Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

Background

Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.

Methodology/Principal Findings

C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor∶recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.

Conclusions/Significance

This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.     

Molecular Characterization of Clostridium botulinum Isolates from Foodborne Outbreaks in Thailand, 2010

Background

Thailand has had several foodborne outbreaks of botulism, one of the biggest being in 2006 when laboratory investigations identified the etiologic agent as Clostridium botulinum type A. Identification of the etiologic agent from outbreak samples is laborious using conventional microbiological methods and the neurotoxin mouse bioassay. Advances in molecular techniques have added enormous information regarding the etiology of outbreaks and characterization of isolates. We applied these methods in three outbreaks of botulism in Thailand in 2010.

Methodology/Principal Findings

A total of 19 cases were involved (seven each in Lampang and Saraburi and five in Maehongson provinces). The first outbreak in Lampang province in April 2010 was associated with C. botulinum type F, which was detected by conventional methods. Outbreaks in Saraburi and Maehongson provinces occurred in May and December were due to C. botulinum type A1(B) and B that were identified by conventional methods and molecular techniques, respectively. The result of phylogenetic sequence analysis showed that C. botulinum type A1(B) strain Saraburi 2010 was close to strain Iwate 2007. Molecular analysis of the third outbreak in Maehongson province showed C. botulinum type B8, which was different from B1–B7 subtype. The nontoxic component genes of strain Maehongson 2010 revealed that ha33, ha17 and botR genes were close to strain Okra (B1) while ha70 and ntnh genes were close to strain 111 (B2).

Conclusion/Significance

This study demonstrates the utility of molecular genotyping of C. botulinum and how it contributes to our understanding the epidemiology and variation of boNT gene. Thus, the recent botulism outbreaks in Thailand were induced by various C. botulinum types.     

Tailored ?-Cyclodextrin Blocks the Translocation Pores of Binary Exotoxins from C. Botulinum and C. Perfringens and Protects Cells from Intoxication

Background

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin are binary exotoxins, which ADP-ribosylate actin in the cytosol of mammalian cells and thereby destroy the cytoskeleton. C2 and iota toxin consists of two individual proteins, an enzymatic active (A-) component and a separate receptor binding and translocation (B-) component. The latter forms a complex with the A-component on the surface of target cells and after receptor-mediated endocytosis, it mediates the translocation of the A-component from acidified endosomal vesicles into the cytosol. To this end, the B-components form heptameric pores in endosomal membranes, which serve as translocation channels for the A-components.

Methodology/Principal Findings

Here we demonstrate that a 7-fold symmetrical positively charged ß-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-ß-cyclodextrin, protects cultured cells from intoxication with C2 and iota toxins in a concentration-dependent manner starting at low micromolar concentrations. We discovered that the compound inhibited the pH-dependent membrane translocation of the A-components of both toxins in intact cells. Consistently, the compound strongly blocked transmembrane channels formed by the B-components of C2 and iota toxin in planar lipid bilayers in vitro. With C2 toxin, we consecutively ruled out all other possible inhibitory mechanisms showing that the compound did not interfere with the binding of the toxin to the cells or with the enzyme activity of the A-component.

Conclusions/Significance

The described ß-cyclodextrin derivative was previously identified as one of the most potent inhibitors of the binary lethal toxin of Bacillus anthracis both in vitro and in vivo, implying that it might represent a broad-spectrum inhibitor of binary pore-forming exotoxins from pathogenic bacteria.     

Genotype-phenotype correlation in Japanese patients with familial Mediterranean fever: differences in genotype and clinical features between Japanese and Mediterranean populations

Introduction

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease characterized by recurrent self-limiting fever and serositis that mainly affects Mediterranean populations. Many patients with FMF have been reported in Japan due to increasing recognition of this condition and the availability of genetic analysis for the gene responsible, MEFV. The present study was performed to elucidate the clinical characteristics of Japanese FMF patients and to examine the precise genotype-phenotype correlation in a large cohort of Japanese FMF patients.

Methods

We analyzed the MEFV genotypes and clinical manifestations in 116 patients clinically diagnosed as having FMF and with at least one mutation.

Results

The most frequent mutation in Japanese patients was E148Q (40.2%), followed by M694I (21.0%), L110P (18.8%), P369S (5.4%), and R408Q (5.4%). In contrast, common mutations seen in Mediterranean patients, such as M694V, V726A, and M680I, were not detected in this population. The clinical features with M694I were associated with more severe clinical course compared to those seen with E148Q. P369S/R408Q showed variable phenotypes with regard to both clinical manifestations and severity. Patients with M694I showed a very favorable response to colchicine therapy, while those with P369S and R408Q did not.

Conclusions

Clinical features and efficacy of treatment in Japanese FMF patients vary widely according to the specific MEFV gene mutation, and therefore genetic analysis should be performed for diagnosis in cases of Japanese FMF.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0439-7) contains supplementary material, which is available to authorized users.     

Comparison of M.I.C.E. and Etest with CLSI Agar Dilution for Antimicrobial Susceptibility Testing against Oxacillin-Resistant Staphylococcus spp

Objective

The main objective of this study was to comparatively evaluate the performance of M.I.C.E. and Etest methodologies to that of agar dilution for determining the antimicrobial susceptibility profile of oxacillin-resistant Staphylococcus spp.

Methods

A total of 100 oxacillin-resistant Staphylococcus spp. isolates were collected from hospitalized patients at a teaching hospital. Antimicrobial susceptibility testing for vancomycin, teicoplanin and linezolid was performed using the reference CLSI agar dilution method (2009), Etest and M.I.C.E. methodologies. The MIC values were interpreted according to CLSI susceptibility breakpoints and compared by regression analysis.

Results

In general, the essential agreement (±1-log₂) between M.I.C.E. and CLSI agar dilution was 93.0%, 84.0% and 77.0% for linezolid, teicoplanin and vancomycin, respectively. Essential agreement rates between M.I.C.E. and Etest were excellent (>90.0%) for all antibiotics tested. Both strips (M.I.C.E. and Etest) yielded two very major errors for linezolid. Unacceptable minor rates were observed for teicoplanin against CoNS and for vancomycin against S. aureus.

Conclusions

According to our results, linezolid and teicoplanin MICs against all staphylococci and S. aureus, respectively, were more accurately predicted by M.I.C.E. strips. However, the Etest showed better performance than M.I.C.E. for predicting vancomycin MICs against all staphylococci. Thus, microbiologists must be aware of the different performance of commercially available gradient strips against staphylococci.     

In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

Background

Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate.

Methodology/Principal Findings

To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation.

Conclusions/Significance

The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study.     

Common genetic variations in the NALP3 inflammasome are associated with delayed apoptosis of human neutrophils

Background

Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1β production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages.

Methods and Findings

Patients carrying the Q705K/C10X polymorphisms displayed significantly delayed spontaneous as well as microbe-induced apoptosis compared to matched controls. Western blotting revealed increased levels and phosphorylation of Akt and Mcl-1 in the patients'' neutrophils. In contrast to macrophages from healthy controls, macrophages from the patients produced lower amounts of TNF; suggesting impaired macrophage clearance response.

Conclusions

The Q705K/C10X polymorphisms are associated with delayed apoptosis of neutrophils. These findings are explained by altered involvement of different regulators of apoptosis, resulting in an anti-apoptotic profile. Moreover, the macrophage response to ingestion of microbe-induced apoptotic neutrophils is altered in the patients. Taken together, the patients display impaired turnover and clearance of apoptotic neutrophils, pointing towards a dysregulated innate immune response that influences the resolution of inflammation. The future challenge is to understand how microbes affect the activation of inflammasomes, and why this interaction will develop into severe inflammatory disease in certain individuals.     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.     

Selective and specific internalization of clostridial C3 ADP‐ribosyltransferases into macrophages and monocytes

The C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono‐ADP‐ribosylate and thereby inactivate RhoA, ‐B and ‐C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP‐ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL‐60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface‐bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.     

Risk factors of Coxiella burnetii (Q fever) seropositivity in veterinary medicine students

Background

Q fever is an occupational risk for veterinarians, however little is known about the risk for veterinary medicine students. This study aimed to assess the seroprevalence of Coxiella burnetii among veterinary medicine students and to identify associated risk factors.

Methods

A cross-sectional study with questionnaire and blood sample collection was performed among all veterinary medicine students studying in the Netherlands in 2006. Serum samples (n = 674), representative of all study years and study directions, were analyzed for C. burnetii IgG and IgM phase I and II antibodies with an immunofluorescence assay (IFA). Seropositivity was defined as IgG phase I and/or II titer of 1∶32 and above.

Results

Of the veterinary medicine students 126 (18.7%) had IgG antibodies against C. burnetii. Seropositivity associated risk factors identified were the study direction ‘farm animals’ (Odds Ratio (OR) 3.27 [95% CI 2.14–5.02]), advanced year of study (OR year 6: 2.31 [1.22–4.39] OR year 3–5 1.83 [1.07–3.10]) having had a zoonosis during the study (OR 1.74 [1.07–2.82]) and ever lived on a ruminant farm (OR 2.73 [1.59–4.67]). Stratified analysis revealed study direction ‘farm animals’ to be a study-related risk factor apart from ever living on a farm. In addition we identified a clear dose-response relation for the number of years lived on a farm with C. burnetii seropositivity.

Conclusions

C. burnetii seroprevalence is considerable among veterinary medicine students and study related risk factors were identified. This indicates Q fever as an occupational risk for veterinary medicine students.     

Human Immunodeficiency Virus Type-1 (HIV-1) Continues to Evolve in Presence of Broadly Neutralizing Antibodies More than Ten Years after Infection

Background

The evolution of HIV-1 and its immune escape to autologous neutralizing antibodies (Nabs) during the acute/early phases of infection have been analyzed in depth in many studies. In contrast, little is known about neither the long-term evolution of the virus in patients who developed broadly Nabs (bNabs) or the mechanism of escape in presence of these bNabs.

Results

We have studied the viral population infecting a long term non progressor HIV-1 infected patient who had developed broadly neutralizing antibodies toward all tier 2/3 viruses (6 clades) tested, 9 years after infection, and was then followed up over 7 years. The autologous neutralization titers of the sequential sera toward env variants representative of the viral population significantly increased during the follow-up period. The most resistant pseudotyped virus was identified at the last visit suggesting that it represented a late emerging escape variant. We identified 5 amino acids substitutions that appeared associated with escape to broadly neutralizing antibodies. They were V319I/S, R/K355T, R/W429G, Q460E and G/T463E, in V3, C3 and V5 regions.

Conclusion

This study showed that HIV-1 may continue to evolve in presence of both broadly neutralizing antibodies and increasing autologous neutralizing activity more than 10 years post-infection.     

Identification of Genes from the Fungal Pathogen Cryptococcus neoformans Related to Transmigration into the Central Nervous System

Background

A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis.

Methodology/Principal Findings

Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the “Trojan horse” model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA.

Conclusions/Significance

This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry.     

No immune responses by the expression of the yeast Ndi1 protein in rats

Background

The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host''s immune system.

Methodology/Principal Findings

In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol.

Conclusions/Significance

We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.     

Glucuronidated quercetin lowers blood pressure in spontaneously hypertensive rats via deconjugation

Background

Chronic oral quercetin reduces blood pressure and restores endothelial dysfunction in hypertensive animals. However, quercetin (aglycone) is usually not present in plasma, because it is rapidly metabolized into conjugated, mostly inactive, metabolites. The aim of the study is to analyze whether deconjugation of these metabolites is involved in the blood pressure lowering effect of quercetin.

Methodology/Principal Findings

We have analyzed the effects on blood pressure and vascular function in vitro of the conjugated metabolites of quercetin (quercetin-3-glucuronide, Q3GA; isorhamnetin-3-glucuronide, I3GA; and quercetin-3′-sulfate, Q3''S) in spontaneously hypertensive rats (SHR). Q3GA and I3GA (1 mg/kg i.v.), but not Q3''S, progressively reduced mean blood pressure (MBP), measured in conscious SHR. The hypotensive effect of Q3GA was abolished in SHR treated with the specific inhibitor of β-glucuronidase, saccharic acid 1,4-lactone (SAL, 10 mg/ml). In mesenteric arteries, unlike quercetin, Q3GA had no inhibitory effect in the contractile response to phenylephrine after 30 min of incubation. However, after 1 hour of incubation Q3GA strongly reduced this contractile response and this effect was prevented by SAL. Oral administration of quercetin (10 mg/Kg) induced a progressive decrease in MBP, which was also suppressed by SAL.

Conclusions

Conjugated metabolites are involved in the in vivo antihypertensive effect of quercetin, acting as molecules for the plasmatic transport of quercetin to the target tissues. Quercetin released from its glucuronidated metabolites could be responsible for its vasorelaxant and hypotensive effect.     

Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.     

Candida albicans induces cyclo-oxygenase 2 expression and prostaglandin E2 production in synovial fibroblasts through an extracellular-regulated kinase 1/2 dependent pathway

Introduction

Synovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicans-induced septic arthritis is largely unknown.

Methods

Primary cultures of rat synovial fibroblasts were infected with C. albicans (ATCC90028). Immunocytochemistry, western blotting, and RT-PCR were performed to assess cyclo-oxygenase 2 induction. Phosphorylation of extracellular-regulated kinase (ERK1/2) following infection in the absence or presence of U0126 was assessed by western blotting whilst prostaglandin E2 production was measured by ELISA. Nuclear factor κB (NFκB) translocation was evaluated by an electrophoretic mobility shift assay.

Results

Infection of synovial fibroblasts with C. albicans resulted in cyclo-oxygenase 2 expression and prostaglandin E2 production. Cyclo-oxygenase 2 expression and prostaglandin E2 production was dependent upon extracellular-regulated kinase 1/2 phosphorylation, associated with activation of NFκB and significantly elevated in the presence of laminarin, an inhibitor of dectin-1 activity. Synovial fibroblasts adjacent to C. albicans hyphae aggregates appeared to be the major contributors to the increased levels of cyclo-oxygenase 2 and phosphorylated extracellular-regulated kinase 1/2.

Conclusions

C. albicans infection of synovial fibroblasts in vitro results in upregulation of cyclo-oxygenase 2 and prostaglandin E2 by mechanisms that may involve activation of extracellular-regulated kinase 1/2 and are associated with NFκB activation.