2C family, and likely an anti‐anti‐σ factor. This reveals that the initial input in the Gac system comes from several signalling pathways, and the final output is adjusted by a differential control on rsmY and rsmZ. This is exemplified by the RetS‐dependent but HptB‐independent control on T6SS. We also demonstrate a redundant action of the two sRNAs on T3SS gene expression, while the impact on pel gene expression is additive. These features underpin a novel mechanism in the fine‐tuned regulation of gene expression.